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Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin

Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of... Roseocin, the two-peptide lantibiotic from Streptomyces roseosporus, carries extensive intramolecular (methyl) lanthionine bridging in the peptides and exhibits synergistic antibacterial activity against clinically relevant Gram- positive pathogens. Both peptides have a conserved leader but a diverse core region. The biosynthesis of roseocin involves post-translational modification of the two precursor peptides by a single promiscuous lanthipeptide synthetase, RosM, to install an indispensable disulfide bond in the Rosα core along with four and six thioether rings in Rosα and Rosβ cores, respectively. RosM homologs in the phylum actinobacteria were identified here to reveal twelve other members of the roseocin family which diverged into three types of biosynthetic gene clusters (BGCs). Further, the evolutionary rate among the BGC variants and analysis of variability within the core peptide versus leader peptide revealed a phylum-dependent lanthipeptide evolution. Analysis of horizontal gene transfer revealed its role in the generation of core peptide diversity. The naturally occurring diverse congeners of roseocin peptides identified from the mined novel BGCs were carefully aligned to identify the conserved sites and the substitutions in the core peptide region. These selected sites in the Rosα peptide were mutated for permitted substitutions, expressed heterologously in E. coli, and post-translationally modified by RosM in vivo. Despite a limited number of generated variants, two variants, RosαL8F and RosαL8W exhibited significantly improved inhibitory activity in a species-dependent manner compared to the wild-type roseocin. Our study proves that a natural repository of evolved variants of roseocin is present in nature and the key variations can be used to generate improved variants. Keypoints • Taxonomically distant species have a wide diversity of roseocin family lantibiotics. • The phylogeny-guided approach opens new possibilities for RiPPs bioengineering. • Bioactive variants of roseocin were generated using semi-in vitro reconstitution. Keywords Actinobacteria, Lantibiotics, Phylum, Post-translational modification, Variants Sandeep Chaudhary and Shweta Kishen have contributed equally *Correspondence: Dipti Sareen diptsare@pu.ac.in Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Chaudhary et al. AMB Express (2023) 13:34 Page 2 of 20 is required. A detailed description of the mechanism and Introduction structure of the lanthipeptide synthetases is provided by Nature has bestowed upon the bacterial species an Repka et al. 2017. arsenal of antimicrobial moieties to benefit the host in The LanMs are of particular interest not only because intraspecies or interspecies competition under the limited of their bifunctional domain structure but also their natural resources of an ecological niche. The human substrate promiscuity. Evolutionary studies have shown race has benefited profoundly from these microbial a significant divergence among LanMs based on the conflicts with the discovery of novel antimicrobials to natural substrate promiscuity, defined by the number combat bacterial infections. However, extensive and of precursor peptides encoded within a single genome indiscriminate use of antimicrobials has fuelled the rapid (Zhang et  al. 2012). Highly promiscuous LanMs possess spread of antimicrobial resistance (AMR), rendering a CCG motif, instead of the CHG motif, for binding antimicrobials of similar type ineffective. One of the 2+ essential Zn ion at the active site (Zhang et  al. 2014). ways to solve this crisis is to discover other alternative ProcM is the first example of a CCG motif LanM with classes of antimicrobials having high efficacy against an ability to process 29 diverse precursor peptides, these multi-drug-resistant (MDR) pathogens along genetically encoded and spread throughout the genome with a scope for possible bioengineering in the future. of Prochlorococcus MIT9313 (Li et  al. 2010). In an Lantibiotics (or antimicrobial lanthipeptides) are one exciting study by Yang et al. 2018, the substrate tolerance such class that belong to ribosomally synthesized and and catalytic fidelity of ProcM were utilized to generate a post-translationally modified peptides (RiPPs), and their library of 1.07 × 10 non-native variants. The 36 peptides heterologous expression in E. coli based platforms has examined out of this library (made from ProcA2.8, facilitated the formation of new-to-nature molecules by which is one of the 30 naturally encoded substrates of site-directed/random mutagenesis and combinatorial ProcM) were all found to have a faithful installation of biosynthesis (Iacovelli et al. 2022). the two-ring scaffold, thus establishing the possibility Lanthipeptides are decorated with characteristic of generating lanthipeptide libraries with a wide range thioether rings that render them proteolytically stable of chemical diversity by utilizing such promiscuous and target-accessible (Montalbán-López et  al. 2021). LanMs. Another recently reported CCG motif containing The synthesis of a lanthipeptide begins from the precur - LanM, SyncM processes 79 naturally encoded precursor sor peptide (LanA), a genetically encoded linear peptide peptides and is the most promiscuous lanthipeptide (Fig.  1A), which is a variable component in the biosyn- synthetase known so far (Arias-Orozco et al. 2021). thetic gene clusters (BGCs), along with the lanthipep- Unlike CCG motif LanMs, comparatively limited tide synthetase, immunity proteins, and other tailoring promiscuity is exhibited by CHG motif containing enzymes. The precursor peptide has an N-terminal leader LanMs, which are involved in the biosynthesis of two- peptide and a C-terminal core, separated by a proteolytic component lantibiotics. These lantibiotics bifurcate the site for removal of the leader peptide following the com- action of a single-component lantibiotic into alpha- and plete post-translational modifications (PTMs) of the core beta-peptides in their synergistic antimicrobial mode of peptide (McAuliffe et  al. 2001; Oman and van der Donk action (Oman and Van Der Donk 2009; Bakhtiary et  al. 2010). A lanthipeptide leader region is essential in guid- 2017). The alpha-peptide is responsible for anchoring to ing the core peptide at the active site of the lanthipep- lipid II in the outer leaflet of the cytoplasmic membrane, tide synthetase for ring installation (Lubelski et al. 2009). and the beta-peptide potentiates the alpha:lipid II The lanthionine rings are installed in two steps; firstly, complex to form a heteromeric complex leading to the the dehydration of Ser/Thr residues occurs, which is fol - rapid cell lysis (Bakhtiary et  al. 2017; Oman et  al. 2011). lowed by intra-peptide Michael addition of cysteine resi- Roseocin is a recent example of a two-component dues (cyclization) to form lanthionine/methyllanthionine lantibiotic that results from the post-translational rings (Fig.  1B) (Xie et  al. 2004). Based on the structure modification of two precursor peptides by a single and function of lanthipeptide synthetase, four lanthipep- promiscuous CHG-type RosM (Singh et  al. 2020). tide classes (class I-IV) have been defined (Arnison et al. Such promiscuity is rare among other two-component 2013; Repka et al. 2017). To process class I lanthipeptides, lantibiotics, as all the lacticin 3147 family lantibiotics two separate dehydration (LanB) and cyclization (LanC) have two separate dedicated CHG type LanMs for the enzymes are encoded in the BGC. For class II lanthipep- two precursors (Mcclerren et  al. 2006; Xin et  al. 2016). tides, a single bifunctional domain-containing enzyme, The only characterized examples of single CHG-type LanM is present with an N-terminal dehydratase and a LanM and two precursors are cytolysin (Coburn and C-terminal cyclase domain. For class III and IV lanthip- Gilmore 2003), carnolysin (Lohans et  al. 2014) and eptides, a single enzyme (LanKC and LanL, respectively) bicereucin (Huo and Van Der Donk 2016), but all of with trifunctional domains, i.e. lyase, kinase, and cyclase Chaudhar y et al. AMB Express (2023) 13:34 Page 3 of 20 Fig. 1 Biosynthesis of the two-component lantibiotic roseocin. A Roseocin is constituted of two precursor peptides, RosA1β and RosA2α, which are post-translationally modified by RosM in two steps (dehydration followed by cyclization). This is followed by leader removal in vitro using commercially available proteases like endoproteinase GluC or proteinase K (Singh et al. 2020). The ring topology of roseocin peptides is supported by mass spectrometry and bioinformatic analysis as per the current study. B Common PTMs in the biosynthesis of lanthipeptide involves the dehydration of serine and threonine amino acid residues to 2,3-dehydroalanine (Dha) and 2,3-dehydrobutyrine (Dhb), respectively. Michael-type addition of Cys to Dha leads to lanthionine (Lan), while addition to Dhb results in a methyllanthionine (MeLan) ring. Abu aminobutyrate, Ala Alanine, SIVR semi-in vitro reconstitution, HP Hypothetical Protein, RosTp dual function peptidase-domain containing transporter, RosF group of immunity proteins (in red colour), TR Transcriptional Regulator these belong to the phylum r fi micutes. However roseocin, Instead of random mutagenesis, we utilized the sequence identified from S. roseosporus NRRL 11379 is the sole diversity among the identified roseocin homologs. The example, from the phylum actinobacteria, of single study of phylogenetically related BGCs is a promising LanM and two precursors that lack homology to any way to obtain an extensive collection of divergent of the known two-component lantibiotics (Singh et  al. congeners resulting from an evolutionary pressure 2020). Owing to the antimicrobial potential of roseocin (Wang et  al. 2022a). In a quest to identify homologs of against MDR strains and having previously developed its roseocin, we devised a methodology using lanthipeptide heterologous expression system in E. coli, we wanted to synthetases, which have evolved in a phylum-dependent explore its variants with improved antibacterial activity. manner (Zhang et  al. 2012; Yu et  al. 2013). An earlier Chaudhary et al. AMB Express (2023) 13:34 Page 4 of 20 study by Walker et  al. 2020 utilized the LanC domain bootstrap values in MEGA X using an outlier group. of lanthipeptide synthetase as a query and classified Homologs of RosM in Archaea (taxid:2157) were placed lanthipeptides by sequence similarity network in various as the outlier group. The final phylogenetic tree was subtypes, thus identifying > 8000 members of all the presented with iTOL v4 (Letunic and Bork 2019). four known classes of lanthipeptides. The extensive lanthipeptide analysis done by the group proved that Precursor peptides identification and biosynthetic gene the divergence in the lanthipeptide classes is ancient cluster (BGC) analysis and supports the hypothesis that the lanthipeptide The genome sequence of the hits was obtained in FASTA synthetases of different classes may have evolved through format from the Genbank database and analyzed with convergent evolution. BAGEL4 (Hart and Moffat 2016) and antiSMASH 5.0 We utilized the phylogenetic distribution of RosM (Blin et  al. 2019) web servers. Precursor peptides were homologs as a guide to explore the evolution of roseocin. identified manually by subjecting intergenic regions to All the roseocin homologs identified using this approach NCBI ORF finder. Putative functions were confirmed lacked the already known lipid II binding motif (Grein based on CDD (Lu et  al. 2020) and TMHMM server et  al. 2019) but were found to have a novel conserved analysis (https:// servi ces. healt htech. dtu. dk/ servi ce. php? motif in the two peptides. Considering that a naturally TMHMM-2.0). selected, more potent roseocin homolog may exist in nature, we selected the mutations that should not alter Tanglegram of evolutionary trees the Rosα peptide thioether rings, but could significantly Available 16S rRNA sequence was obtained from SILVA influence its activity. Only the site-specific mutants were reference database SSU r114 (Quast et  al. 2013) to plot generated by an efficient one step site-directed single site the species tree. Nucleotide sequences of lanM and plasmid mutagenesis protocol, followed by their success- lanA were fetched from the Genbank. The species and ful PTMs by RosM in vivo. The semi-in vitro reconstitu - gene tree were constructed with Maxiumum Likelihood tion (SIVR) strategy established earlier (Singh et al. 2020) (ML) method and subjected to the tanglegram algorithm was followed to obtain the bioactive core peptides for (Scornavacca et  al. 2011) incorporated in Dendroscope the determination of minimum inhibitory concentration ver. 3.7.2. Species vs gene tree tanglegram was used to (MIC) and structural characterization by mass spectrom- find horizontal gene transfer (HGT), while gene vs gene etry. Our study incorporated multiple approaches for tree was used to test the coevolution of LanM and leader investigating the lanthipeptide evolution i.e., an analysis or core peptides in the same operon. of evolution rates between BGC types, analysis of varia- bility within core peptide sequences versus leader peptide Variability analysis sequences, and an assessment of whether horizontal gene Variability among the set of genes was visualized in col- transfer is a major source of diversity. Assuming the BGC our gradient matrix or graphical form using the sequence as a single evolutionary unit, many overlooked aspects of demarcation tool version1.2 (SDT v1.2) (Muhire et  al. lanthipeptide evolution are revealed here that can aid the 2014). Shannon entropy for sequences aligned with evolution-based studies of lanthipeptides in future. clustal omega was calculated using a protein variability server (Garcia-Boronat et al. 2008). Evolutionary pressure Materials and methods (purifying, neutral, or positive) was tested using the Nei- Genome mining for RosM like LanMs and phylogenetic Gojobori method in a codon-based Z-test of selection in analysis MEGA X. The only sample under purifying or neutral RosM sequence (WP_010071701.1) was used as a query selection (p value < 0.05) was considered for the next step for blastp search on NCBI Genbank non-redundant to calculate the substitution rate (ῳ = d /d , i.e., the ratio N S protein sequences database (May, 2020). The top 100 hits of the rate of nonsynonymous substitutions (dN) and were aligned with MUSCLE (Edgar 2004) using MEGA X rate of synonymous substitutions (dS) per site in YN00 of (Kumar et  al. 2018) and processed by GBlocks ver0.91.1 PAML package (Pamilo and Bianchi 1993; Yang 2007). (Talavera and Castresana 2007) to select conserved domains for phylogenetic analysis by Bayesian method Variants generation by site‑directed mutagenesis (Huelsenbeck and Ronquist 2001), available on an Site-directed mutagenesis of selected roseocin alpha open server NGPhylogeny.fr (Lemoine et  al. 2019). The precursor peptide residues was carried out using phylogenetic tree was scrutinized to eliminate redundant Agilent’s QuikChange site-directed mutagenesis kit. hits from the same clade and genus. The remaining hits Primers and methodology were designed using the were pruned and analyzed using the maximum likelihood procedure described by Liu and Naismith 2008 study (ML) method and JTT matrix-based model with 500 (Additional file  1: Figure S11). Desalted primers at a Chaudhar y et al. AMB Express (2023) 13:34 Page 5 of 20 0.05  µM scale were obtained from Sigma Genosys. 2020). Log phase culture was diluted with Mueller The T (melting temperature of the primer-primer Hinton Broth (MHB) to obtain 2 × 10   CFU/mL. The m pp overlapping region) and T (melting temperature of bacterial suspension and the lantibiotic were added to m no non-overlapping primer region) were calculated for each 96-well microplates at a ratio of 1:1. The microplates were primer (Additional file  1: Table  S7). PCR was carried incubated overnight at 37 ºC. The lowest concentration out using Bio-Rad MyCycler thermocycler. The PCR at which no visible growth was observed, was considered products were treated with 5 units of DpnI at 37  °C for MIC. one hour, and the reaction was stopped by heating at 75  °C for 15  min. The PCR product was transformed Results into chemically competent E. coli DH5α. The mutations Phylogenetic analysis for selection of diverse RosM were confirmed through Sanger sequencing, and the homologs plasmid was retransformed into E. coli BL21(DE3) for RosM (WP_010071701.1) installs thioether rings on two protein over-expression and in  vivo post-translational peptides (Rosα and Rosβ) that differ structurally and modification by RosM (Singh et  al. 2020), as per the functionally but display synergistic antimicrobial activity details mentioned in Additional file 1. as roseocin. Due to the unique natural promiscuity of RosM, it was speculated that RosM may have evolved Reduction and alkylation of peptides distinctively and hence was subjected to query search For confirming ring formation and availability of free in the GenBank database, which resulted in hits from cysteine(s), endoproteinase GluC (NEB, #P8100S) a wide range of actinobacteria species and four other cleaved peptides (30  µM) in 50  mM Tris–HCl pH 8.0 phyla (Additional file  1: Figure S1A). In the top 100 were incubated with 1  mM TCEP (Tris(2-carboxyethyl) hits, a sequence identity ranging from 33.8–99.0% phosphine) at 37 ºC for 30  min to reduce the disulfide and conservation in CHG motif in the active site bond. Following reduction the peptide was alkylated by was observed. The majority of the hits represented the addition of 10 mM IAA (Iodoacetamide) for 90 min. actinobacteria (n = 45) and cyanobacteria (n = 29) Reaction was set up in 60 µL volume in 1.5  mL MCTs. (Additional file  1: Figure S1A). As expected, amino acid The samples were desalted using Pierce C18 spin sequence identity criteria for genome mining generated column (#89873) before sample analysis with MALDI- an uneven distribution of hits, making it challenging to TOF MS. evaluate all the hits for novel BGCs. However, a Bayesian phylogenetic analysis of the obtained hits led to the MALDI‑TOF MS analysis phylum-wise clade formation along with the formation of Matrix-assisted laser desorption/ionization time-of-flight subclades having BGCs of similar properties (Additional mass spectrometry (MALDI-TOF MS) was carried out file  1: Figure S1B), thus helping in the systematic on AB Sciex TOF/TOF 5800 system maintained at CIF, evaluation of distantly related RosM homologs. NABI, Mohali. The proteolytic digest was processed with Interestingly, the RosM query search did not result in any Pierce C-18 spin column and mixed with α-cyano- hits from r fi micutes, the only phylum with the lacticin 4-hydroxycinnamic acid (1  mg/mL) for analysis in the 3147-like two-component lantibiotics discovered so reflectron mode. The mass spectra were calibrated using far (Zhang et  al. 2012). This observation indicated the a mass standards kit for the calibration of AB scion TOF/ independent evolution of roseocin family from lacticin TOF instruments (#4333604). The TOF/TOF explorer family two-component lantibiotics. was used for data acquisition, and SeeMS (Chambers To understand the features of the respective BGCs, et  al. 2012) and mMass programs (Niedermeyer and we analyzed the genome sequences of RosM hits with Strohalm 2012) were used for data analysis. To get a deep BAGEL 4 (Hart and Moffat 2016) and antiSMASH 5.0 insight into the thioether rings pattern, we did MS/MS (Blin et al. 2019) webservers. Both software identified the analysis for all the variants. BGC cluster boundaries including all the major genes of the BGC, but showed limitations in defining the genes Antimicrobial activity analysis encoding precursor peptides. Hence, we located the The minimum inhibitory concentration of wild-type putative precursor peptide genes on the GenBank file or roseocin and its variants was determined with micro subjected intergenic gap regions to NCBI ORF-finder, enabling us to identify the specific precursor peptide broth-dilution method. Sterile 96-well microtiter plates encoding genes. As a major advantage of mining in a were treated with 200 µL of 1% (w/v) bovine serum phylogeny-guided manner, identical lanthipeptide pre albumin (BSA) in 1 × phosphate-buffered saline (PBS) - solution at 37 ºC for 30  min. After this, wells were cursors (termed redundant hits) were easily identified in washed with 1 × PBS to remove excess BSA (Ellis et  al. an initial analysis across the BGCs of the same subclade. Chaudhary et al. AMB Express (2023) 13:34 Page 6 of 20 Roseocin family BGCs identified in actinobacteria For example, in BGC analysis across actinobacteria, 18 Actinobacteria showed the presence of three divergent out of 22 hits from the Streptomyces genus and 10 out of subclades (Fig. 2), each displaying a characteristic pattern 12 species from the Micromonospora genus encoded an of genetic arrangement within the BGC. Initially, BGCs identical precursor (Additional file  1: Figure S1B). Such seemed unrelated owing to a difference in the organiza - redundant hits were eliminated to limit the sample size tion of genes, sequence, and the number of lanthipeptide and prevent skewing the final sequence alignment. Phylo - precursors, with some encoding more than one CHG- genetic branch lengths were observed as < 0.05 in LanMs type LanMs (Fig.  2). However, further analysis showed of actinobacteria which corresponded to BGCs encoding that all BGCs encode precursor peptides homologous identical precursors (Additional file  1: Figure S1). Hence, to either Rosα or Rosβ (Additional file  1: Figure S3A and such RosM hits were removed from the rest of the phyla. B, respectively). Based on the chronological order in the Finally, 42 RosM homologs from five phyla (Additional phylogenetic tree, these 13 BGCs were classified as type file  1: Table  S1) were selected for phylogenetic analysis 1–3, to represent their respective subclades (Figs.  2 and using an appropriate outgroup for rooting. Unrooted 3A). Roseocin was grouped as a member of the type 2 trees, like in Additional file  1: Figure S1B, are only use- class, having stringent conservation of roseocin BGC ful for visualization of the relatedness of sequences of features among the other mined members of the same different clades, while only a rooted tree provides insight subclade. Precursor peptides of type 2 BGCs also display into evolution. A careful selection of outgroups was fol- features that agree with the earlier postulated structure lowed, as suggested by Adamek et al. 2019, being neither of roseocin (Singh et  al. 2020) and hence were used as too distant nor too close to the ingroups of the dataset a platform for designing the variants of Rosα (explained under study. In a recent genome mining study (Makarova in later sections). However, precursor peptides in type 1 et  al. 2019), archaea have been shown to contain lan- and 3 BGCs showed a more significant variation in the thipeptide BGCs across the species of the Halorussus amino acids that might result in a different ring topology genus. Interestingly, these archaeal lanthipeptide BGCs of these lanthipeptides (Additional file  1: Figure S3). The are of class II type with a single CCG motif LanM and type 1 BGCs containing three instead of two precursor an unknown class of lanthipeptides. We selected three genes deviate from the usual two-component lantibiot- BGCs from the Halorussus genus in the archaea database ics (Fig. 3A). Such kind of BGCs have been characterized and placed them as the outgroup to plot the maximum earlier in the lacticin 3147 family (Xin et  al. 2016; Zhao likelihood (ML) phylogenetic tree using a 500 bootstrap and Van Der Donk 2016). There, additional precur - value (Fig. 2). sor was found to be a result of the duplication of one of BGC analysis of each of the 42 RosM hits from the final the two genes. Contrary to this, we did not observe any phylogenetic tree (Additional file  1: Figure S2) showed core sequence similarity in the third precursor peptide of a gradual shift in the genomic location of minimally type 1 subclade (designated as LanA2A) to either of the required biosynthetic genes, lanA (lanthipeptide other two lanthipeptides (Additional file  2), which ruled precursor), lanM and a bifunctional lanTp (peptidase out an evolutionary gene duplication event. Phylogenetic domain-containing transporter) alongside the subclades analysis showed that LanA2A is closely related to alpha (Fig.  2). In most BGCs, precursor genes were found homologs (Fig.  3B). This indicates that LanA2A is either upstream to lanM, which probably is the natural an alpha peptide that synergizes with a common beta- temporal order of their synthesis. The common observed peptide, or it may be a constituent of a novel three-com- feature in most of the precursor peptide genes was the ponent synergistic system. As an advantage of random NHLP (nitrile hydratase leader peptide) family signature genome mining for lanthipeptide, many small-sized rose- (Haft et al. 2010) in their leader region and a single lanM ocin homologs were also found in the study by Walker gene of CHG-type present for their processing (Fig.  2). et al. 2020 (Additional file  2). To classify such small-sized But more than one lanM genes carrying BGCs were also homologs into type 1–3 subclade, BGC analysis was done found across actinobacterial and cyanobacterial species. in the current study (Additional file  1: Figure S5) and var- In cyanobacteria, these BGCs showed precursor peptides ious peculiar attributes like missing or duplicated genes, having conservation in leader regions from two divergent multiple LanMs, etc. were noted and hence, they could types of leader families, i.e. NHLP and N11P (Nif11 not be categorized as either of the members of type 1–3 derived peptides) family, discussed later in detail (Fig. 4). subclade. These genes probably might be of lower signifi - Overall, these conservations and variations made it cance and might have come into temporary existence to intriguing to study the dataset further for the conserved get eliminated during the natural selection for the most features of lanthipeptide evolution. potential genes. Chaudhar y et al. AMB Express (2023) 13:34 Page 7 of 20 Halorussussp. HD8-83 Halorussussalinus Halorussussp. RC-68 Acnoalloteichus ordicus Streptomyces rhizosphaericus Type 1 (2) Agromycesaureus Pseudo. massilense Ornithinicoccushortensis Nonomuraea sp. 6K102 Type 2 (8) Glycomyces harbinensis Streptomyces spinoverrucosus Streptomyces sp. AD196-02 Streptomyces roseosporus Catellatospora methionotrophica Micromonospora arida Type 3 (3) Micromonospora noduli Archangium gephyra Meliangiumboletus Viosangiumsp. GDMCC 1.1324 (5) Cystobacterfuscus DSM 2262 Cystobacterfuscus DSM 52655 Thermosporothrix hazakensis (2) Thermogemmaspora carboxidivorans Acidobacteriabacterium AA17 Blastocatellia bacterium AA13 (3) Acidobacteriabacterium AA12 Stanieriacyanosphaera Nostocales cyanobacterium HT-58-2 (2) Nostoc sp. PCC 7524 Fischerella muscicola (3) Fischerellathermalis Nitrolancea hollandica Alkalinema sp. CACIAM 70d Synechocyss sp. PCC 7509 (2) Chamaesiphon minutus Chlorogloeopsisfritschii Nostoc sp. MBR 210 Nostoc piscinale Nostoc minutum NIES-26 Coleofasciculuschthonoplastes (10) Tolypothrixsp. PCC 7601 Tolypothrixsp. PCC 7910 Hapalosiphon sp. MRB220 Nostoc sp. 106C Nostoc carneum Archaea Actinobacteria Proteobacteria Chloroflexi Acidobacteria Cyanobacteria CHG type NHLP leader C39 HlyD, type I N11P leader CCG type LanM LanM peptidase secretion protein Fig. 2 Phylogenetic tree of 42 selected RosM homologs showed conservation of gene locus and characteristic features along the phylogenetic tree. Roseocin family constituted the BGCs from actinobacteria, having three types of BGCs’ organization (type 1–3), each forming a separate subclade. CHG motif LanM for processing of NHLP type leader sequence was found in all the BGCs except in a subclade of cyanobacteria (Synechocystis sp. PCC 7509 and C. minutus) where conservation of two types of LanMs (CHG and CCG motif ) and two types of leader sequence (NHLP and N11P) were found in a single BGC. LanMs from the BGCs of Halorussus genus were placed in the root. Value from 500 replicates bootstrap test is indicated on each branch. The numbers given in the bracket are the number of members of that particular subclade. NHLP nitrile hydratase leader peptide, N11P Nif11 derived peptides The type 3 BGCs of roseocin family, consisting of two this separate LanM might have resulted from a recent LanMs and two precursor peptides (Fig.  3A), were con- LanM gene duplication event (Additional file  1: Fig- fined to the Micromonosporaceae family (Fig.  2). These ure S4), unlike the two LanMs in the lacticin 3147 fam- BGCs encoded a supersized homolog of Rosα, with the ily, which have low sequence identity (24–29%) and one highest number of thioether-forming moieties (13 Ser/ LanM has evolved specificity for modification of only one Thr and 9 Cys residues) in a single precursor peptide of the two precursors (Mcclerren et  al. 2006). A similar (Fig.  3E; Rosα has 5 Ser/Thr and 6 Cys residues). Such a sequence identity score in the pairwise alignment of lan- huge precursor peptide probably necessitates a dedicated thipeptide leaders (Additional file  1: Table S2) is surpris- LanM for efficient post-translational modification in ing and can make sense only under the coevolutionary parallel to the LanM for the beta peptide. High pairwise phenomenon, a perspective discussed in detail in the fol- sequence identity of LanMs of the Micromonosporaceae lowing sections. family (~ 50%) (Additional file  1: Table S2) indicates that Chaudhary et al. AMB Express (2023) 13:34 Page 8 of 20 Fig. 3 Diversity among the 13 representative members of the roseocin family. A Three common types of BGCs encode roseocin homologs, type-1, type-2, and type-3 BGC examples are of S. rhizosphericus, S. roseosporus NRRL 11379 and C. methinotrophica, respectively. B Phylogenetic tree of lanthipeptide core sequences with ML method with bootstrap values of 500 replicates. The exceptional third precursor (LanA2A) of the single LanM-three precursor i.e. type-1 BGC, is phylogenetically related to alpha peptides. Colour coding in Fig. 3B is red: alpha peptides; green: beta peptides; blue: third precursor core region. C and D Variation in the core peptide sequences as a function of Shannon entropy in the roseocin alpha and beta homologs, respectively. The alpha peptides contain a S/TxxxxTxGCC motif at the N-terminal end, and beta homologs contain a GS/ TxxxxS/TxGCC motif at the C-terminal end. E A gigantic Rosα homolog from the Micromonosporaceae family contains nine Cys and thirteen Ser/Thr residues that may form as many lanthionine rings. Rosα of Streptomyces roseosporus (S. roseo) contains an indispensable disulphide bond and four (methyl)lanthionine rings (dotted lines depict the proposed ring topology in Rosα, Singh et al. 2020); M. arida-Micromonospora arida. LanA precursor peptide, LanM modification enzyme, HP Hypothetical Protein, LanT dual function peptidase-domain containing transporter As discussed earlier, alpha peptide initiates the interac- the roseocin homologs, a Shannon entropy (SE) analysis tion with the bacterial membrane by targeting lipid II, a was done. Lower SE value (< 2.0) indicates higher con- key step in the mechanism of action of two-component servation of amino acid residues through evolution (Gar- lantibiotics (Bakhtiary et  al. 2017; Oman et  al. 2011). cia-Boronat et  al. 2008). A conservation of a ten amino Most of the alpha peptides characterized to date pos- acid long stretch, S/TxxxxTxGCC, at the N-terminus of sess an Asp/Glu residue containing lipid II binding motif Rosα homologs (Fig.  3C) and an 11 amino acid stretch, (CTxTxD/EC), which is absent in Rosα peptide (Singh GS/TxxxxS/TxGCC at the C-terminus of Rosβ homologs et  al. 2020). Using the knowledge generated in the cur- (Fig.  3D) was observed. Both the motifs were proposed rent study on the diversity of the roseocin family, it seems to have a structure with overlapping lanthionine rings in necessary to look for a novel motif for a similar or diver- our earlier study (Singh et  al. 2020). Such a ring struc- gent action mechanism. To understand the variability ture at the N-terminus of Rosα homologs is analogous and conservation of amino acid substitutions among all to the nisin-like peptides (having two N-terminal rings, Chaudhar y et al. AMB Express (2023) 13:34 Page 9 of 20 Fig. 4 Novel BGCs, encoding diverse lanthipeptide core sequences, consist of two LanMs for processing two types of precursor peptides. A Two BGCs encoding NHLP and N11P family lanthipeptide leaders in their precursor peptides with the corresponding synthetases, i.e. CHG motif and CCG motif LanM, were identified in Synechococcales. B Sequence logos of NHLP family and N11P family lanthipeptide leader sequences using the precursor sequences from the above two BGCs. C Sequence alignment of cyclase domain of putative LanMs from characteristic BGC of Synechococcales showed a difference in catalytic motif. RosM like LanMs has a CHG motif, while ProcM like LanMs have a CCG motif. D Sequence identity percentage in the pairwise alignment of the 12 lanthipeptide precursors’ leader sequences (lower half ) and core sequences (upper half ). Diversity among lanthipeptide core sequences was high, irrespective of leader conservation. E Multiple sequence alignment of lanthipeptide core sequences depicts natural diversity. LanA precursor peptide, LanM modification enzyme, HP Hypothetical Protein, LanT dual function peptidase-domain containing transporter Chaudhary et al. AMB Express (2023) 13:34 Page 10 of 20 proven to be responsible for target binding), instead of (Nif11 derived peptides; cl06756 subfamily TIGR03798) an Asp/Glu residue-specific target binding motif of the are the two well-characterized lanthipeptide leader two-component lacticin 3147-family lantibiotics (Cooper types that have evolved from nitrile hydratase enzyme et  al. 2008; Bakhtiary et al. 2017). Increased SE (> 2.0) in and Nif11 proteins, respectively (Haft et  al. 2010). the other amino acid sequence positions (Fig.  3C, 3D, Usually, a single type of lanthipeptide leader, i.e. either and Additional file  1: Figure S3A, S3B) revealed the innu- of the NHLP or N11P, is observed in a BGC (Zhang merable combinations experimented by nature, as is evi- et  al. 2014). However, an exception was observed in the dent by the changes in the number of Ser/Thr and Cys cyanobacteria (Fig.  2), which earlier were the source of residues of the core region among the Rosα and Rosβ prochlorosin family lanthipeptides as well (Cubillos- homologs. Except for the stretches mentioned above, Ruiz et  al. 2017). A non-conventional BGC with both substitutions were allowed at all the amino acid positions. types of lanthipeptide leader (NHLP and N11P), along Further, plausible exchange of indispensable disulfide of with two LanMs in a single BGC, for the maturation of Rosα with thioether ring in Streptomyces rhizosphaericus three and nine precursor peptides (Fig.  4A and B) was (Additional file  1: Figure S3A); exchangeable lanthionine identified by a manual search of the nearby ORFs. This (Lan) and methyllanthionine (MeLan) rings; insertion/ type of BGC was found confined to Synechococcales and deletion of one or more thioether rings suggests the included Synechocystis sp. PCC 7509 and Chamaesiphon enormous scope of modular engineering of both, Rosα minutus as member species (Fig.  4A). As N11P family and Rosβ peptides (Additional file  1: Figure S3). The pres - lanthipeptides are only associated with the CCG motif ence of a conserved motif and variability in the rest of the LanM, we speculated one of the LanMs to be the CCG core region probably results from balanced combinatorial motif LanM. Surprisingly, sequence alignment showed chemistry, operating parallelly with the conserved motif- the presence of ProcM-like CCG motif LanM in the oriented evolution of lanthipeptides. same BGC besides a CHG motif LanM (Fig.  4C). This However, the rest of the BGCs from other phyla unprecedented example of association between two showed no significant core sequence conservation. The leader types and two LanM types in a single BGC lanthipeptides of proteobacteria, chloroflexi, acidobacteria, indicates another evolved mechanism of diversity- and cyanobacteria phyla seldom have significant antimicro - oriented BGCs in cyanobacterial species that could bial activity (Mohr et  al. 2015; Cubillos-Ruiz et  al. 2017; be a better way of efficient biosynthesis of diverse Bothwell et  al. 2021). Nevertheless, we proceeded fur- lanthipeptide core sequences (Fig. 4D and E). ther and discovered many overlooked aspects of lanthi- Three lanthipeptide precursor sequences (2 + 1) in peptide BGCs, providing new insights into lanthipeptide C. minutus and nine (4 + 5) in Synechocystis sp. PCC evolution. 7509 represents an intermediate number of diverse sequences observed earlier for prochlorosin-like genes (Cubillos-Ruiz et  al. 2017). A truncated gene found in A new diversity‑oriented class of lanthipeptides C. minutus genome (Additional file  2) could result from in cyanobacteria mutations like frameshift or early stop codon, preventing Unlike significant conservation observed above in the the synthesis of a functional ORF. Such pseudogenes core region of the roseocin-like lanthipeptides, diversity- are a common feature of the prochlorosin family oriented evolution is characterized by the generation of lanthipeptides and support the ongoing diversification of a vast variety of lanthipeptide core sequences with no precursor genes in a diversity-oriented manner (Cubillos- conservation at all (Zhang et al. 2012; Cubillos-Ruiz et al. Ruiz et  al. 2017). In the C. minutus genome, four more 2017). So far, prochlorosin-like BGCs are the only exam- distantly located N11P-type lanthipeptide precursors ple which have evolved a highly promiscuous LanM (with were found, which might also be associated with this CCG motif ) for the maturation of diverse lanthipeptide BGC (Additional file  1: Figure S2). Intrigued by the novel sequences in marine cyanobacteria i.e. Synechococcus and mechanism of diversity generation in Synechococcales, we Prochlorococcus (Li et  al. 2010; Mukherjee and Van Der further analyzed the other BGCs to identify conserved Donk 2014). Similarly, in our dataset, freshwater cyano- features of relevance. bacterium species from Synechococcales also showed the diversity-oriented lanthipeptide BGCs, but with a novel, exquisitely divergent mechanism (Fig. 4). Coevolution of lanthipeptide leader and lanthipeptide In the current study, despite the expansion of hits synthetase among different phyla across different phyla, obtained BGCs had CHG- Lanthipeptide precursor is derived from an assimila- type LanM, for processing NHLP-type lanthipeptide tion of a protein tailored as a leader sequence with an precursors (Fig.  2). NHLP family (or nitrile hydratase independently evolving core sequence rich in Ser/Thr leader peptide; cl22942 subfamily TIGR03898) and N11P and Cys residues (Haft et al. 2010; Zhang et al. 2012). In Chaudhar y et al. AMB Express (2023) 13:34 Page 11 of 20 Fig. 5 Conservation of lanthipeptide leader sequence over diverse core sequences. A Multiple sequence alignment using MUSCLE of all the identified 68 precursor peptides from 42 BGCs. All leader sequences are from the NHLP family of lanthipeptide leaders. Conserved residue positions are highlighted. B Pairwise identity among 68 lanthipeptide leader sequences (lower half ) and core sequences (upper half ) showed high similarity among leader over core sequences (except lanthipeptide core sequences of the roseocin family having core conservation). C Cumulative frequency of pairwise sequence identity among the lanthipeptide leader and core sequences, respectively. 80% of lanthipeptide leader pairwise alignment showed > 39% identity. However, only 10% of lanthipeptide core pairwise alignment fulfilled the same criteria (mainly of the roseocin family) Chaudhary et al. AMB Express (2023) 13:34 Page 12 of 20 a previous study by Zhang et  al. 2014, the ProcM (hav- S6B). Earlier, the conservation of two leader family types ing CCG motif ) was used for genome mining, and thus and two different types of LanMs in a single BGC of C. obtained BGCs showed highly varying lanthipeptide minutus and Synechocystis sp. (Fig.  4) also suggested an leader families. However, in our study, despite the diver- essential linkage between the leader and lanthipeptide sity among BGCs from a wider range of phyla, high synthetases. conservation among the leader region of the precursor To explore further, we determined the mutation peptides was observed (Figs.  2 and 5A). The only excep - rates of both the lanM and the lanthipeptide leader tion was cyanobacterial species (Fig.  4), which could be genetic region by calculating the d /d ratio, which is N S unearthed by manual inspection of ORFs that other- the ratio of the rate of nonsynonymous to synonymous wise would have been missed (Singh and Sareen 2014; mutations. The lanM and lanthipeptide leader genetic Zhang et al. 2014). Thus, we found that all leader peptide regions from 13 BGCs of actinobacteria (roseocin fam- sequences belonged to the NHLP family (Fig. 5A). In 80% ily) and 9 BGCs of cyanobacteria were selected for the of pairwise sequence alignments of leader sequences, separate analysis of two phyla. The calculated d /d for N S we observed > 39% sequence identity; while only in 10% lanM and leader peptide exhibited distinct patterns for of the core sequences pairwise alignments, an identity the two phyla (Fig.  6A). The d /d ratio was in agree- N S of > 39% was observed (Fig.  5B and C). These identity ment with the phylum-wise evolution of lanthipeptide scores support the fact that conservation in leader pep- synthetases (Zhang et al. 2012), as well as the coevolu- tides does not restrict the lanthipeptide core diversifi - tion of lanthipeptide synthetases and leader sequences. cation even among different phyla. Variability pattern It has been proposed earlier by Cubillos-Ruiz et  al. was also plotted for all the 42 LanMs (Additional file  1: 2017, that a lower d /d ratio is confined only to the N S Figure S6A) which surprisingly had an overlap with the lanMs having the CCG motif of the prochlorosin fam- variability in leader regions (Additional file  1: Figure ily (or ProcMs), suggesting an evolutionary locked state Fig. 6 Coevolution of lanthipeptide leader and synthetase in a phylum-dependent manner. A Pairwise d /d distribution of lanthipeptide leader N S and lanM from actinobacteria (13 BGCs of roseocin family) and cyanobacteria phylum (9 BGCs) showed variation in different phyla suggesting coevolution of the lanthipeptide leader and synthetases. B Standard box plot of the evolutionary rates of CCG motif prochlorosin family LanMs (Cubillos-Ruiz et al. 2017) (Additional file 1: Table S3) and CHG motif LanMs of cyanobacteria (from the current study) showed a similar pattern (median 0.21 and 0.24, respectively), while the CHG motif lanMs of roseocin family in actinobacteria has a higher value (median 0.48). In the standard box plot, the lower and upper shows the first and third quartile values, respectively, separated by the median value. The error bar plots the minimum and maximum values. C Phylogenetic tree of CCG motif ProcMs (Cubillos-Ruiz et al. 2017) and CHG motif LanMs of cyanobacteria and actinobacteria (from current study). Prochlorosin family LanMs displayed significant divergence from CHG motif LanMs of cyanobacteria and actinobacteria Chaudhar y et al. AMB Express (2023) 13:34 Page 13 of 20 + + + + Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] mono. m/z 3848.76 mono. m/z 3960.76 mono. m/z 3887.75 mono. m/z 3999.75 LL88FF LL88W W + + + + Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] mono. m/z 3913.81 mono. m/z 4025.81 mono. m/z 3843.75 mono. m/z 3955.75 S S1122W W VV2222Q Q Fig. 7 MALDI-TOF MS of endoproteinase GluC treated Rosα after TCEP only and after TCEP + IAA alkylation assay. Additional file 1: Table S5 represents the calculated and observed mass of the respective species of each variant generated that favors the catalytic promiscuity for the process- diverged during evolution into two subclades of CHG ing of diverse precursors. Interestingly, cyanobacterial and CCG motif LanMs, both being locked into a simi- lanMs with CHG motif (hence we proposed the name, lar evolutionary conserved state and probably having a CyanMs) found in the current study also displayed similar level of substrate tolerance. The reason for such a lower d /d ratio, i.e. 0.24 (Fig.  7B), suggesting an a divergence is not clear, but the significance of phy - N S evolutionary linkage between CyanMs and ProcMs. lum in deciding the fate of lanthipeptide synthetases However, phylogenetic analysis showed a significant enforces the phylum-dependent effect on the evolution divergence of ProcMs from CyanMs (Fig. 7C) even with of lanthipeptides than proposed earlier (Cubillos-Ruiz similar d /d values (Fig.  7B). This indicates that lan - et al. 2017). N S thipeptide synthetases of the cyanobacteria must have Chaudhary et al. AMB Express (2023) 13:34 Page 14 of 20 Production of evolutionarily selected natural variants disorder to order upon target binding. We hypothesize of Rosα that the four variable residues in this stretch (3-GVLG- The gene-encoded nature of lantibiotics offers an oppor - 6) probably indicate disorder among the fully conserved tunity to bioengineer the peptide components for ring-forming residues. Hence, alterations in this motif obtaining roseocin variants with improved bioactivity/ can significantly affect the binding affinity of Rosα to its physicochemical parameters. The evolutionarily selected target and consequently the antimicrobial activity/MIC natural variants of Roseocin peptides helped us identify of roseocin. Leu8 of this motif seems to be significant due the structural regions amenable to amino acid substitu- to its relatively high conservation, with the only naturally tions. Keeping in mind the earlier common observation allowed substitutions being L8I, L8F, and L8W. Leu8 was on substitutions that disrupt (methyl)lanthionine forma- thus mutated to F and W. Serine at position 12 being the tion, ring size or lanthionine location leading to struc- residue that we characterized earlier to have escaped tural alteration with loss of bioactivity (Barbosa et  al. dehydration (Singh et al. 2020) and W being the only nat- 2019; Bédard et al. 2019; Field et al. 2015; Rahman et al. urally allowed substitution, we tried substituting S12 for 2021), and the limitation of PTM enzymes that gener- W. V22Q substitution was selected for it might affect the ally do not accommodate all amino acid substitutions overall hydrophobicity of the peptide and hence its inter- (Cooper et al. 2008), only a few naturally allowed substi- action with the target. tutions were tried to prove our concept. These evolutionarily selected Rosα variants were gener - We focused specifically on the actinobacteria clade of ated by site-directed mutagenesis and tested for the PTM the roseocin family phylogenetic tree, as the sequences by RosM in vivo in E. coli BL21(DE3). Notably, unlike the arising from BGCs sharing close common ancestors and, other studies where low expression of the lantibiotic vari- thus, the same mode of action are likely to group into the ants is often observed (Field et al. 2013; Geng and Smith same clade. For generating naturally inspired roseocin 2018), phylogeny-guided mutations generated in Rosα variants, we identified the most conserved and divergent led to a substantial yield of the bioengineered variants to sites in roseocin peptides’ homologs (Fig. 3C and D). Var- allow their further analysis. All four RP-HPLC purified iants were restricted to the type 2 subclade only (roseocin full-length peptide variants were analyzed by ESI–MS being its member; Fig.  3A), as types 1 and 3 exhibited (Additional file  1: Figure S8; Table  S4). As was reported significant divergence in BGC structure along with a earlier for the wild-type Rosα, that underwent four-fold divergence in the sequence of both alpha and beta com- dehydration (Singh et  al. 2020), observed mass peaks in ponents. Further, highly divergent variations of type 1 Rosα L8F, L8W, S12W, and V22Q agreed with the calcu- and 3 subclades from the roseocin peptides might hinder lated mass having four-fold dehydrations and one disul- the post-translational modifications by RosM. A consen - phide bond (Additional file  1: Table  S4). An additional sus sequence was obtained using the alignment of Rosα peak of + 89  Da (double-charged ion for 178  Da) mass and Rosβ homologs from the type 2 subclade for the nat- shift indicated the presence of species with N-terminal urally permissible mutations (Additional file  1: Figure S7). gluconoylation. The RP-HPLC purified peptide variants As alpha peptide plays a pivotal role in the mechanism were subjected to leader cleavage with endoprotein- of two-component lantibiotics (Oman et  al. 2011; Bakh- ase GluC and analyzed with MALDI-TOF MS and M S tiary et  al. 2017), Rosα peptide variants were designed (Fig.  7). The MALDI-TOF MS data of the four variants first. Among Rosα homologs, essential amino acid resi - (after TCEP treatment) agreed with the calculated m/z dues required for (methyl)lanthionine rings installation of the reduced peptide i.e. 2  Da higher than the theo- showed high conservation along with the two cysteines retical monoisotopic mass, indicating a reduction of involved in the disulphide bond formation. Another con- the disulfide bond between Cys13 and Cys33 (Fig.  7). served stretch i.e. S/TxxxxTxGCC was clearly notice- Alkylation with IAA confirmed the disulfide bond and able, which we speculate to be a putative target binding assessed the presence of free cysteines, if any, arising motif. Two critical observations favour this speculation: due to incomplete dehydration or cyclizations. A mass first, the ‘ring within a ring’ structure of this N-terminal shift of 57  Da was expected per available free cysteine region, proposed by us due to the absence of fragment residue. It was observed that the natural variants were ions in the MALDI-TOF MS/MS analysis (Singh et  al. fully dehydrated and cyclized to possess four (methyl) 2020). Second, from the pockets and mouth information lanthionine rings and a disulfide bond (Fig.  7, Additional revealed by the homology modelling of 37 characterized file  1: Table  S5). The MS analysis of the leader-cleaved lantibiotics (Chakraborty et  al. 2019), the disordered alkylated peptides further revealed protection imparted residues (the ones involved in protein–protein interac- by (methyl)lanthionine rings. The absence of b and y tions or target binding) were found within the pockets/ fragment ions for the stretches 2-SGVLGTLGCC-11 rings and hence are thought to undergo a transition from and 18-SGWTVCGLAC-27 confirmed thioether ring Chaudhar y et al. AMB Express (2023) 13:34 Page 15 of 20 Fig. 8 Tandem MS fragmentation pattern of alkylated Rosα variants confirms the similar ring topology in all the variants as that of wild-type Rosα, published earlier (Singh et al. 2020). Underlined regions correspond to leader sequence overhang that remained attached after endoproteinase GluC proteolytic digestion Table 1 Minimal inhibitory concentration (MIC) of wild-type concentration (MIC) was determined to compare the roseocin and its variants efficacy of variants with respect to wild-type Rosα against M. luteus ATCC 10240 and methicillin-sensitive Lantibiotic MIC against M. luteus MIC against MSSA ATCC 10240 (µM) ATCC 25923 (µM) Staphylococcus aureus (MSSA) ATCC  25923 (Table  1). Variants generated by substitution within the N-termi- Roseocin (wild-type) 0.5 2 nal conserved motif of Rosα i.e. L8F and L8W showed Roseocin L8F 0.5 0.5 improvement in the MIC. RosαL8F exhibited four-fold Roseocin L8W 0.25 2 lower MIC against MSSA ATCC 25923, while RosαL8W Roseocin S12W 0.5 > 16 showed two-fold lower MIC against M. luteus ATCC Roseocin V22Q > 8 > 16 10240. However, no improvement with RosαS12W and Nisin 0.0625 2 a loss of potency in RosαV22Q were observed. Consid- ering individual roseocin homologs have many simulta- neous amino acid alterations instead of a single residue protected regions in all the variants (Fig.  8), similar to (Additional file  1: Figure S3), some mutations like V22Q wild-type Rosα (Singh et  al. 2020). The fragment ions might depend upon those other amino acids to exert a (Additional file  1: Table  S6) confirmed the dehydration positive effect on antimicrobial activity. The MIC data of Ser2, Thr7, Ser18, Thr21 and the undehydrated status shows that our strategy of mutants generation to obtain of Ser12. The mass shift of 57 Da in y4-y22 indicated the only the bioactive variants was successful and hence can alkylation of Cys33 and a shift of + 114  Da in the frag- be extrapolated to obtain multi-residue variants to match ment ion next to y22 the disulfide bond partner as Cys13. the natural congeners. The thus-engineered best variant The observed fragment ions confirmed the presence of can be subjected to detailed structural and functional a disulphide and 4 (methyl)lanthionine rings in all the characterization for development into a new drug. Rosα variants (Fig.  7). All the variants were tested for their bioactivity in combination with wild-type Rosβ by Phylogeny‑based ring structure prediction. agar diffusion assay against M. luteus ATCC 10240, after Structure elucidation of lanthipeptides is often hindered leader cleavage by proteinase K (Additional file  1: Figure owing to low yield and complexity in the NMR data S9). Bioactivity was observed with all the variants (Addi- acquisition and solving the data. Tandem MS has tional file  1: Figure S9) however, minimum inhibitory Chaudhary et al. AMB Express (2023) 13:34 Page 16 of 20 been widely successful in depicting the structure with to the genome mining study of Walker et al. 2020, which a low amount of lanthipeptides. However, a lack of identified and classified > 8000 precursor peptide hits, fragmentation is observed in the region with overlapping including the members of the roseocin family. Cyclase rings (Garg et al. 2012; Singh et al. 2020), like that in wild- domain (being a common feature among all the currently type Rosα peptide and its variants (Fig. 8). The structural known classes of lanthipeptide synthetases) was used features of roseocin peptides could be solved only to as a query [i.e. LANC_like (PF05147) hidden Markov the extent of alternative possibilities in the ring pattern model (HMM)] from the Protein family (Pfam) database of Rosα, while in Rosβ, only the first two rings A and B (Finn et  al. 2016), which resulted in 12,705 proteins. could be figured out by tandem MS. As an unanticipated Further classification into Class I–IV lanthipeptide advantage of the current phylogenetic study, the ring BGCs was achieved by analyzing the genomic content pattern in the overlapping regions of both the roseocin of each of these for the presence of Pfam HMM for the peptides could further be unfolded, as it seemed that dehydratase domain/LanM/protein kinase (Class III and they are dispensable, probably to gain some advantage in IV). Further on, genes for precursor peptides (being the terms of antimicrobial activity after the ring alteration/ most diverse and hence the least similar) were identified structure modification. in a representative sample from the BGC of each class Since (methyl)lanthionine rings are the result of bond by manual examination of the lanthipeptide-specific formation between a conserved Thr/Ser and a Cys features and genetic distance from the processing residue, the simultaneous deletion of both the Ser/Thr enzyme. Unlike our study, the phylogenetic distance and and Cys residues was used as the hallmark for deducing BGCs organization formed the basis of the grouping the ring pattern. Rosα peptide contains four (methyl) and subgrouping to understand lanthipeptide diversity. lanthionine rings (labelled A to D) and one disulfide However, the proposed class II29/31 for Rosα homologs bond. Based on earlier tandem MS study, Rosα is a excluded roseocin family members of the type 3 subclade, globular-shaped peptide with two pairs of overlapping from the Micromonosporaceae family while at the same regions, i.e. N-terminal rings A and B, and the C-terminal time included many redundant hits of the roseocin rings C and D (Singh et al. 2020). Ring A and B are part of family. For Rosβ homologs, class II2 was proposed that the essential motif of Rosα and its homologs; therefore, included hits from unrelated distant phyla, r fi micutes the mutation in this region was neither expected and cyanobacteria (roseocin being a member of nor found in any of the homologs. However, in the actinobacteria). Thus, the sequence logo generated from C-terminal region, simultaneous deletion of constituent there, varies hugely in member species and conservation residue partners was observed in Rosα homolog from patterns to ours. However, a larger sample size in Walker Nonomuraea (Additional file  1: Figure S10A), showing et  al. 2020 study helped in identifying five new BGCs the possible location of ring C. of the type 2 subclade of the roseocin family (single Rosβ structure has been proposed earlier as a linear LanM-two precursor BGC; Additional file  2), making type 2 the largest subclade of roseocin family (including peptide with a total of six (methyl)lanthionine rings eight from the current study) causing it to have total 13 (labelled A to F). The rings (A and B) were identifiable members (Additional file  1: Figure S3). A consensus logo by tandem MS (Singh et al. 2020), while the rest are part obtained by the multiple sequence alignment of these 13 of the overlapping ring structure. Rings E and F seem indispensable, hence, their ring topology could not be members has formed the basis of our amino acid residue predicted here. Ring C location has been predicted by identification to generate Rosα variants (Additional missing constituent residue partners in M. Muleris, A. file  1: Figure S7A). Overall, the combined approach of radicidentis etc. (Additional file  1: Figure S10B). This left genome mining tools with the evolutionary principles only one possibility of ring D for which missing residue can rapidly and conclusively classify the members of all partners can also be confirmed in Rosβ homologs of S. the lantibiotic families, along with designing the natural rhizosphaericus and A. fjordicus (Additional file  1: Figure variants to select candidates with better efficacy. S10B). Recently, by coupling genome mining methods to identify evolutionarily related polymyxin family-like BGCs from ~ 10,858 sequenced bacterial genomes, Wang Discussion et  al. 2022b successfully identified macolacin, a colistin- Phylogeny-guided mining approach has been successfully like antibiotic that is active against colistin-resistant applied to discover new NRPS/PKS natural products from Gram-negative pathogens. Since the colistin resistance microbial genomes and metagenomes (Kang 2017), but it is mediated by either mcr-1 or intrinsic PEtN transferase is for the first time being applied here for two-component genes (eptA), they reasoned that a solution might have lantibiotics. Phylogeny-guided approach for mining evolved through natural selection to circumvent this roseocin family lantibiotics displays a comparative view Chaudhar y et al. AMB Express (2023) 13:34 Page 17 of 20 seems to evolve as a result of the selection pressure of troubling resistance mechanism and bioinformatically the target structure (Zhang et  al. 2012). However, no searched for the naturally evolved colistin congeners. similar evolutionary pressure over the leader sequence Using the phylogeny-guided genome mining approach, could be postulated. The role of lanthipeptide leader in another nature-inspired lipopeptide antibiotic, cilagicin interaction of the precursor peptide with lanthipeptide was discovered that exhibits a distinct mode of action synthetase is known to impel the core and enzyme into (Wang et  al. 2022a). Out of the ~ 10,000 sequenced a conformationally constraint structure for successful bacterial genomes, a phylogenetic tree was constructed post-translational modifications (Li et  al. 2017). Recent using sequences of condensation starter (Cs) domain studies have confirmed the significance of the leader that installs the N-terminal lipid in lipopeptides. The peptide in successful modification of cognate and clades that fell out as a separate group were selected noncognate precursor peptidesd by lanthipeptide to identify cryptic BGCs, as a potential source of an synthetase LanM (Burkhart et al. 2017; Viel and Kuipers uncharacterized lipopeptide antibiotic. The structure 2022). Findings, like a highly conserved lanthipeptide of the encoded product was bioinformatically predicted leader with diversity in the core peptide (Cubillos- and chemically synthesized, thus producing a synthetic- Ruiz et  al. 2017); vital conservation of FxLx motif in bioinformatic natural product (syn-BNP), cilagicin. lanthipeptide leaders for class I lanthipeptide synthetases Phylogenetic analysis of lanthipeptide synthetases (Abts et  al. 2013); chimeric leader peptide for post- has formed the basis of our current study to identify translational modifications by two different classes of roseocin family lantibiotics and therefore gene synthetases (Burkhart et  al. 2017) and the unique layout crossover events of lanthipeptide synthetases during of mersacidin leader for MrsM (Viel and Kuipers 2022) evolution can be a significant determinant of the fate of suggests the unique function of leader region in post- lanthipeptides diversification in nature. Gene crossovers translational maturation of the bioactive core peptide. of lanthipeptide synthetases can be predicted using a Our study adds an evolutionary logic by showing a tanglegram, which indicates a difference in gene topology coevolutionary relationship between lanthipeptide (lanM in our case) vis-à-vis species evolution tree (16S leader and lanthipeptide synthetase in a phylum- rRNA). Thus, a comparative phylogenetic analysis of dependent manner (Fig.  6). Fewer crossovers in the the 16S rRNA-based species tree with the lanM gene tanglegram of phylogenetic trees of lanthipeptide leader tree was plotted in the dendroscope (Scornavacca et  al. and LanM sequences (Additional file  1: Figure S14), 2011), allowing swapping of the branches from both of also point towards the coevolution of lanthipeptide the trees at the possible closest distance and HGT events leader and lanthipeptide synthetases to maintain the were inferred by crossover lines between the gene and required interactions for post-translational modification species tree (Zhang et  al. 2016). Remarkably, the final in the core sequence. This observation opens up new tanglegram showed only intra-phyla gene crossover possibilities for future combinatorial biosynthesis and events (Additional file  1: Figure S12), which supports emphasizes using the phylogenetically related pair of the phylum-dependent evolution of lanthipeptide lanthipeptide leader and synthetase to produce novel synthetases and hence a probable dispersion of the cognate and non-cognate lantibiotics efficiently. lanthipeptides within the same phylum. To check the Some BGCs, identified here are the only representatives BGC’s organization in correlation to the 16S rRNA from actinobacteria and cyanobacteria (while archaeal of their group that can be explored by in  vitro studies 16S rRNA was placed as an outgroup), two different for novel lanthipeptides. Chloroflexi and acidobacteria ML phylogenetic trees were also plotted (Additional constitute bacterial species from extreme ecological file  1: Figure S13). In the LanM-based phylogenetic conditions and the identified precursor peptides were tree, a systematic classification of the BGC’s structure found enriched with thioether-forming moieties (Ser/ was obtained for both actinobacteria and cyanobacteria Thr and Cys) that, in addition to bioactivity may provide (Fig.  2), which is not apparent in the 16S rRNA-based enhanced stability at extremes of temperature and phylogenetic trees (Additional file  1: Figure S13). Again, pH (Additional file  1: Figure S2, Additional file  2), an the intra-phyla gene crossover events (Additional important feature desired in industrial applications. file  1: Figure S12) could be the precise reason for the Extremophiles can be a source for thermostable PTM difference in the BGC’s classification in the two kinds of machinery for bioengineering. Another important phylogenetic trees. observed feature was the highly conserved dual transport The biosynthesis of a lanthipeptide involves the secretion system of HylD membrane protein and the installation of thioether ring topology in a selective double glycine peptidase domain containing LanTp in stereochemistry (Mukherjee and Van Der Donk 2014). proteobacteria and cyanobacteria subclades (Additional The ring topology in the core sequence of a lanthipeptide file  1: Figure S2). It has been proposed that HylD plays Chaudhary et al. AMB Express (2023) 13:34 Page 18 of 20 a vital role in exporting lanthipeptide across the outer diversity. Figure S13. Maximum likelihood phylogenetic tree of 16S membrane of Gram-negative bacteria (Haft et  al. 2010). rRNA from bacterial species of (A) actinobacteria and (B) cyanobacteria, in correlation of their BGCs organization. Figure S14. (A, B) Tanglegram The dual transport system is a notable feature among between the phylogenetic tree of LanM and their associated lanthipep- BGCs identified in Gram-negative bacteria (Mohr et  al. tide leader/core in Dendroscope. 4. Table S1. 42 selected hits from RosM 2015). search in the NCBI database. Table S2. A comparison of the roseocin and lacticin 3147 families of two LanM-two precursor genes. Table S3. Prochlorosin synthetase (ProcM)-like LanMs in Accession number of ProcM family LanMs from Cubillos-Ruiz et al. 2017. cyanobacteria are a vital component of diversity- Table S4. Calculated (by Expasy) and observed (by ESI-MS) average mass oriented lanthipeptide evolution due to its unparalleled of full length (with leader region) post-translationally modified Rosα-wild type and its variants. Selected positions for mutant generation are bold capacity to process diverse types of lanthipeptide and italicized. Table S5. Calculated (by Expasy) and observed (by MALDI- cores (Cubillos-Ruiz et  al. 2017). However, in our TOF MS) monoisotopic mass of Rosα variants after leader cleavage using dataset, freshwater cyanobacterium species from endoproteinase GluC (leaving an overhang of four amino acid residues) reduced with TCEP only & TCEP reduced peptides alkylated with IAA. Synechococcales showed an association of ProcM-like Table S6. b and y ions obtained in MS-MS fragmentation of Rosα variants. LanMs (CCG motif) with RosM-like LanMs (CHG Table S7. The list of primers used for the SDM-PCR in the generation of motif) within a single BGC suggesting a novel and variants (F-forward and R-reverse primer). exquisitely divergent mechanism of diversity-oriented Additional file 2. Amino acid sequence of lanthipeptide precursors asso - ciated with (A) 42 BGCs of Figure 2 (main text), and (B) additional roseocin evolution in lanthipeptides from cyanobacteria (Fig. 4). homologs from Walker et al, 2020 study. Further, a similar evolutionary rate among the LanM with CCG and CHG motif across the cyanobacteria Acknowledgements phylum (Fig.  6B) increases the possibility of similar AB Sciex TOF/TOF 5800 system maintained at CIF, NABI, Mohali and ESI-MS promiscuity in substrate tolerance. The two earlier system maintained at SAIF in CIL, Panjab University, Chandigarh are duly genome mining studies on BGCs of Synechocystis sp. acknowledged. PCC 7509 and Chamaesiphon minutus missed the Author contributions underlying association of two diverse leader types and SC, SK, DS conceived and designed the study. SC conducted the LanM classes in a single BGC (Singh and Sareen 2014; bioinformatics study. SK designed and generated the variants and did the mass spectrometry measurements. RP, SJ, KB conducted the purification Zhang et al. 2014), which strengthens the perspective of experiments. SC wrote the initial manuscript. MS analysed the data and edited studies done in a phylogeny-guided manner to discover the manuscript. DS arranged the funding, approved the final results, edited of novel lanthipeptides. the manuscript. The authors read and approved the manuscript. Funding Supplementary Information SC received CSIR-SRF fellowship (09/135(0773)/2017-EMR-I) by the The online version contains supplementary material available at https:// doi. Council of Scientific and Industrial Research, New Delhi. SK (JRF) and DS org/ 10. 1186/ s13568- 023- 01536-9. (PI) duly acknowledge the grant received from DST-SERB (Grant Number: CRG/2018/004218). Financial assistance received from University Grants Commission-Special Assistance Programme (UGC-SAP) (DRS Phase-I) is duly Additional file 1: Figure S1. (A) Pie chart of the obtained hits, with acknowledged. most of the hits being from actinobacteria and cyanobacteria phylum. (B) Phylum-dependent clade formation of 100 RosM homologs in Bayesian Availability of data and materials analysis. Figure S2. Complete BGCs of selected 42 LanMs based on BAGEL Original raw dataset can be accessed in Mendeley dataset: https:// data. mende 4 and antiSMASH 5.0 prediction. Annotation is based on CDD analysis. ley. com/ datas ets/ nbwzz 4gg6v/ draft?a= a6d56 111- 2ed8- 4895- b578- 47ff2 Figure S3. Multiple sequence alignment of roseocin family lanthipeptide c474b de.. precursor sequences to determine the conserved motif. Figure S4. Bayes- ian analysis of two LanMs of the same biosynthetic gene cluster from roseocin and lacticin 3147 families. Figure S5. (A) Distantly related BGCs Declarations of roseocin family from Walker et al. 2020 study, with multiple numbers of precursors in gene clusters. (B) Rosα homologs of these BGC are unusu- Ethics approval and consent to participate ally short but contain the conserved motif S/TxxxxTxGCC. Figure S6. (A) Not applicable. Pairwise sequence alignment and percent identity of 42 LanM sequences (B) and the comparative cumulative frequency of identity percentage Consent for publication among leader, core, and LanM protein sequences of 42 BGCs. Figure S7. Not applicable. Sequence logo from multiple sequence alignment (A) Rosα and (B) Rosβ homologs from type 2 subclade showed highly conserved positions and Competing Interests possible sites of evolutionary variation. Figure S8. ESI-MS data showed All the authors declare no competing financial interests. that variants of Rosα were post-translationally modified by RosM, in E. coli BL21(DE3). Figure S9. Evaluation of antimicrobial activity in synergism Author details with Rosβ after leader removal with proteinase K. Figure S10. Multiple Department of Biochemistry, Panjab University, Chandigarh 160014, sequence alignment of Rosα homologs to predict the ring topology in (A) India. Department of Biosciences and Bioengineering, Indian Institute Rosα and (B) Rosβ. Figure S11. Schematic representation of the primer of Technology Roorkee, Roorkee 247667, India. design for site-directed mutagenesis. The arrowheads represent the site of mutation in the primer-primer overlapping region. Figure S12. A tangle- Received: 26 February 2023 Accepted: 4 March 2023 gram between the 16S rRNA and the lanM gene tree indicates that intra- phyla horizontal gene transfer (HGT ) is a major source of lanthipeptide Chaudhar y et al. AMB Express (2023) 13:34 Page 19 of 20 References Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy Abts A, Montalban-Lopez M, Kuipers OP, Smits SH, Schmitt L (2013) NisC binds and high throughput. Nucleic Acids Res 32:1792–1797. https:// doi. org/ the FxLx motif of the nisin leader peptide. 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Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin

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2191-0855
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10.1186/s13568-023-01536-9
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Abstract

Roseocin, the two-peptide lantibiotic from Streptomyces roseosporus, carries extensive intramolecular (methyl) lanthionine bridging in the peptides and exhibits synergistic antibacterial activity against clinically relevant Gram- positive pathogens. Both peptides have a conserved leader but a diverse core region. The biosynthesis of roseocin involves post-translational modification of the two precursor peptides by a single promiscuous lanthipeptide synthetase, RosM, to install an indispensable disulfide bond in the Rosα core along with four and six thioether rings in Rosα and Rosβ cores, respectively. RosM homologs in the phylum actinobacteria were identified here to reveal twelve other members of the roseocin family which diverged into three types of biosynthetic gene clusters (BGCs). Further, the evolutionary rate among the BGC variants and analysis of variability within the core peptide versus leader peptide revealed a phylum-dependent lanthipeptide evolution. Analysis of horizontal gene transfer revealed its role in the generation of core peptide diversity. The naturally occurring diverse congeners of roseocin peptides identified from the mined novel BGCs were carefully aligned to identify the conserved sites and the substitutions in the core peptide region. These selected sites in the Rosα peptide were mutated for permitted substitutions, expressed heterologously in E. coli, and post-translationally modified by RosM in vivo. Despite a limited number of generated variants, two variants, RosαL8F and RosαL8W exhibited significantly improved inhibitory activity in a species-dependent manner compared to the wild-type roseocin. Our study proves that a natural repository of evolved variants of roseocin is present in nature and the key variations can be used to generate improved variants. Keypoints • Taxonomically distant species have a wide diversity of roseocin family lantibiotics. • The phylogeny-guided approach opens new possibilities for RiPPs bioengineering. • Bioactive variants of roseocin were generated using semi-in vitro reconstitution. Keywords Actinobacteria, Lantibiotics, Phylum, Post-translational modification, Variants Sandeep Chaudhary and Shweta Kishen have contributed equally *Correspondence: Dipti Sareen diptsare@pu.ac.in Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Chaudhary et al. AMB Express (2023) 13:34 Page 2 of 20 is required. A detailed description of the mechanism and Introduction structure of the lanthipeptide synthetases is provided by Nature has bestowed upon the bacterial species an Repka et al. 2017. arsenal of antimicrobial moieties to benefit the host in The LanMs are of particular interest not only because intraspecies or interspecies competition under the limited of their bifunctional domain structure but also their natural resources of an ecological niche. The human substrate promiscuity. Evolutionary studies have shown race has benefited profoundly from these microbial a significant divergence among LanMs based on the conflicts with the discovery of novel antimicrobials to natural substrate promiscuity, defined by the number combat bacterial infections. However, extensive and of precursor peptides encoded within a single genome indiscriminate use of antimicrobials has fuelled the rapid (Zhang et  al. 2012). Highly promiscuous LanMs possess spread of antimicrobial resistance (AMR), rendering a CCG motif, instead of the CHG motif, for binding antimicrobials of similar type ineffective. One of the 2+ essential Zn ion at the active site (Zhang et  al. 2014). ways to solve this crisis is to discover other alternative ProcM is the first example of a CCG motif LanM with classes of antimicrobials having high efficacy against an ability to process 29 diverse precursor peptides, these multi-drug-resistant (MDR) pathogens along genetically encoded and spread throughout the genome with a scope for possible bioengineering in the future. of Prochlorococcus MIT9313 (Li et  al. 2010). In an Lantibiotics (or antimicrobial lanthipeptides) are one exciting study by Yang et al. 2018, the substrate tolerance such class that belong to ribosomally synthesized and and catalytic fidelity of ProcM were utilized to generate a post-translationally modified peptides (RiPPs), and their library of 1.07 × 10 non-native variants. The 36 peptides heterologous expression in E. coli based platforms has examined out of this library (made from ProcA2.8, facilitated the formation of new-to-nature molecules by which is one of the 30 naturally encoded substrates of site-directed/random mutagenesis and combinatorial ProcM) were all found to have a faithful installation of biosynthesis (Iacovelli et al. 2022). the two-ring scaffold, thus establishing the possibility Lanthipeptides are decorated with characteristic of generating lanthipeptide libraries with a wide range thioether rings that render them proteolytically stable of chemical diversity by utilizing such promiscuous and target-accessible (Montalbán-López et  al. 2021). LanMs. Another recently reported CCG motif containing The synthesis of a lanthipeptide begins from the precur - LanM, SyncM processes 79 naturally encoded precursor sor peptide (LanA), a genetically encoded linear peptide peptides and is the most promiscuous lanthipeptide (Fig.  1A), which is a variable component in the biosyn- synthetase known so far (Arias-Orozco et al. 2021). thetic gene clusters (BGCs), along with the lanthipep- Unlike CCG motif LanMs, comparatively limited tide synthetase, immunity proteins, and other tailoring promiscuity is exhibited by CHG motif containing enzymes. The precursor peptide has an N-terminal leader LanMs, which are involved in the biosynthesis of two- peptide and a C-terminal core, separated by a proteolytic component lantibiotics. These lantibiotics bifurcate the site for removal of the leader peptide following the com- action of a single-component lantibiotic into alpha- and plete post-translational modifications (PTMs) of the core beta-peptides in their synergistic antimicrobial mode of peptide (McAuliffe et  al. 2001; Oman and van der Donk action (Oman and Van Der Donk 2009; Bakhtiary et  al. 2010). A lanthipeptide leader region is essential in guid- 2017). The alpha-peptide is responsible for anchoring to ing the core peptide at the active site of the lanthipep- lipid II in the outer leaflet of the cytoplasmic membrane, tide synthetase for ring installation (Lubelski et al. 2009). and the beta-peptide potentiates the alpha:lipid II The lanthionine rings are installed in two steps; firstly, complex to form a heteromeric complex leading to the the dehydration of Ser/Thr residues occurs, which is fol - rapid cell lysis (Bakhtiary et  al. 2017; Oman et  al. 2011). lowed by intra-peptide Michael addition of cysteine resi- Roseocin is a recent example of a two-component dues (cyclization) to form lanthionine/methyllanthionine lantibiotic that results from the post-translational rings (Fig.  1B) (Xie et  al. 2004). Based on the structure modification of two precursor peptides by a single and function of lanthipeptide synthetase, four lanthipep- promiscuous CHG-type RosM (Singh et  al. 2020). tide classes (class I-IV) have been defined (Arnison et al. Such promiscuity is rare among other two-component 2013; Repka et al. 2017). To process class I lanthipeptides, lantibiotics, as all the lacticin 3147 family lantibiotics two separate dehydration (LanB) and cyclization (LanC) have two separate dedicated CHG type LanMs for the enzymes are encoded in the BGC. For class II lanthipep- two precursors (Mcclerren et  al. 2006; Xin et  al. 2016). tides, a single bifunctional domain-containing enzyme, The only characterized examples of single CHG-type LanM is present with an N-terminal dehydratase and a LanM and two precursors are cytolysin (Coburn and C-terminal cyclase domain. For class III and IV lanthip- Gilmore 2003), carnolysin (Lohans et  al. 2014) and eptides, a single enzyme (LanKC and LanL, respectively) bicereucin (Huo and Van Der Donk 2016), but all of with trifunctional domains, i.e. lyase, kinase, and cyclase Chaudhar y et al. AMB Express (2023) 13:34 Page 3 of 20 Fig. 1 Biosynthesis of the two-component lantibiotic roseocin. A Roseocin is constituted of two precursor peptides, RosA1β and RosA2α, which are post-translationally modified by RosM in two steps (dehydration followed by cyclization). This is followed by leader removal in vitro using commercially available proteases like endoproteinase GluC or proteinase K (Singh et al. 2020). The ring topology of roseocin peptides is supported by mass spectrometry and bioinformatic analysis as per the current study. B Common PTMs in the biosynthesis of lanthipeptide involves the dehydration of serine and threonine amino acid residues to 2,3-dehydroalanine (Dha) and 2,3-dehydrobutyrine (Dhb), respectively. Michael-type addition of Cys to Dha leads to lanthionine (Lan), while addition to Dhb results in a methyllanthionine (MeLan) ring. Abu aminobutyrate, Ala Alanine, SIVR semi-in vitro reconstitution, HP Hypothetical Protein, RosTp dual function peptidase-domain containing transporter, RosF group of immunity proteins (in red colour), TR Transcriptional Regulator these belong to the phylum r fi micutes. However roseocin, Instead of random mutagenesis, we utilized the sequence identified from S. roseosporus NRRL 11379 is the sole diversity among the identified roseocin homologs. The example, from the phylum actinobacteria, of single study of phylogenetically related BGCs is a promising LanM and two precursors that lack homology to any way to obtain an extensive collection of divergent of the known two-component lantibiotics (Singh et  al. congeners resulting from an evolutionary pressure 2020). Owing to the antimicrobial potential of roseocin (Wang et  al. 2022a). In a quest to identify homologs of against MDR strains and having previously developed its roseocin, we devised a methodology using lanthipeptide heterologous expression system in E. coli, we wanted to synthetases, which have evolved in a phylum-dependent explore its variants with improved antibacterial activity. manner (Zhang et  al. 2012; Yu et  al. 2013). An earlier Chaudhary et al. AMB Express (2023) 13:34 Page 4 of 20 study by Walker et  al. 2020 utilized the LanC domain bootstrap values in MEGA X using an outlier group. of lanthipeptide synthetase as a query and classified Homologs of RosM in Archaea (taxid:2157) were placed lanthipeptides by sequence similarity network in various as the outlier group. The final phylogenetic tree was subtypes, thus identifying > 8000 members of all the presented with iTOL v4 (Letunic and Bork 2019). four known classes of lanthipeptides. The extensive lanthipeptide analysis done by the group proved that Precursor peptides identification and biosynthetic gene the divergence in the lanthipeptide classes is ancient cluster (BGC) analysis and supports the hypothesis that the lanthipeptide The genome sequence of the hits was obtained in FASTA synthetases of different classes may have evolved through format from the Genbank database and analyzed with convergent evolution. BAGEL4 (Hart and Moffat 2016) and antiSMASH 5.0 We utilized the phylogenetic distribution of RosM (Blin et  al. 2019) web servers. Precursor peptides were homologs as a guide to explore the evolution of roseocin. identified manually by subjecting intergenic regions to All the roseocin homologs identified using this approach NCBI ORF finder. Putative functions were confirmed lacked the already known lipid II binding motif (Grein based on CDD (Lu et  al. 2020) and TMHMM server et  al. 2019) but were found to have a novel conserved analysis (https:// servi ces. healt htech. dtu. dk/ servi ce. php? motif in the two peptides. Considering that a naturally TMHMM-2.0). selected, more potent roseocin homolog may exist in nature, we selected the mutations that should not alter Tanglegram of evolutionary trees the Rosα peptide thioether rings, but could significantly Available 16S rRNA sequence was obtained from SILVA influence its activity. Only the site-specific mutants were reference database SSU r114 (Quast et  al. 2013) to plot generated by an efficient one step site-directed single site the species tree. Nucleotide sequences of lanM and plasmid mutagenesis protocol, followed by their success- lanA were fetched from the Genbank. The species and ful PTMs by RosM in vivo. The semi-in vitro reconstitu - gene tree were constructed with Maxiumum Likelihood tion (SIVR) strategy established earlier (Singh et al. 2020) (ML) method and subjected to the tanglegram algorithm was followed to obtain the bioactive core peptides for (Scornavacca et  al. 2011) incorporated in Dendroscope the determination of minimum inhibitory concentration ver. 3.7.2. Species vs gene tree tanglegram was used to (MIC) and structural characterization by mass spectrom- find horizontal gene transfer (HGT), while gene vs gene etry. Our study incorporated multiple approaches for tree was used to test the coevolution of LanM and leader investigating the lanthipeptide evolution i.e., an analysis or core peptides in the same operon. of evolution rates between BGC types, analysis of varia- bility within core peptide sequences versus leader peptide Variability analysis sequences, and an assessment of whether horizontal gene Variability among the set of genes was visualized in col- transfer is a major source of diversity. Assuming the BGC our gradient matrix or graphical form using the sequence as a single evolutionary unit, many overlooked aspects of demarcation tool version1.2 (SDT v1.2) (Muhire et  al. lanthipeptide evolution are revealed here that can aid the 2014). Shannon entropy for sequences aligned with evolution-based studies of lanthipeptides in future. clustal omega was calculated using a protein variability server (Garcia-Boronat et al. 2008). Evolutionary pressure Materials and methods (purifying, neutral, or positive) was tested using the Nei- Genome mining for RosM like LanMs and phylogenetic Gojobori method in a codon-based Z-test of selection in analysis MEGA X. The only sample under purifying or neutral RosM sequence (WP_010071701.1) was used as a query selection (p value < 0.05) was considered for the next step for blastp search on NCBI Genbank non-redundant to calculate the substitution rate (ῳ = d /d , i.e., the ratio N S protein sequences database (May, 2020). The top 100 hits of the rate of nonsynonymous substitutions (dN) and were aligned with MUSCLE (Edgar 2004) using MEGA X rate of synonymous substitutions (dS) per site in YN00 of (Kumar et  al. 2018) and processed by GBlocks ver0.91.1 PAML package (Pamilo and Bianchi 1993; Yang 2007). (Talavera and Castresana 2007) to select conserved domains for phylogenetic analysis by Bayesian method Variants generation by site‑directed mutagenesis (Huelsenbeck and Ronquist 2001), available on an Site-directed mutagenesis of selected roseocin alpha open server NGPhylogeny.fr (Lemoine et  al. 2019). The precursor peptide residues was carried out using phylogenetic tree was scrutinized to eliminate redundant Agilent’s QuikChange site-directed mutagenesis kit. hits from the same clade and genus. The remaining hits Primers and methodology were designed using the were pruned and analyzed using the maximum likelihood procedure described by Liu and Naismith 2008 study (ML) method and JTT matrix-based model with 500 (Additional file  1: Figure S11). Desalted primers at a Chaudhar y et al. AMB Express (2023) 13:34 Page 5 of 20 0.05  µM scale were obtained from Sigma Genosys. 2020). Log phase culture was diluted with Mueller The T (melting temperature of the primer-primer Hinton Broth (MHB) to obtain 2 × 10   CFU/mL. The m pp overlapping region) and T (melting temperature of bacterial suspension and the lantibiotic were added to m no non-overlapping primer region) were calculated for each 96-well microplates at a ratio of 1:1. The microplates were primer (Additional file  1: Table  S7). PCR was carried incubated overnight at 37 ºC. The lowest concentration out using Bio-Rad MyCycler thermocycler. The PCR at which no visible growth was observed, was considered products were treated with 5 units of DpnI at 37  °C for MIC. one hour, and the reaction was stopped by heating at 75  °C for 15  min. The PCR product was transformed Results into chemically competent E. coli DH5α. The mutations Phylogenetic analysis for selection of diverse RosM were confirmed through Sanger sequencing, and the homologs plasmid was retransformed into E. coli BL21(DE3) for RosM (WP_010071701.1) installs thioether rings on two protein over-expression and in  vivo post-translational peptides (Rosα and Rosβ) that differ structurally and modification by RosM (Singh et  al. 2020), as per the functionally but display synergistic antimicrobial activity details mentioned in Additional file 1. as roseocin. Due to the unique natural promiscuity of RosM, it was speculated that RosM may have evolved Reduction and alkylation of peptides distinctively and hence was subjected to query search For confirming ring formation and availability of free in the GenBank database, which resulted in hits from cysteine(s), endoproteinase GluC (NEB, #P8100S) a wide range of actinobacteria species and four other cleaved peptides (30  µM) in 50  mM Tris–HCl pH 8.0 phyla (Additional file  1: Figure S1A). In the top 100 were incubated with 1  mM TCEP (Tris(2-carboxyethyl) hits, a sequence identity ranging from 33.8–99.0% phosphine) at 37 ºC for 30  min to reduce the disulfide and conservation in CHG motif in the active site bond. Following reduction the peptide was alkylated by was observed. The majority of the hits represented the addition of 10 mM IAA (Iodoacetamide) for 90 min. actinobacteria (n = 45) and cyanobacteria (n = 29) Reaction was set up in 60 µL volume in 1.5  mL MCTs. (Additional file  1: Figure S1A). As expected, amino acid The samples were desalted using Pierce C18 spin sequence identity criteria for genome mining generated column (#89873) before sample analysis with MALDI- an uneven distribution of hits, making it challenging to TOF MS. evaluate all the hits for novel BGCs. However, a Bayesian phylogenetic analysis of the obtained hits led to the MALDI‑TOF MS analysis phylum-wise clade formation along with the formation of Matrix-assisted laser desorption/ionization time-of-flight subclades having BGCs of similar properties (Additional mass spectrometry (MALDI-TOF MS) was carried out file  1: Figure S1B), thus helping in the systematic on AB Sciex TOF/TOF 5800 system maintained at CIF, evaluation of distantly related RosM homologs. NABI, Mohali. The proteolytic digest was processed with Interestingly, the RosM query search did not result in any Pierce C-18 spin column and mixed with α-cyano- hits from r fi micutes, the only phylum with the lacticin 4-hydroxycinnamic acid (1  mg/mL) for analysis in the 3147-like two-component lantibiotics discovered so reflectron mode. The mass spectra were calibrated using far (Zhang et  al. 2012). This observation indicated the a mass standards kit for the calibration of AB scion TOF/ independent evolution of roseocin family from lacticin TOF instruments (#4333604). The TOF/TOF explorer family two-component lantibiotics. was used for data acquisition, and SeeMS (Chambers To understand the features of the respective BGCs, et  al. 2012) and mMass programs (Niedermeyer and we analyzed the genome sequences of RosM hits with Strohalm 2012) were used for data analysis. To get a deep BAGEL 4 (Hart and Moffat 2016) and antiSMASH 5.0 insight into the thioether rings pattern, we did MS/MS (Blin et al. 2019) webservers. Both software identified the analysis for all the variants. BGC cluster boundaries including all the major genes of the BGC, but showed limitations in defining the genes Antimicrobial activity analysis encoding precursor peptides. Hence, we located the The minimum inhibitory concentration of wild-type putative precursor peptide genes on the GenBank file or roseocin and its variants was determined with micro subjected intergenic gap regions to NCBI ORF-finder, enabling us to identify the specific precursor peptide broth-dilution method. Sterile 96-well microtiter plates encoding genes. As a major advantage of mining in a were treated with 200 µL of 1% (w/v) bovine serum phylogeny-guided manner, identical lanthipeptide pre albumin (BSA) in 1 × phosphate-buffered saline (PBS) - solution at 37 ºC for 30  min. After this, wells were cursors (termed redundant hits) were easily identified in washed with 1 × PBS to remove excess BSA (Ellis et  al. an initial analysis across the BGCs of the same subclade. Chaudhary et al. AMB Express (2023) 13:34 Page 6 of 20 Roseocin family BGCs identified in actinobacteria For example, in BGC analysis across actinobacteria, 18 Actinobacteria showed the presence of three divergent out of 22 hits from the Streptomyces genus and 10 out of subclades (Fig. 2), each displaying a characteristic pattern 12 species from the Micromonospora genus encoded an of genetic arrangement within the BGC. Initially, BGCs identical precursor (Additional file  1: Figure S1B). Such seemed unrelated owing to a difference in the organiza - redundant hits were eliminated to limit the sample size tion of genes, sequence, and the number of lanthipeptide and prevent skewing the final sequence alignment. Phylo - precursors, with some encoding more than one CHG- genetic branch lengths were observed as < 0.05 in LanMs type LanMs (Fig.  2). However, further analysis showed of actinobacteria which corresponded to BGCs encoding that all BGCs encode precursor peptides homologous identical precursors (Additional file  1: Figure S1). Hence, to either Rosα or Rosβ (Additional file  1: Figure S3A and such RosM hits were removed from the rest of the phyla. B, respectively). Based on the chronological order in the Finally, 42 RosM homologs from five phyla (Additional phylogenetic tree, these 13 BGCs were classified as type file  1: Table  S1) were selected for phylogenetic analysis 1–3, to represent their respective subclades (Figs.  2 and using an appropriate outgroup for rooting. Unrooted 3A). Roseocin was grouped as a member of the type 2 trees, like in Additional file  1: Figure S1B, are only use- class, having stringent conservation of roseocin BGC ful for visualization of the relatedness of sequences of features among the other mined members of the same different clades, while only a rooted tree provides insight subclade. Precursor peptides of type 2 BGCs also display into evolution. A careful selection of outgroups was fol- features that agree with the earlier postulated structure lowed, as suggested by Adamek et al. 2019, being neither of roseocin (Singh et  al. 2020) and hence were used as too distant nor too close to the ingroups of the dataset a platform for designing the variants of Rosα (explained under study. In a recent genome mining study (Makarova in later sections). However, precursor peptides in type 1 et  al. 2019), archaea have been shown to contain lan- and 3 BGCs showed a more significant variation in the thipeptide BGCs across the species of the Halorussus amino acids that might result in a different ring topology genus. Interestingly, these archaeal lanthipeptide BGCs of these lanthipeptides (Additional file  1: Figure S3). The are of class II type with a single CCG motif LanM and type 1 BGCs containing three instead of two precursor an unknown class of lanthipeptides. We selected three genes deviate from the usual two-component lantibiot- BGCs from the Halorussus genus in the archaea database ics (Fig. 3A). Such kind of BGCs have been characterized and placed them as the outgroup to plot the maximum earlier in the lacticin 3147 family (Xin et  al. 2016; Zhao likelihood (ML) phylogenetic tree using a 500 bootstrap and Van Der Donk 2016). There, additional precur - value (Fig. 2). sor was found to be a result of the duplication of one of BGC analysis of each of the 42 RosM hits from the final the two genes. Contrary to this, we did not observe any phylogenetic tree (Additional file  1: Figure S2) showed core sequence similarity in the third precursor peptide of a gradual shift in the genomic location of minimally type 1 subclade (designated as LanA2A) to either of the required biosynthetic genes, lanA (lanthipeptide other two lanthipeptides (Additional file  2), which ruled precursor), lanM and a bifunctional lanTp (peptidase out an evolutionary gene duplication event. Phylogenetic domain-containing transporter) alongside the subclades analysis showed that LanA2A is closely related to alpha (Fig.  2). In most BGCs, precursor genes were found homologs (Fig.  3B). This indicates that LanA2A is either upstream to lanM, which probably is the natural an alpha peptide that synergizes with a common beta- temporal order of their synthesis. The common observed peptide, or it may be a constituent of a novel three-com- feature in most of the precursor peptide genes was the ponent synergistic system. As an advantage of random NHLP (nitrile hydratase leader peptide) family signature genome mining for lanthipeptide, many small-sized rose- (Haft et al. 2010) in their leader region and a single lanM ocin homologs were also found in the study by Walker gene of CHG-type present for their processing (Fig.  2). et al. 2020 (Additional file  2). To classify such small-sized But more than one lanM genes carrying BGCs were also homologs into type 1–3 subclade, BGC analysis was done found across actinobacterial and cyanobacterial species. in the current study (Additional file  1: Figure S5) and var- In cyanobacteria, these BGCs showed precursor peptides ious peculiar attributes like missing or duplicated genes, having conservation in leader regions from two divergent multiple LanMs, etc. were noted and hence, they could types of leader families, i.e. NHLP and N11P (Nif11 not be categorized as either of the members of type 1–3 derived peptides) family, discussed later in detail (Fig. 4). subclade. These genes probably might be of lower signifi - Overall, these conservations and variations made it cance and might have come into temporary existence to intriguing to study the dataset further for the conserved get eliminated during the natural selection for the most features of lanthipeptide evolution. potential genes. Chaudhar y et al. AMB Express (2023) 13:34 Page 7 of 20 Halorussussp. HD8-83 Halorussussalinus Halorussussp. RC-68 Acnoalloteichus ordicus Streptomyces rhizosphaericus Type 1 (2) Agromycesaureus Pseudo. massilense Ornithinicoccushortensis Nonomuraea sp. 6K102 Type 2 (8) Glycomyces harbinensis Streptomyces spinoverrucosus Streptomyces sp. AD196-02 Streptomyces roseosporus Catellatospora methionotrophica Micromonospora arida Type 3 (3) Micromonospora noduli Archangium gephyra Meliangiumboletus Viosangiumsp. GDMCC 1.1324 (5) Cystobacterfuscus DSM 2262 Cystobacterfuscus DSM 52655 Thermosporothrix hazakensis (2) Thermogemmaspora carboxidivorans Acidobacteriabacterium AA17 Blastocatellia bacterium AA13 (3) Acidobacteriabacterium AA12 Stanieriacyanosphaera Nostocales cyanobacterium HT-58-2 (2) Nostoc sp. PCC 7524 Fischerella muscicola (3) Fischerellathermalis Nitrolancea hollandica Alkalinema sp. CACIAM 70d Synechocyss sp. PCC 7509 (2) Chamaesiphon minutus Chlorogloeopsisfritschii Nostoc sp. MBR 210 Nostoc piscinale Nostoc minutum NIES-26 Coleofasciculuschthonoplastes (10) Tolypothrixsp. PCC 7601 Tolypothrixsp. PCC 7910 Hapalosiphon sp. MRB220 Nostoc sp. 106C Nostoc carneum Archaea Actinobacteria Proteobacteria Chloroflexi Acidobacteria Cyanobacteria CHG type NHLP leader C39 HlyD, type I N11P leader CCG type LanM LanM peptidase secretion protein Fig. 2 Phylogenetic tree of 42 selected RosM homologs showed conservation of gene locus and characteristic features along the phylogenetic tree. Roseocin family constituted the BGCs from actinobacteria, having three types of BGCs’ organization (type 1–3), each forming a separate subclade. CHG motif LanM for processing of NHLP type leader sequence was found in all the BGCs except in a subclade of cyanobacteria (Synechocystis sp. PCC 7509 and C. minutus) where conservation of two types of LanMs (CHG and CCG motif ) and two types of leader sequence (NHLP and N11P) were found in a single BGC. LanMs from the BGCs of Halorussus genus were placed in the root. Value from 500 replicates bootstrap test is indicated on each branch. The numbers given in the bracket are the number of members of that particular subclade. NHLP nitrile hydratase leader peptide, N11P Nif11 derived peptides The type 3 BGCs of roseocin family, consisting of two this separate LanM might have resulted from a recent LanMs and two precursor peptides (Fig.  3A), were con- LanM gene duplication event (Additional file  1: Fig- fined to the Micromonosporaceae family (Fig.  2). These ure S4), unlike the two LanMs in the lacticin 3147 fam- BGCs encoded a supersized homolog of Rosα, with the ily, which have low sequence identity (24–29%) and one highest number of thioether-forming moieties (13 Ser/ LanM has evolved specificity for modification of only one Thr and 9 Cys residues) in a single precursor peptide of the two precursors (Mcclerren et  al. 2006). A similar (Fig.  3E; Rosα has 5 Ser/Thr and 6 Cys residues). Such a sequence identity score in the pairwise alignment of lan- huge precursor peptide probably necessitates a dedicated thipeptide leaders (Additional file  1: Table S2) is surpris- LanM for efficient post-translational modification in ing and can make sense only under the coevolutionary parallel to the LanM for the beta peptide. High pairwise phenomenon, a perspective discussed in detail in the fol- sequence identity of LanMs of the Micromonosporaceae lowing sections. family (~ 50%) (Additional file  1: Table S2) indicates that Chaudhary et al. AMB Express (2023) 13:34 Page 8 of 20 Fig. 3 Diversity among the 13 representative members of the roseocin family. A Three common types of BGCs encode roseocin homologs, type-1, type-2, and type-3 BGC examples are of S. rhizosphericus, S. roseosporus NRRL 11379 and C. methinotrophica, respectively. B Phylogenetic tree of lanthipeptide core sequences with ML method with bootstrap values of 500 replicates. The exceptional third precursor (LanA2A) of the single LanM-three precursor i.e. type-1 BGC, is phylogenetically related to alpha peptides. Colour coding in Fig. 3B is red: alpha peptides; green: beta peptides; blue: third precursor core region. C and D Variation in the core peptide sequences as a function of Shannon entropy in the roseocin alpha and beta homologs, respectively. The alpha peptides contain a S/TxxxxTxGCC motif at the N-terminal end, and beta homologs contain a GS/ TxxxxS/TxGCC motif at the C-terminal end. E A gigantic Rosα homolog from the Micromonosporaceae family contains nine Cys and thirteen Ser/Thr residues that may form as many lanthionine rings. Rosα of Streptomyces roseosporus (S. roseo) contains an indispensable disulphide bond and four (methyl)lanthionine rings (dotted lines depict the proposed ring topology in Rosα, Singh et al. 2020); M. arida-Micromonospora arida. LanA precursor peptide, LanM modification enzyme, HP Hypothetical Protein, LanT dual function peptidase-domain containing transporter As discussed earlier, alpha peptide initiates the interac- the roseocin homologs, a Shannon entropy (SE) analysis tion with the bacterial membrane by targeting lipid II, a was done. Lower SE value (< 2.0) indicates higher con- key step in the mechanism of action of two-component servation of amino acid residues through evolution (Gar- lantibiotics (Bakhtiary et  al. 2017; Oman et  al. 2011). cia-Boronat et  al. 2008). A conservation of a ten amino Most of the alpha peptides characterized to date pos- acid long stretch, S/TxxxxTxGCC, at the N-terminus of sess an Asp/Glu residue containing lipid II binding motif Rosα homologs (Fig.  3C) and an 11 amino acid stretch, (CTxTxD/EC), which is absent in Rosα peptide (Singh GS/TxxxxS/TxGCC at the C-terminus of Rosβ homologs et  al. 2020). Using the knowledge generated in the cur- (Fig.  3D) was observed. Both the motifs were proposed rent study on the diversity of the roseocin family, it seems to have a structure with overlapping lanthionine rings in necessary to look for a novel motif for a similar or diver- our earlier study (Singh et  al. 2020). Such a ring struc- gent action mechanism. To understand the variability ture at the N-terminus of Rosα homologs is analogous and conservation of amino acid substitutions among all to the nisin-like peptides (having two N-terminal rings, Chaudhar y et al. AMB Express (2023) 13:34 Page 9 of 20 Fig. 4 Novel BGCs, encoding diverse lanthipeptide core sequences, consist of two LanMs for processing two types of precursor peptides. A Two BGCs encoding NHLP and N11P family lanthipeptide leaders in their precursor peptides with the corresponding synthetases, i.e. CHG motif and CCG motif LanM, were identified in Synechococcales. B Sequence logos of NHLP family and N11P family lanthipeptide leader sequences using the precursor sequences from the above two BGCs. C Sequence alignment of cyclase domain of putative LanMs from characteristic BGC of Synechococcales showed a difference in catalytic motif. RosM like LanMs has a CHG motif, while ProcM like LanMs have a CCG motif. D Sequence identity percentage in the pairwise alignment of the 12 lanthipeptide precursors’ leader sequences (lower half ) and core sequences (upper half ). Diversity among lanthipeptide core sequences was high, irrespective of leader conservation. E Multiple sequence alignment of lanthipeptide core sequences depicts natural diversity. LanA precursor peptide, LanM modification enzyme, HP Hypothetical Protein, LanT dual function peptidase-domain containing transporter Chaudhary et al. AMB Express (2023) 13:34 Page 10 of 20 proven to be responsible for target binding), instead of (Nif11 derived peptides; cl06756 subfamily TIGR03798) an Asp/Glu residue-specific target binding motif of the are the two well-characterized lanthipeptide leader two-component lacticin 3147-family lantibiotics (Cooper types that have evolved from nitrile hydratase enzyme et  al. 2008; Bakhtiary et al. 2017). Increased SE (> 2.0) in and Nif11 proteins, respectively (Haft et  al. 2010). the other amino acid sequence positions (Fig.  3C, 3D, Usually, a single type of lanthipeptide leader, i.e. either and Additional file  1: Figure S3A, S3B) revealed the innu- of the NHLP or N11P, is observed in a BGC (Zhang merable combinations experimented by nature, as is evi- et  al. 2014). However, an exception was observed in the dent by the changes in the number of Ser/Thr and Cys cyanobacteria (Fig.  2), which earlier were the source of residues of the core region among the Rosα and Rosβ prochlorosin family lanthipeptides as well (Cubillos- homologs. Except for the stretches mentioned above, Ruiz et  al. 2017). A non-conventional BGC with both substitutions were allowed at all the amino acid positions. types of lanthipeptide leader (NHLP and N11P), along Further, plausible exchange of indispensable disulfide of with two LanMs in a single BGC, for the maturation of Rosα with thioether ring in Streptomyces rhizosphaericus three and nine precursor peptides (Fig.  4A and B) was (Additional file  1: Figure S3A); exchangeable lanthionine identified by a manual search of the nearby ORFs. This (Lan) and methyllanthionine (MeLan) rings; insertion/ type of BGC was found confined to Synechococcales and deletion of one or more thioether rings suggests the included Synechocystis sp. PCC 7509 and Chamaesiphon enormous scope of modular engineering of both, Rosα minutus as member species (Fig.  4A). As N11P family and Rosβ peptides (Additional file  1: Figure S3). The pres - lanthipeptides are only associated with the CCG motif ence of a conserved motif and variability in the rest of the LanM, we speculated one of the LanMs to be the CCG core region probably results from balanced combinatorial motif LanM. Surprisingly, sequence alignment showed chemistry, operating parallelly with the conserved motif- the presence of ProcM-like CCG motif LanM in the oriented evolution of lanthipeptides. same BGC besides a CHG motif LanM (Fig.  4C). This However, the rest of the BGCs from other phyla unprecedented example of association between two showed no significant core sequence conservation. The leader types and two LanM types in a single BGC lanthipeptides of proteobacteria, chloroflexi, acidobacteria, indicates another evolved mechanism of diversity- and cyanobacteria phyla seldom have significant antimicro - oriented BGCs in cyanobacterial species that could bial activity (Mohr et  al. 2015; Cubillos-Ruiz et  al. 2017; be a better way of efficient biosynthesis of diverse Bothwell et  al. 2021). Nevertheless, we proceeded fur- lanthipeptide core sequences (Fig. 4D and E). ther and discovered many overlooked aspects of lanthi- Three lanthipeptide precursor sequences (2 + 1) in peptide BGCs, providing new insights into lanthipeptide C. minutus and nine (4 + 5) in Synechocystis sp. PCC evolution. 7509 represents an intermediate number of diverse sequences observed earlier for prochlorosin-like genes (Cubillos-Ruiz et  al. 2017). A truncated gene found in A new diversity‑oriented class of lanthipeptides C. minutus genome (Additional file  2) could result from in cyanobacteria mutations like frameshift or early stop codon, preventing Unlike significant conservation observed above in the the synthesis of a functional ORF. Such pseudogenes core region of the roseocin-like lanthipeptides, diversity- are a common feature of the prochlorosin family oriented evolution is characterized by the generation of lanthipeptides and support the ongoing diversification of a vast variety of lanthipeptide core sequences with no precursor genes in a diversity-oriented manner (Cubillos- conservation at all (Zhang et al. 2012; Cubillos-Ruiz et al. Ruiz et  al. 2017). In the C. minutus genome, four more 2017). So far, prochlorosin-like BGCs are the only exam- distantly located N11P-type lanthipeptide precursors ple which have evolved a highly promiscuous LanM (with were found, which might also be associated with this CCG motif ) for the maturation of diverse lanthipeptide BGC (Additional file  1: Figure S2). Intrigued by the novel sequences in marine cyanobacteria i.e. Synechococcus and mechanism of diversity generation in Synechococcales, we Prochlorococcus (Li et  al. 2010; Mukherjee and Van Der further analyzed the other BGCs to identify conserved Donk 2014). Similarly, in our dataset, freshwater cyano- features of relevance. bacterium species from Synechococcales also showed the diversity-oriented lanthipeptide BGCs, but with a novel, exquisitely divergent mechanism (Fig. 4). Coevolution of lanthipeptide leader and lanthipeptide In the current study, despite the expansion of hits synthetase among different phyla across different phyla, obtained BGCs had CHG- Lanthipeptide precursor is derived from an assimila- type LanM, for processing NHLP-type lanthipeptide tion of a protein tailored as a leader sequence with an precursors (Fig.  2). NHLP family (or nitrile hydratase independently evolving core sequence rich in Ser/Thr leader peptide; cl22942 subfamily TIGR03898) and N11P and Cys residues (Haft et al. 2010; Zhang et al. 2012). In Chaudhar y et al. AMB Express (2023) 13:34 Page 11 of 20 Fig. 5 Conservation of lanthipeptide leader sequence over diverse core sequences. A Multiple sequence alignment using MUSCLE of all the identified 68 precursor peptides from 42 BGCs. All leader sequences are from the NHLP family of lanthipeptide leaders. Conserved residue positions are highlighted. B Pairwise identity among 68 lanthipeptide leader sequences (lower half ) and core sequences (upper half ) showed high similarity among leader over core sequences (except lanthipeptide core sequences of the roseocin family having core conservation). C Cumulative frequency of pairwise sequence identity among the lanthipeptide leader and core sequences, respectively. 80% of lanthipeptide leader pairwise alignment showed > 39% identity. However, only 10% of lanthipeptide core pairwise alignment fulfilled the same criteria (mainly of the roseocin family) Chaudhary et al. AMB Express (2023) 13:34 Page 12 of 20 a previous study by Zhang et  al. 2014, the ProcM (hav- S6B). Earlier, the conservation of two leader family types ing CCG motif ) was used for genome mining, and thus and two different types of LanMs in a single BGC of C. obtained BGCs showed highly varying lanthipeptide minutus and Synechocystis sp. (Fig.  4) also suggested an leader families. However, in our study, despite the diver- essential linkage between the leader and lanthipeptide sity among BGCs from a wider range of phyla, high synthetases. conservation among the leader region of the precursor To explore further, we determined the mutation peptides was observed (Figs.  2 and 5A). The only excep - rates of both the lanM and the lanthipeptide leader tion was cyanobacterial species (Fig.  4), which could be genetic region by calculating the d /d ratio, which is N S unearthed by manual inspection of ORFs that other- the ratio of the rate of nonsynonymous to synonymous wise would have been missed (Singh and Sareen 2014; mutations. The lanM and lanthipeptide leader genetic Zhang et al. 2014). Thus, we found that all leader peptide regions from 13 BGCs of actinobacteria (roseocin fam- sequences belonged to the NHLP family (Fig. 5A). In 80% ily) and 9 BGCs of cyanobacteria were selected for the of pairwise sequence alignments of leader sequences, separate analysis of two phyla. The calculated d /d for N S we observed > 39% sequence identity; while only in 10% lanM and leader peptide exhibited distinct patterns for of the core sequences pairwise alignments, an identity the two phyla (Fig.  6A). The d /d ratio was in agree- N S of > 39% was observed (Fig.  5B and C). These identity ment with the phylum-wise evolution of lanthipeptide scores support the fact that conservation in leader pep- synthetases (Zhang et al. 2012), as well as the coevolu- tides does not restrict the lanthipeptide core diversifi - tion of lanthipeptide synthetases and leader sequences. cation even among different phyla. Variability pattern It has been proposed earlier by Cubillos-Ruiz et  al. was also plotted for all the 42 LanMs (Additional file  1: 2017, that a lower d /d ratio is confined only to the N S Figure S6A) which surprisingly had an overlap with the lanMs having the CCG motif of the prochlorosin fam- variability in leader regions (Additional file  1: Figure ily (or ProcMs), suggesting an evolutionary locked state Fig. 6 Coevolution of lanthipeptide leader and synthetase in a phylum-dependent manner. A Pairwise d /d distribution of lanthipeptide leader N S and lanM from actinobacteria (13 BGCs of roseocin family) and cyanobacteria phylum (9 BGCs) showed variation in different phyla suggesting coevolution of the lanthipeptide leader and synthetases. B Standard box plot of the evolutionary rates of CCG motif prochlorosin family LanMs (Cubillos-Ruiz et al. 2017) (Additional file 1: Table S3) and CHG motif LanMs of cyanobacteria (from the current study) showed a similar pattern (median 0.21 and 0.24, respectively), while the CHG motif lanMs of roseocin family in actinobacteria has a higher value (median 0.48). In the standard box plot, the lower and upper shows the first and third quartile values, respectively, separated by the median value. The error bar plots the minimum and maximum values. C Phylogenetic tree of CCG motif ProcMs (Cubillos-Ruiz et al. 2017) and CHG motif LanMs of cyanobacteria and actinobacteria (from current study). Prochlorosin family LanMs displayed significant divergence from CHG motif LanMs of cyanobacteria and actinobacteria Chaudhar y et al. AMB Express (2023) 13:34 Page 13 of 20 + + + + Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] mono. m/z 3848.76 mono. m/z 3960.76 mono. m/z 3887.75 mono. m/z 3999.75 LL88FF LL88W W + + + + Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] Calc.[M-4H2O+H] Calc.[M-4H2O-2H+2IAA+H] mono. m/z 3913.81 mono. m/z 4025.81 mono. m/z 3843.75 mono. m/z 3955.75 S S1122W W VV2222Q Q Fig. 7 MALDI-TOF MS of endoproteinase GluC treated Rosα after TCEP only and after TCEP + IAA alkylation assay. Additional file 1: Table S5 represents the calculated and observed mass of the respective species of each variant generated that favors the catalytic promiscuity for the process- diverged during evolution into two subclades of CHG ing of diverse precursors. Interestingly, cyanobacterial and CCG motif LanMs, both being locked into a simi- lanMs with CHG motif (hence we proposed the name, lar evolutionary conserved state and probably having a CyanMs) found in the current study also displayed similar level of substrate tolerance. The reason for such a lower d /d ratio, i.e. 0.24 (Fig.  7B), suggesting an a divergence is not clear, but the significance of phy - N S evolutionary linkage between CyanMs and ProcMs. lum in deciding the fate of lanthipeptide synthetases However, phylogenetic analysis showed a significant enforces the phylum-dependent effect on the evolution divergence of ProcMs from CyanMs (Fig. 7C) even with of lanthipeptides than proposed earlier (Cubillos-Ruiz similar d /d values (Fig.  7B). This indicates that lan - et al. 2017). N S thipeptide synthetases of the cyanobacteria must have Chaudhary et al. AMB Express (2023) 13:34 Page 14 of 20 Production of evolutionarily selected natural variants disorder to order upon target binding. We hypothesize of Rosα that the four variable residues in this stretch (3-GVLG- The gene-encoded nature of lantibiotics offers an oppor - 6) probably indicate disorder among the fully conserved tunity to bioengineer the peptide components for ring-forming residues. Hence, alterations in this motif obtaining roseocin variants with improved bioactivity/ can significantly affect the binding affinity of Rosα to its physicochemical parameters. The evolutionarily selected target and consequently the antimicrobial activity/MIC natural variants of Roseocin peptides helped us identify of roseocin. Leu8 of this motif seems to be significant due the structural regions amenable to amino acid substitu- to its relatively high conservation, with the only naturally tions. Keeping in mind the earlier common observation allowed substitutions being L8I, L8F, and L8W. Leu8 was on substitutions that disrupt (methyl)lanthionine forma- thus mutated to F and W. Serine at position 12 being the tion, ring size or lanthionine location leading to struc- residue that we characterized earlier to have escaped tural alteration with loss of bioactivity (Barbosa et  al. dehydration (Singh et al. 2020) and W being the only nat- 2019; Bédard et al. 2019; Field et al. 2015; Rahman et al. urally allowed substitution, we tried substituting S12 for 2021), and the limitation of PTM enzymes that gener- W. V22Q substitution was selected for it might affect the ally do not accommodate all amino acid substitutions overall hydrophobicity of the peptide and hence its inter- (Cooper et al. 2008), only a few naturally allowed substi- action with the target. tutions were tried to prove our concept. These evolutionarily selected Rosα variants were gener - We focused specifically on the actinobacteria clade of ated by site-directed mutagenesis and tested for the PTM the roseocin family phylogenetic tree, as the sequences by RosM in vivo in E. coli BL21(DE3). Notably, unlike the arising from BGCs sharing close common ancestors and, other studies where low expression of the lantibiotic vari- thus, the same mode of action are likely to group into the ants is often observed (Field et al. 2013; Geng and Smith same clade. For generating naturally inspired roseocin 2018), phylogeny-guided mutations generated in Rosα variants, we identified the most conserved and divergent led to a substantial yield of the bioengineered variants to sites in roseocin peptides’ homologs (Fig. 3C and D). Var- allow their further analysis. All four RP-HPLC purified iants were restricted to the type 2 subclade only (roseocin full-length peptide variants were analyzed by ESI–MS being its member; Fig.  3A), as types 1 and 3 exhibited (Additional file  1: Figure S8; Table  S4). As was reported significant divergence in BGC structure along with a earlier for the wild-type Rosα, that underwent four-fold divergence in the sequence of both alpha and beta com- dehydration (Singh et  al. 2020), observed mass peaks in ponents. Further, highly divergent variations of type 1 Rosα L8F, L8W, S12W, and V22Q agreed with the calcu- and 3 subclades from the roseocin peptides might hinder lated mass having four-fold dehydrations and one disul- the post-translational modifications by RosM. A consen - phide bond (Additional file  1: Table  S4). An additional sus sequence was obtained using the alignment of Rosα peak of + 89  Da (double-charged ion for 178  Da) mass and Rosβ homologs from the type 2 subclade for the nat- shift indicated the presence of species with N-terminal urally permissible mutations (Additional file  1: Figure S7). gluconoylation. The RP-HPLC purified peptide variants As alpha peptide plays a pivotal role in the mechanism were subjected to leader cleavage with endoprotein- of two-component lantibiotics (Oman et  al. 2011; Bakh- ase GluC and analyzed with MALDI-TOF MS and M S tiary et  al. 2017), Rosα peptide variants were designed (Fig.  7). The MALDI-TOF MS data of the four variants first. Among Rosα homologs, essential amino acid resi - (after TCEP treatment) agreed with the calculated m/z dues required for (methyl)lanthionine rings installation of the reduced peptide i.e. 2  Da higher than the theo- showed high conservation along with the two cysteines retical monoisotopic mass, indicating a reduction of involved in the disulphide bond formation. Another con- the disulfide bond between Cys13 and Cys33 (Fig.  7). served stretch i.e. S/TxxxxTxGCC was clearly notice- Alkylation with IAA confirmed the disulfide bond and able, which we speculate to be a putative target binding assessed the presence of free cysteines, if any, arising motif. Two critical observations favour this speculation: due to incomplete dehydration or cyclizations. A mass first, the ‘ring within a ring’ structure of this N-terminal shift of 57  Da was expected per available free cysteine region, proposed by us due to the absence of fragment residue. It was observed that the natural variants were ions in the MALDI-TOF MS/MS analysis (Singh et  al. fully dehydrated and cyclized to possess four (methyl) 2020). Second, from the pockets and mouth information lanthionine rings and a disulfide bond (Fig.  7, Additional revealed by the homology modelling of 37 characterized file  1: Table  S5). The MS analysis of the leader-cleaved lantibiotics (Chakraborty et  al. 2019), the disordered alkylated peptides further revealed protection imparted residues (the ones involved in protein–protein interac- by (methyl)lanthionine rings. The absence of b and y tions or target binding) were found within the pockets/ fragment ions for the stretches 2-SGVLGTLGCC-11 rings and hence are thought to undergo a transition from and 18-SGWTVCGLAC-27 confirmed thioether ring Chaudhar y et al. AMB Express (2023) 13:34 Page 15 of 20 Fig. 8 Tandem MS fragmentation pattern of alkylated Rosα variants confirms the similar ring topology in all the variants as that of wild-type Rosα, published earlier (Singh et al. 2020). Underlined regions correspond to leader sequence overhang that remained attached after endoproteinase GluC proteolytic digestion Table 1 Minimal inhibitory concentration (MIC) of wild-type concentration (MIC) was determined to compare the roseocin and its variants efficacy of variants with respect to wild-type Rosα against M. luteus ATCC 10240 and methicillin-sensitive Lantibiotic MIC against M. luteus MIC against MSSA ATCC 10240 (µM) ATCC 25923 (µM) Staphylococcus aureus (MSSA) ATCC  25923 (Table  1). Variants generated by substitution within the N-termi- Roseocin (wild-type) 0.5 2 nal conserved motif of Rosα i.e. L8F and L8W showed Roseocin L8F 0.5 0.5 improvement in the MIC. RosαL8F exhibited four-fold Roseocin L8W 0.25 2 lower MIC against MSSA ATCC 25923, while RosαL8W Roseocin S12W 0.5 > 16 showed two-fold lower MIC against M. luteus ATCC Roseocin V22Q > 8 > 16 10240. However, no improvement with RosαS12W and Nisin 0.0625 2 a loss of potency in RosαV22Q were observed. Consid- ering individual roseocin homologs have many simulta- neous amino acid alterations instead of a single residue protected regions in all the variants (Fig.  8), similar to (Additional file  1: Figure S3), some mutations like V22Q wild-type Rosα (Singh et  al. 2020). The fragment ions might depend upon those other amino acids to exert a (Additional file  1: Table  S6) confirmed the dehydration positive effect on antimicrobial activity. The MIC data of Ser2, Thr7, Ser18, Thr21 and the undehydrated status shows that our strategy of mutants generation to obtain of Ser12. The mass shift of 57 Da in y4-y22 indicated the only the bioactive variants was successful and hence can alkylation of Cys33 and a shift of + 114  Da in the frag- be extrapolated to obtain multi-residue variants to match ment ion next to y22 the disulfide bond partner as Cys13. the natural congeners. The thus-engineered best variant The observed fragment ions confirmed the presence of can be subjected to detailed structural and functional a disulphide and 4 (methyl)lanthionine rings in all the characterization for development into a new drug. Rosα variants (Fig.  7). All the variants were tested for their bioactivity in combination with wild-type Rosβ by Phylogeny‑based ring structure prediction. agar diffusion assay against M. luteus ATCC 10240, after Structure elucidation of lanthipeptides is often hindered leader cleavage by proteinase K (Additional file  1: Figure owing to low yield and complexity in the NMR data S9). Bioactivity was observed with all the variants (Addi- acquisition and solving the data. Tandem MS has tional file  1: Figure S9) however, minimum inhibitory Chaudhary et al. AMB Express (2023) 13:34 Page 16 of 20 been widely successful in depicting the structure with to the genome mining study of Walker et al. 2020, which a low amount of lanthipeptides. However, a lack of identified and classified > 8000 precursor peptide hits, fragmentation is observed in the region with overlapping including the members of the roseocin family. Cyclase rings (Garg et al. 2012; Singh et al. 2020), like that in wild- domain (being a common feature among all the currently type Rosα peptide and its variants (Fig. 8). The structural known classes of lanthipeptide synthetases) was used features of roseocin peptides could be solved only to as a query [i.e. LANC_like (PF05147) hidden Markov the extent of alternative possibilities in the ring pattern model (HMM)] from the Protein family (Pfam) database of Rosα, while in Rosβ, only the first two rings A and B (Finn et  al. 2016), which resulted in 12,705 proteins. could be figured out by tandem MS. As an unanticipated Further classification into Class I–IV lanthipeptide advantage of the current phylogenetic study, the ring BGCs was achieved by analyzing the genomic content pattern in the overlapping regions of both the roseocin of each of these for the presence of Pfam HMM for the peptides could further be unfolded, as it seemed that dehydratase domain/LanM/protein kinase (Class III and they are dispensable, probably to gain some advantage in IV). Further on, genes for precursor peptides (being the terms of antimicrobial activity after the ring alteration/ most diverse and hence the least similar) were identified structure modification. in a representative sample from the BGC of each class Since (methyl)lanthionine rings are the result of bond by manual examination of the lanthipeptide-specific formation between a conserved Thr/Ser and a Cys features and genetic distance from the processing residue, the simultaneous deletion of both the Ser/Thr enzyme. Unlike our study, the phylogenetic distance and and Cys residues was used as the hallmark for deducing BGCs organization formed the basis of the grouping the ring pattern. Rosα peptide contains four (methyl) and subgrouping to understand lanthipeptide diversity. lanthionine rings (labelled A to D) and one disulfide However, the proposed class II29/31 for Rosα homologs bond. Based on earlier tandem MS study, Rosα is a excluded roseocin family members of the type 3 subclade, globular-shaped peptide with two pairs of overlapping from the Micromonosporaceae family while at the same regions, i.e. N-terminal rings A and B, and the C-terminal time included many redundant hits of the roseocin rings C and D (Singh et al. 2020). Ring A and B are part of family. For Rosβ homologs, class II2 was proposed that the essential motif of Rosα and its homologs; therefore, included hits from unrelated distant phyla, r fi micutes the mutation in this region was neither expected and cyanobacteria (roseocin being a member of nor found in any of the homologs. However, in the actinobacteria). Thus, the sequence logo generated from C-terminal region, simultaneous deletion of constituent there, varies hugely in member species and conservation residue partners was observed in Rosα homolog from patterns to ours. However, a larger sample size in Walker Nonomuraea (Additional file  1: Figure S10A), showing et  al. 2020 study helped in identifying five new BGCs the possible location of ring C. of the type 2 subclade of the roseocin family (single Rosβ structure has been proposed earlier as a linear LanM-two precursor BGC; Additional file  2), making type 2 the largest subclade of roseocin family (including peptide with a total of six (methyl)lanthionine rings eight from the current study) causing it to have total 13 (labelled A to F). The rings (A and B) were identifiable members (Additional file  1: Figure S3). A consensus logo by tandem MS (Singh et al. 2020), while the rest are part obtained by the multiple sequence alignment of these 13 of the overlapping ring structure. Rings E and F seem indispensable, hence, their ring topology could not be members has formed the basis of our amino acid residue predicted here. Ring C location has been predicted by identification to generate Rosα variants (Additional missing constituent residue partners in M. Muleris, A. file  1: Figure S7A). Overall, the combined approach of radicidentis etc. (Additional file  1: Figure S10B). This left genome mining tools with the evolutionary principles only one possibility of ring D for which missing residue can rapidly and conclusively classify the members of all partners can also be confirmed in Rosβ homologs of S. the lantibiotic families, along with designing the natural rhizosphaericus and A. fjordicus (Additional file  1: Figure variants to select candidates with better efficacy. S10B). Recently, by coupling genome mining methods to identify evolutionarily related polymyxin family-like BGCs from ~ 10,858 sequenced bacterial genomes, Wang Discussion et  al. 2022b successfully identified macolacin, a colistin- Phylogeny-guided mining approach has been successfully like antibiotic that is active against colistin-resistant applied to discover new NRPS/PKS natural products from Gram-negative pathogens. Since the colistin resistance microbial genomes and metagenomes (Kang 2017), but it is mediated by either mcr-1 or intrinsic PEtN transferase is for the first time being applied here for two-component genes (eptA), they reasoned that a solution might have lantibiotics. Phylogeny-guided approach for mining evolved through natural selection to circumvent this roseocin family lantibiotics displays a comparative view Chaudhar y et al. AMB Express (2023) 13:34 Page 17 of 20 seems to evolve as a result of the selection pressure of troubling resistance mechanism and bioinformatically the target structure (Zhang et  al. 2012). However, no searched for the naturally evolved colistin congeners. similar evolutionary pressure over the leader sequence Using the phylogeny-guided genome mining approach, could be postulated. The role of lanthipeptide leader in another nature-inspired lipopeptide antibiotic, cilagicin interaction of the precursor peptide with lanthipeptide was discovered that exhibits a distinct mode of action synthetase is known to impel the core and enzyme into (Wang et  al. 2022a). Out of the ~ 10,000 sequenced a conformationally constraint structure for successful bacterial genomes, a phylogenetic tree was constructed post-translational modifications (Li et  al. 2017). Recent using sequences of condensation starter (Cs) domain studies have confirmed the significance of the leader that installs the N-terminal lipid in lipopeptides. The peptide in successful modification of cognate and clades that fell out as a separate group were selected noncognate precursor peptidesd by lanthipeptide to identify cryptic BGCs, as a potential source of an synthetase LanM (Burkhart et al. 2017; Viel and Kuipers uncharacterized lipopeptide antibiotic. The structure 2022). Findings, like a highly conserved lanthipeptide of the encoded product was bioinformatically predicted leader with diversity in the core peptide (Cubillos- and chemically synthesized, thus producing a synthetic- Ruiz et  al. 2017); vital conservation of FxLx motif in bioinformatic natural product (syn-BNP), cilagicin. lanthipeptide leaders for class I lanthipeptide synthetases Phylogenetic analysis of lanthipeptide synthetases (Abts et  al. 2013); chimeric leader peptide for post- has formed the basis of our current study to identify translational modifications by two different classes of roseocin family lantibiotics and therefore gene synthetases (Burkhart et  al. 2017) and the unique layout crossover events of lanthipeptide synthetases during of mersacidin leader for MrsM (Viel and Kuipers 2022) evolution can be a significant determinant of the fate of suggests the unique function of leader region in post- lanthipeptides diversification in nature. Gene crossovers translational maturation of the bioactive core peptide. of lanthipeptide synthetases can be predicted using a Our study adds an evolutionary logic by showing a tanglegram, which indicates a difference in gene topology coevolutionary relationship between lanthipeptide (lanM in our case) vis-à-vis species evolution tree (16S leader and lanthipeptide synthetase in a phylum- rRNA). Thus, a comparative phylogenetic analysis of dependent manner (Fig.  6). Fewer crossovers in the the 16S rRNA-based species tree with the lanM gene tanglegram of phylogenetic trees of lanthipeptide leader tree was plotted in the dendroscope (Scornavacca et  al. and LanM sequences (Additional file  1: Figure S14), 2011), allowing swapping of the branches from both of also point towards the coevolution of lanthipeptide the trees at the possible closest distance and HGT events leader and lanthipeptide synthetases to maintain the were inferred by crossover lines between the gene and required interactions for post-translational modification species tree (Zhang et  al. 2016). Remarkably, the final in the core sequence. This observation opens up new tanglegram showed only intra-phyla gene crossover possibilities for future combinatorial biosynthesis and events (Additional file  1: Figure S12), which supports emphasizes using the phylogenetically related pair of the phylum-dependent evolution of lanthipeptide lanthipeptide leader and synthetase to produce novel synthetases and hence a probable dispersion of the cognate and non-cognate lantibiotics efficiently. lanthipeptides within the same phylum. To check the Some BGCs, identified here are the only representatives BGC’s organization in correlation to the 16S rRNA from actinobacteria and cyanobacteria (while archaeal of their group that can be explored by in  vitro studies 16S rRNA was placed as an outgroup), two different for novel lanthipeptides. Chloroflexi and acidobacteria ML phylogenetic trees were also plotted (Additional constitute bacterial species from extreme ecological file  1: Figure S13). In the LanM-based phylogenetic conditions and the identified precursor peptides were tree, a systematic classification of the BGC’s structure found enriched with thioether-forming moieties (Ser/ was obtained for both actinobacteria and cyanobacteria Thr and Cys) that, in addition to bioactivity may provide (Fig.  2), which is not apparent in the 16S rRNA-based enhanced stability at extremes of temperature and phylogenetic trees (Additional file  1: Figure S13). Again, pH (Additional file  1: Figure S2, Additional file  2), an the intra-phyla gene crossover events (Additional important feature desired in industrial applications. file  1: Figure S12) could be the precise reason for the Extremophiles can be a source for thermostable PTM difference in the BGC’s classification in the two kinds of machinery for bioengineering. Another important phylogenetic trees. observed feature was the highly conserved dual transport The biosynthesis of a lanthipeptide involves the secretion system of HylD membrane protein and the installation of thioether ring topology in a selective double glycine peptidase domain containing LanTp in stereochemistry (Mukherjee and Van Der Donk 2014). proteobacteria and cyanobacteria subclades (Additional The ring topology in the core sequence of a lanthipeptide file  1: Figure S2). It has been proposed that HylD plays Chaudhary et al. AMB Express (2023) 13:34 Page 18 of 20 a vital role in exporting lanthipeptide across the outer diversity. Figure S13. Maximum likelihood phylogenetic tree of 16S membrane of Gram-negative bacteria (Haft et  al. 2010). rRNA from bacterial species of (A) actinobacteria and (B) cyanobacteria, in correlation of their BGCs organization. Figure S14. (A, B) Tanglegram The dual transport system is a notable feature among between the phylogenetic tree of LanM and their associated lanthipep- BGCs identified in Gram-negative bacteria (Mohr et  al. tide leader/core in Dendroscope. 4. Table S1. 42 selected hits from RosM 2015). search in the NCBI database. Table S2. A comparison of the roseocin and lacticin 3147 families of two LanM-two precursor genes. Table S3. Prochlorosin synthetase (ProcM)-like LanMs in Accession number of ProcM family LanMs from Cubillos-Ruiz et al. 2017. cyanobacteria are a vital component of diversity- Table S4. Calculated (by Expasy) and observed (by ESI-MS) average mass oriented lanthipeptide evolution due to its unparalleled of full length (with leader region) post-translationally modified Rosα-wild type and its variants. Selected positions for mutant generation are bold capacity to process diverse types of lanthipeptide and italicized. Table S5. Calculated (by Expasy) and observed (by MALDI- cores (Cubillos-Ruiz et  al. 2017). However, in our TOF MS) monoisotopic mass of Rosα variants after leader cleavage using dataset, freshwater cyanobacterium species from endoproteinase GluC (leaving an overhang of four amino acid residues) reduced with TCEP only & TCEP reduced peptides alkylated with IAA. Synechococcales showed an association of ProcM-like Table S6. b and y ions obtained in MS-MS fragmentation of Rosα variants. LanMs (CCG motif) with RosM-like LanMs (CHG Table S7. The list of primers used for the SDM-PCR in the generation of motif) within a single BGC suggesting a novel and variants (F-forward and R-reverse primer). exquisitely divergent mechanism of diversity-oriented Additional file 2. Amino acid sequence of lanthipeptide precursors asso - ciated with (A) 42 BGCs of Figure 2 (main text), and (B) additional roseocin evolution in lanthipeptides from cyanobacteria (Fig. 4). homologs from Walker et al, 2020 study. Further, a similar evolutionary rate among the LanM with CCG and CHG motif across the cyanobacteria Acknowledgements phylum (Fig.  6B) increases the possibility of similar AB Sciex TOF/TOF 5800 system maintained at CIF, NABI, Mohali and ESI-MS promiscuity in substrate tolerance. The two earlier system maintained at SAIF in CIL, Panjab University, Chandigarh are duly genome mining studies on BGCs of Synechocystis sp. acknowledged. PCC 7509 and Chamaesiphon minutus missed the Author contributions underlying association of two diverse leader types and SC, SK, DS conceived and designed the study. SC conducted the LanM classes in a single BGC (Singh and Sareen 2014; bioinformatics study. SK designed and generated the variants and did the mass spectrometry measurements. RP, SJ, KB conducted the purification Zhang et al. 2014), which strengthens the perspective of experiments. SC wrote the initial manuscript. MS analysed the data and edited studies done in a phylogeny-guided manner to discover the manuscript. DS arranged the funding, approved the final results, edited of novel lanthipeptides. the manuscript. The authors read and approved the manuscript. Funding Supplementary Information SC received CSIR-SRF fellowship (09/135(0773)/2017-EMR-I) by the The online version contains supplementary material available at https:// doi. Council of Scientific and Industrial Research, New Delhi. SK (JRF) and DS org/ 10. 1186/ s13568- 023- 01536-9. (PI) duly acknowledge the grant received from DST-SERB (Grant Number: CRG/2018/004218). Financial assistance received from University Grants Commission-Special Assistance Programme (UGC-SAP) (DRS Phase-I) is duly Additional file 1: Figure S1. (A) Pie chart of the obtained hits, with acknowledged. most of the hits being from actinobacteria and cyanobacteria phylum. (B) Phylum-dependent clade formation of 100 RosM homologs in Bayesian Availability of data and materials analysis. Figure S2. Complete BGCs of selected 42 LanMs based on BAGEL Original raw dataset can be accessed in Mendeley dataset: https:// data. mende 4 and antiSMASH 5.0 prediction. Annotation is based on CDD analysis. ley. com/ datas ets/ nbwzz 4gg6v/ draft?a= a6d56 111- 2ed8- 4895- b578- 47ff2 Figure S3. Multiple sequence alignment of roseocin family lanthipeptide c474b de.. precursor sequences to determine the conserved motif. Figure S4. Bayes- ian analysis of two LanMs of the same biosynthetic gene cluster from roseocin and lacticin 3147 families. Figure S5. (A) Distantly related BGCs Declarations of roseocin family from Walker et al. 2020 study, with multiple numbers of precursors in gene clusters. (B) Rosα homologs of these BGC are unusu- Ethics approval and consent to participate ally short but contain the conserved motif S/TxxxxTxGCC. Figure S6. (A) Not applicable. Pairwise sequence alignment and percent identity of 42 LanM sequences (B) and the comparative cumulative frequency of identity percentage Consent for publication among leader, core, and LanM protein sequences of 42 BGCs. Figure S7. Not applicable. Sequence logo from multiple sequence alignment (A) Rosα and (B) Rosβ homologs from type 2 subclade showed highly conserved positions and Competing Interests possible sites of evolutionary variation. Figure S8. ESI-MS data showed All the authors declare no competing financial interests. that variants of Rosα were post-translationally modified by RosM, in E. coli BL21(DE3). Figure S9. Evaluation of antimicrobial activity in synergism Author details with Rosβ after leader removal with proteinase K. Figure S10. Multiple Department of Biochemistry, Panjab University, Chandigarh 160014, sequence alignment of Rosα homologs to predict the ring topology in (A) India. Department of Biosciences and Bioengineering, Indian Institute Rosα and (B) Rosβ. Figure S11. Schematic representation of the primer of Technology Roorkee, Roorkee 247667, India. design for site-directed mutagenesis. The arrowheads represent the site of mutation in the primer-primer overlapping region. Figure S12. A tangle- Received: 26 February 2023 Accepted: 4 March 2023 gram between the 16S rRNA and the lanM gene tree indicates that intra- phyla horizontal gene transfer (HGT ) is a major source of lanthipeptide Chaudhar y et al. AMB Express (2023) 13:34 Page 19 of 20 References Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy Abts A, Montalban-Lopez M, Kuipers OP, Smits SH, Schmitt L (2013) NisC binds and high throughput. Nucleic Acids Res 32:1792–1797. https:// doi. org/ the FxLx motif of the nisin leader peptide. 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Journal

AMB ExpressSpringer Journals

Published: Mar 20, 2023

Keywords: Actinobacteria; Lantibiotics; Phylum; Post-translational modification; Variants

References