Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/springer-journals/rego-a-novel-locus-in-the-regulation-of-tetrapyrrole-biosynthesis-in-e0hx6i5HKl
- Publisher
- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- ISSN
- 1590-4261
- eISSN
- 1869-2044
- DOI
- 10.1186/s13213-022-01709-0
- Publisher site
- See Article on Publisher Site
Abstract
Purpose A new locus, regO, involved in the regulation of photosynthesis gene expression in response to oxygen and light, has been studied in Rhodosprillum rubrum ATCC1117 (Rsp. rubrum) for identification of its function. Methods Inactivation of regO by interposon mutagenesis resulted in the inability of cells to grow photosynthetically, (i.e. become PS ). Protein domain analysis of RegO using the BLAST engine was also performed. Results The mutant strain was able to grow only anaerobically in the dark in the presence of DMSO as an external electron acceptor. Under these conditions, the mutant strain produced substantially lower amounts of photosyn‑ thetic membranes, indicating that regO is involved in the regulation of photosynthetic gene expression in response to anaerobiosis. The Rsp. rubrum REGO disrupted mutant recovered the synthesis of photosynthetic membranes and retained regulation by light and/or oxygen tension when wild‑type regO was provided in-trans. Protein domain analysis of RegO revealed that it encodes a multi‑ domain sensor histidine kinase (HK). The signal‑ input domains, or PAS domains, bear strong similarities to putative heme‑bound sensors involved in sensing light, redox potential, and/or oxygen. The output HK domain exhibits strong homology to sensor domains from bacterial two‑ component systems involved in signal transduction in response to the same environmental signals. Conclusion regO is coding for a sensor histidine kinase that belongs to bacterial two‑ component systems respon‑ sible for signal transduction in response to light and oxygen, particularly in the absence of oxygen. It is believed to be involved in the regulation of tetrapyrrole biosynthesis, which was shown as a lack of photosynthetic membranes – . in the mutant strain REGO Unlike other sensor kinase homologues from related anoxygenic phototrophic bacte‑ rial species, although functionally similar to RegB and PrrB, RegO is predicted to lack transmembrane domains and is thus expected to be a cytosolic member of a two‑ component signal transduction system. RegO also differs from its functional homologues, Reg B/PrrB sensor protein kinases, of the two component systems in that it lacks the second component of this two‑ component signal transduction system found in the neighboring genes. That encouraged us to give it the name RegO, indicating the lack of a cognate response regulator similar to Reg A/PrrA on other closely related anoxygenic Rhodobacter species. Keywords Sensor histidine kinase, Rhodosprillum rubrum, Tetrapyrrole biosynthesis, Two‑ component systems Introduction *Correspondence: In order to survive in a continuously varying environ- Manar Mansour ment, living organisms must be able to adjust their meta- manar.soliman@guc.edu.eg Department of Microbiology & Immunology, the German University bolic functions to their surrounding conditions, with in Cairo (GUC), Cairo, Egypt factors such as the availability of nutrients, oxygen, pH © 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/. Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 2 of 12 value, temperature, and light having direct effects on the at the cell surface leading to the phosphorylation of the development and differentiation of organisms (Armitage protein(Isaacson et al. 2006). In all cases studied so far, 1997). In unicellular organisms, their entire surface is in phosphorylation could be identified as a bimolecular direct contact with their environments and must there- reaction: autophosphorylation leads to the formation fore quickly adapt to the prevailing conditions in order to of a homodimer of the kinase. The phosphorylation of avoid cell damage or death (Lee and Wang 2019). In those the histidine kinase monomer is catalyzed by a second cells, environmental conditions are sensed by a variable monomer of the protein (Creager-Allen et al. 2013). His- number of sensor proteins (receptors) (Stein et al. 2020). tidine-protein kinases can vary greatly in their structure. The activation of these proteins leads to the trans - u Th s, whereas some proteins, as mentioned above, have duction of the respective signals to the so-called effec - a membrane anchoring domain, others, such as NtrB, tor proteins, which in turn can elicit, at variable levels, are cytoplasmic proteins (Martínez-Argudo et al. 2002). the required adaptive effect. The most frequent form of In these cases, the transfer of the phosphate group to modification is by phosphorylation. Whereas eukaryotes an internal aspartate residue in the response regulator use, in the main, the amino acids serine, threonine, and domain is possible. Response regulators are classified tyrosine in phosphorylation, bacterial kinases preferen- into four different families: the CheY-, NtrB-, FixJ-, and tially utilise histidine and aspartate for phosphorylation OmpR-families. The CheY family contains the response (Singh et al. 2003). Although serine-, threonine-, and regulators, which consist of a single domain. All other tyrosine kinases are functionally very similar to histi- response regulators are built of multiple domains. Most dine kinases, decisive differences do exist. Prokaryotic response regulators are transcription regulators. They kinases play a crucial role in signal transduction, and in carry in their C-termini DNA binding domains, which many cases, environmental signals are perceived by the enable them to interact with DNA. CheY, however, does so-called two-component systems (Francis et al. 2018; not carry such a DNA-binding region. Instead, the pro- Francis and Porter 2019). tein receives a phosphate group from the corresponding As their name implies, two-component systems con- sensor kinase CheA in a chemotaxis system. The phos - sist of two individual components: a sensor kinase and a phorylated CheY interacts with the flagellar motor pro - cognate response regulator, which become phosphoryl- tein of E. coli, thereby affecting its swimming motility ated upon sensor activation (Desai and Kenney 2017). (Galperin 2006; West and Stock 2001). The phosphorylation of the response regulator enables All fully sequenced bacterial and archaeal genomes the specific regulation of the target gene(s). In organisms have been found to contain, in their HK sensor proteins, such as Escherichia coli (E. coli), Bacillus subtilis, and specific signalling modules, called PAS domains (Tay - Synechocystis sp., 30 to 40 different two-component sys - lor and Zhulin 1999). PAS domains containing signal- tems have been identified, where each individual system transducing proteins are always located intracellularly responds to a different signal and activates a specific gene (Szurmant et al. 2007). These domains track changes in (Hoch and Varughese 2001). light, redox potential, oxygen, small ligands, and the cell’s The activation of the sensor kinases described above overall energy level. PAS domains may also sense exter- by binding a signalling molecule, either from the external nal environmental factors that cross the cell membrane environment or conveyed from another protein, leads to and/or affect cell metabolism (Möglich et al. 2009). The their autophosphorylation (Stock et al. 2000; Buelow and cytoplasmic location of PAS domains suggests that they Raivio 2010). The histidine-protein kinases of prokary - sense changes in the intracellular environment, but PAS otes are characterised by the presence of a conserved domains can directly sense the environment outside the amino acid sequence of approximately 200 amino acids cell for stimuli that enter the cell, such as light (Taylor that contains an ATP-binding domain. This sequence is and Zhulin 1999). The advantage of detecting oxygen, flanked by other domains that show low homology when light, redox potential, and energy levels for cell survival several histidine kinases are compared (Casino et al. has long been recognised (Yang and Tang 2000). An 2009). These regions possess regulatory functions and intracellular location of single and multiple PAS domains are thus specific for the signal that a given kinase per - was predicted in all analysed sensor proteins (Möglich ceives (Mitrophanov and Groisman 2008; Skerker et al. et al. 2009). 2008). Many histidine-protein kinases possess extensive In some signalling pathways, the signal from the recep- N-terminal domains with long stretches of hydrophobic tor is itself transduced into a different form of energy by amino acids. These regions serve to anchor the protein in a second protein. FixL is an oxygen receptor, in which the cytoplasmic membrane (Mascher et al. 2006). Some oxygen binds directly to a heme that is coordinated to a of these transmembrane kinases possess external sen- histidine residue within a PAS domain (Key and Moffat sor domains, which enable the protein to accept signals 2005). Other PAS proteins, such as Aer, are transducers M ansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 3 of 12 that detect oxygen indirectly by sensing redox changes In this study, we investigated, in Rsp. rubrum, a gene as the electron transport system responds to changes called regO, as suggested by the author, encoding a puta- in oxygen concentration (Taylor 2007). Adaptation of tive sensor HK using interposon mutagenesis and com- the PAS domain structure to sense various stimuli such plementation analysis. We show that the encoded protein as oxygen, ligands, light, and redox potential is found in is functionally analogous to the previously identified sen - prokaryotes, and the presence of divergent PAS domains sor HKs from other anoxygenic phototrophic bacteria, in a single protein may be functionally discriminated to e.g., RegB/PrrB in Rb. capsulatus and Rb. sphaeroides, sense different stimuli (Taylor and Zhulin 1999; Mann respectively and Shapiro 2018). Rhodosprillum rubrum is a facultative anoxygenic pho- Material and methods tosynthetic bacterium that exhibits a versatile metabo- Bacterial strains and growth conditions lism that allows it to adapt to rapidly changing growth The characteristics of the bacterial strains and plasmids conditions in its natural environment and, therefore, has used in this study are listed in Table 1. been utilized as a model organism for cellular redox stud- Wild-type Rsp. rubrum S1 (ATCC 11170) cells were ies (Ghosh et al. 1994; Grammel et al. 2003). grown at 30°C, in Sistrom’s basal medium (Table 2) In the presence of oxygen, Rsp. rubrum performs (M-medium). Cultures were grown aerobically in the aerobic respiration. Under anaerobic conditions, R. sp. dark (chemotrophically) or anaerobically in the light rubrum can, in the presence of light, grow photosyn- (phototrophically). Rsp. rubrum was grown aerobically thetically. Reduction of oxygen partial pressure induces in the dark by inoculating 100 ml of Sistrom’s medium in the synthesis of photosynthetic complexes. In the closely 250 ml baffled Erlenmeyer flasks with vigorous shaking at related species Rhodobacter sphaeroides and Rb. capsu- 200 rpm. latus, the expression of photosynthetic genes and genes To grow R. rubrum anaerobically (phototrophically), required for carbon dioxide fixation are largely con - 100-ml screw-cap Pyrex bottles were filled with Sis - trolled by the well-conserved global two-component trom medium, supplemented with succinate as a carbon systems referred to as RegA/RegB in Rb. capsulatus or source, and incubated in the dark for 24 h to deplete oxy- its homologue PrrA/PrrB in Rb. sphaeroides (Grammel gen from the medium. Cultures were then incubated in et al. 2003). The physical gene organisation of the two light using a 160-W lamp at an intensity of 10 W/m . regulatory circuitries is maintained in both species and Transformed E. coli strains, containing different plas - in other members of purple bacteria (Eraso et al. 2008). mid vectors (see Table 1), were grown in Lysogeny Broth The regulation of photosynthesis gene expression in spe - (LB) medium supplemented, when required, with 100 μg/ cies other than Rhodobacter has also been examined ml ampicillin, 34 μg/ml chloramphenicol, 40 μg/ml strep- (Masuda et al. 1999). tomycin plus spectinomycin, 50 μg/ml kanamycin, or 10 Tetrapyrroles are a “color palette” of physiologically μg/ml tetracycline, for selection. Cells were incubated at packed metal ions that play critical roles in both anabolic 37 °C. and catabolic metabolisms, and the kinds of tetrapyr- roles synthesised or acquired by cells define their meta - bolic capabilities (Sebastien et al. 2010). The presence of Interposon mutagenesis of regO locus two tetrapyrrole species, heme and bacteriochlorophyll Genomic DNA of Rsp. rubrum was isolated by the rapid (BChl), made anoxygenic photosynthesis—their most isolation protocol of genomic DNA of gram-negative distinctive feature—possible. bacteria as described by (Neumann et al. 1992). The pro - Transcription of the tetrapyrrole biosynthesis genes— tocol starts with the lysis of bacterial cells by Lysozyme just like photosynthesis genes—is also responsive to oxy- 1 mg/mL (Roth, Germany) 37 °C for 30 min. Following gen, since the need for heme and BChl is dictated to a the addition of 10% SDS and protinase K 1 mg/ml (Roth, significant degree by what form of energy metabolism is Germany) pre-incubated at 37 °C, the tube was gently used by the cell (Sebastien et al. 2010). inverted to obtain a homogenous solution. NaCl (5 M) in In the presence of high oxygen tensions, heme bio- a ratio of 1/3 volume was then added. After transferring synthesis is necessary in order to form respiratory the clear supernatant into a new tube, an equal volume of cytochromes, the cells have no need for, nor do they pro- chloroform was also added. The tube was gently inverted, duce, BChl. But when oxygen tensions fall, BChl levels and the mixture was incubated at room temperature are estimated to increase more than 100-fold (Kořený for 30–60 min with continuous gentle mixing through- et al. 2021), while at the same time heme production also out the incubation period. The aqueous phase was then increases, as both are required for photosynthesis (Flory transferred after centrifugation to a new tube. The DNA and Donohue 1997). were precipitated by addition of 1 volume isopropanol, Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 4 of 12 Table 1 Bacterial strains and plasmids Bacterial strain Genotype or phenotype Reference/source E. coli q r XL1-Blue recA1, endA1 gyrA96 thi‑1 hsdr17 sup E44 relA1 lac ZΔ M15 Tn 10 ( Tet )] Stratagen Rsp. rubrum Wild type ATCC11170 ‑ r r REGO mutants R. rubrum regO Ω, sm, sp This work r r r REGO pRK415 regO regO Ω, sm, sp , pRK415 Tet regO PCR fragment This work Plasmids pBluescript KSII Cloning vector (Ap ); with T3 and T7 promotors Stratagen r r pHP45Ω Source of the Ω Sm Sp cassete (Prentki and and Henry M. Krisch 1984) r r r + pSUP202 Suicide vector for R. rubrum, ap cm tc; mob (Simon et al. 1983) r + pRK2013 kan; tra (Lois et al. 1993) pRK415 tet l (Lois et al. 1993) pBS‑515 1545 bp PCR fragment from R. rubrum was cloned in pBSKS(+), ap This work r r pBS‑515Ω 3545 bp, PCR fragment of R. rubrum with Ω, sm sp at NdeI site of regO was cloned in This work pBSKS(+), ap r r pPSUP‑515Ω 3500 bp, PCR fragment of R. ` with Ω, sm sp at NdeI site of regOwas cloned in pSUP202, This work r r ap ,cm and BamHI using a compatible buffer according to the man - Table 2 Composition of Sistrom’s basal medium ufacturer’s recommendations (NEB, Germany). The frag - Ingredient Concentration (mM) ment was then cloned into a pBSKs ΙΙ plasmid vector that K HPO 199.7 mM was double digested using the same restriction enzymes 2 4 or KH PO 199.8 mM and overnight ligated by T4 DNA ligase (NEB, Germany) at 2 4 (NH ) SO 37.8 mM 16 °C, E. coli XL1 was then transformed with the new con- 4 2 4 or NH Cl 36.55 mM struct, now called pBS-515. Transformed cells were selected Succinic acid 338.7 mM on LB supplemented with 100 μg/ml ampicillin. l ‑ glutamic acid 5.34 mM A spectinomycin-streptomycin resistant omega Ω cas- l ‑aspartic acid 3 mM sette (Gene 29, 303 313) (Prentki and Krisch 1984) was NaCl 86.2 mM cloned at the NdeI unique site of regO located 930 bp Nitrilotriacetic acid 10.4 mM downstream from the start codon. The NdeI digested MgSO . 7H O 12.17 mM regO in pBSKsII was blunted by DNA polymerase I Kle- 4 2 CaCl . 2H O 2.27 mM now fragment (NEB, Germany) at 21 °C. Cloning of the 2 2 FeSO . 7H O 0.072 mM Ω interposon into the regO at the NdeI blunted site was 4 2 (NH ) Mo 0 0.2 ml performed using overnight blunt end ligation by T4 DNA 4 6 7 24 (1% solution) ligase at 16 °C, followed by transformation of the new Trace Elements Solution 1 ml construct (pBSKsII + regO Ω) into E. coli. XL1. Trans- Vitamins Solution 1 ml formed cells were selected on LB plates supplemented by ampicillin; streptomycin, and spectinomycin. regO Ω was cut out of the pBSKsII plasmid by PvuII, purified using the QIAquick gel extraction kit, and then washed twice with 70% EtOH, vacuum dried and dis- sub-cloned into the suicide plasmid vector pSUP202 pre- solved in 1× TE. viously linearized by EcoRV. All constructs were verified The regO gene was amplified by PCR using, a forward by Sanger sequencing. primer: 5′-AAG CTT GGC CAT GGG CGG CGA GCC CGT CGG TCT G-3′, and a reverse primer: 5′-GGA TCC Mobilization of the new construct by tri‑parental TTC ACC CGC CCC CGT CGA TAA-3′ with HindIII and conjugation into Rsp. Rubrum BamHI restriction sites, (bold underlined), respectively The recipient strain, Rsp. rubrum, was grown on liquid (Invitrogen, USA). PCR products were then purified from culture to log phase, and concentrated to obtain a den- agarose gel by QIAquick gel extraction kit (Qiagen, Ger- sity of (5.0 × 10 ) cells/ml. The donor E. coli XL1 and the many). The PCR product was double digested by HindIII M ansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 5 of 12 helper strains were grown separately, overnight, in liquid generated using Vector NTI–Advance-Version 11 software. LB culture containing the appropriate antibiotics. OD660 Domain structure analysis was performed by SMART (http:// of recipient, donor, and helper cultures were measured, smart. embl- heide lberg. de/ smart/ show_ motifs. pl/ ). Protein mixed in an Eppendorf tube (ratio 1:1 in E. coli mating interaction network was performed using STRING http:// and 1:100 in E. coli -Rsp. rubrum mating). The mating smart. embl- heide lberg. de/ smart/ show_ motifs. pl . 11 (https:// mixtures were carefully suspended in 15–20 μl 0.9% ster-versi on11. string- db. org/ cgi/ netwo rk. pl? taskId= kRRkI T0mYz ile NaCl and spread onto a nitrocellulose filter paper on V2) scan the attached QR code for more details. LB agar plate, incubated at 37 °C for 6 h. Mating mixtures were resuspended and diluted in a 0.9% NaCl solution, and plated onto M plates supplemented with appropriate antibiotics. Selected mutants, Rsp. rubrum, were selected following incubation anaerobically at 30 °C for 3–5 days. Complementation analysis of regO gene Complementation analysis of Rsp. rubrum regO mutants was carried out by cloning the wild-type regO gene into the broad-host-range vector pRK415 and introducing it into the regO disrupted mutant strain REGO . Alignments and gene annotation Database searches were performed using National Center for Biotechnology Information (NCBI) BLASTP (http:// www. Results ncbi. nlm. nih. gov/ BLAST. cgi ), with regO from Rsp. rubrum Gene annotation and alignment of regO ATCC11170 as a query from https:// img. jgi. doe. gov/ cgi- Sequence analysis of published genomes from several bin/m/ main. cgi? secti on= G eneD etail & page= geneD etail & phototrophic bacteria (http:// img. jgi. doe. gov/ cgi- bin/ gene_ oid= 63782 7484. A protein sequence alignments were Fig. 1 Physical and genetic maps of the photosynthetic regulatory gene cluster in different Rhodobacter species compared to Rhodosprillum. ORFs and their directions of transcription are represented by open arrows. Rba., Rhodobacter capsulatus; Rba., Rhodobacter sphaeroides. (drawn by Biorender). Gene organization in the regO gene locus of Rhodosprillum rubrum (Rsp. rubrum); hp, hypothetical protein; uroIII, predicted uroporphyrinogenIII methyltransferase Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 6 of 12 pub/ main. cgi ) revealed that genes encoding two-compo- open reading frame (orf515, Gene ID: 637827484), which nent system proteins are frequently organised in a regula- is predicted to encode a PAS/PAC sensor kinase (Fig. 2). tory locus on the chromosome. In addition to the sensor Whereas regB from other purple bacterial species formed kinase (e.g., regB in Rb. capsulatus or its homologue prrB a divergent transcriptional unit relative to regA and senC in Rb sphaeroides) and its cognate response regulator (Masuda et al. 1999), orf515 is flanked by two hypotheti - (regA or prrA), this regulatory locus also carries a third cal proteins and transcribes in the same orientation as gene, senC (or prrC) with strong homology to scoI (Smith senC (Fig. 1). One striking observation is the presence, et al. 1996), (Happ et al. 2005) encoding inner mitochon- in the same locus, of four other open reading frames that drial membrane protein (Fig. 1). Despite their conserva- have been annotated as being involved in the cobalamin tion in all bacterial species studied so far, a search of the biosynthetic pathway. Those ORFs included a uropor - published genome of Rsp. rubrum for homologues of both phoryinogen III methyltransferase, known to be involved regulatory (reg/prr) genes in the Integrated Microbial in the early stages of the general tetrapyrrole biosynthetic Genome database (http:// img. jgi. doe. gov/ cgi. bin/ pub/ pathway. Local alignment of either the nucleotide- or the main. cgi) returned no results. Apparently, Rsp. rubrum protein sequences of orf515 (hereafter designated regO) ATCC11170 has no sequences coding for PrrA/RegA or against regB, and prrB showed that regO shares no sig- PrrB/RegB proteins (Zeilstra-Ryalls 2009). However, the nificant sequence similarity (13% of similarity with Rb third gene of the abovementioned regulatory locus, senC sphaeroides and 11% of similarity with Rb capsuatus) (prrC), seems to be conserved in Rsp. rubrum and pos- with either genes. sesses strong homology to its Rhodobacter homologues. A BLAST similarity search for RegO homologues in Since the physical organisation of the kinase and regula- the NCBI database returned more than one hundred tor genes is often maintained in two-component systems proteins that share significant similarity with the query (Nijhoff et al. 1984; Stock et al. 1989), we reasoned that, (regO); none of them belonged to either Rb. capsulatus or at least, a regB-homologue might be adjacent to senC. Rb. sphaeroides. The alignment of RegO with the top ten Indeed, sequence analysis of the upstream region of Rsp. protein homologies obtained from the BLAST search is rubrum senC-homologue indicated the presence of an shown in Table 3. Fig. 2 Domain structure of RegO generated by SMART (http:// smart. ambl‑ heide lberg. de/ ). Multi‑ domain hybrid structure of RegO in Rsp. rubrum. RegO possesses two PAS domains, a histidine kinase (HisKA) domain and HATPase domain Table 3 Amino acid lengths, percent identities, and similarities of the proteins with highest homology to RegO and their respective E values, as obtained from the BLAST search Source Length Identity % Similarity % E value −62 Geobacter sp. (strain FRC‑32) 737 38 57 4.0 × 10 −59 Methanosarcina acetivorans C2A 1456 31 51 2.0 × 10 −58 Magnetospirillum magnetotacticum MS‑1 380 39 54 6.0 × 10 −58 Magnetospirillum magneticum AMB‑1 626 36 54 6.0 × 10 −57 Magnetospirillum gryphiswaldense 730 33 49 1.0 × 10 MSR‑1 −57 Candidatus Kuenenia stuttgartiensis 494 33 49 4.0 × 10 −56 Magnetospirillum magneticum AMB‑1 368 40 53 3.0 × 10 −56 Nitrosococcus oceani ATCC 19707 368 36 54 6.0 × 10 −55 Magnetospirillum magnetotacticum MS‑1 625 37 55 1.0 ×10 −55 Candidatus Methanosphaerula palustris 640 45 60 3.0 × 10 M ansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 7 of 12 Fig. 3 Absorption spectra of whole cultures of Rsp. Rubrum. A Absorption spectra of whole cultures of the wild‑type strain of Rsp. rubrum grown phototrophically anaerobically (dashed line), chemophototrophically, grown aerobically (no photosynthetic membranes) (dotted line) and the mutant strain Rsp. rubrum REGO (solid line) grown anaerobically in the dark at 30 °C in presence of DMSO. B (a) Absorption spectrum of whole culture of wild‑type strain of Rsp. rubrum grown phototrophically. (b) Absorption spectrum of Rsp. rubrum of the complementation strpRK ‑REGO grown phototrophically is also shown. (c) Wild‑type Rsp. rubrum grown chemophototrophically in presence of DMSO as an external electron acceptor Table 4 Predicted domains, repeats, motifs and features, into the Rsp. rubrum regO disrupted mutant strain generated using SMART REGO . Transconjuants Rsp. rubrum containing the plas- mid pRK-REGO, complemented the chromosomal dis- Name Begin End E value ruption of regO see (Fig. 3B). PAS 11 77 1.88e−06 PAC 77 120 1.80e−03 Domain structure analysis and hydropathicity of RegO PAS 132 198 1.12e−04 Analysis of the domain structure of RegO using the PAC 204 247 1.04e+01 BLAST engine revealed that the deduced amino acid HisKA 267 335 5.28e−04 sequence possesses multiple domains: two PAS domains HATPa se_c 376 491 1.02e−28 at the C-terminus, a histidine kinase (HisKA) domain, and a HATPase domain at the N-terminus (Fig. 2) (Table 4). PAS domains are known as signalling modules that Phenotype of regO‑disrupted mutant Rsp. rubrum REGO monitor changes in light, redox potential, oxygen, small A RegO interrupted mutant was constructed as described ligands, and the overall energy level of a cell (see “Intro- before. The mutant strain was unable to grow photo - duction” section). The histidine kinase domain includes a synthetically, i.e., became PS at any light intensity. The histidine-containing block, which is known to be the site mutant strain showed reduced synthesis of photosynthetic of autophosphrylation in several two-component regu- pigments compared to those of the wild type, when grown latory systems. Unlike many other sensor modules, PAS anaerobically in the dark in the presence of dimethyl sul- domains are located in the cytosol (Taylor and Zhulin foxide (DMSO) as an external electron acceptor (Fig. 3A). 1999). A Kyte and Dolittle hydropathy plot (Fig. 4) pre- Compared to the wild type, levels of A were dramatically dicts the lack of membrane-spanning domains over the reduced. In Rsp. rubrum, A value is routinely used as entire length of RegO. The same prediction was reached a simple reliable screen for variations in levels of spectral when other hydropathy parameters were used (e.g., using complex-synthesis of the light harvesting system (LH1) TMIM). under all growth conditions (Ghosh et al. 1994). Protein interaction network analysis Complementation analysis of regO A protein-interaction network analysis was generated using DNA fragments containing wild-type regO were cloned the STRING 11 freeware (http:// string- db. org/ ) engine into broad-host-range vector pRK415 and introduced employing a medium confidence scale. The retrieved Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 8 of 12 Fig. 4 Hydropathy plot of RegO, using Kyte and Dolittle hydropathy parameters. Showing that RegO is a cytoplasmic not a membrane bound sensor his Kinase Fig. 5 Protein‑interaction network display, showing possible cognate functional partner proteins of RegO. Generated by STRING V.11 https:// versi on11. string‑ db. org/ cgi/ netwo rk. pl? taskId= n9lju Dp6PK vU scan the attached QR code (designated Rru_A3363 in the figure). Description and designations of the predicted functional proteins are listed in Table 5 M ansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 9 of 12 Table 5 Predicted functional partners of RegO as produced by to a family of genes containing a putative membrane- STRING (version 11). (Scanning the attached QR code directs to spanning domain, among which are the sco1 and sco2 the online analysis page) genes from S. cerevisiae. In the latter organism, these genes are believed to play a role in cytochrome c oxi- Rru_A1654 Response regulator receiver domain protein (CheY ) (139 aa) dase subunit assembly in the mitochondria. However, Rru_A3364 Putative uncharacterized protein (409 aa) in anoxygenic phototrophic bacteria, senC is located Rru_A0665 Response regulator receiver domain protein (CheY ) (148 aa) upstream of the two-component RegB/A activation sys- Rru_A3362 Uroporphyrin‑III C‑methyltransferase ‑like (291 aa) tem but downstream of the cbb3 cytochrome c oxidase. Rru_A3361 Putative uncharacterized protein (255 aa) It was suggested that SenC functions as a transducer of Rru_A3206 Response regulator receiver domain protein (CheY ) (126 aa) the oxygen and therefore the redox signal from Cbb3 to lepA Small GTP‑binding protein domain (621 aa) RegB (Eraso and Kaplan 1994). Despite the predicted Rru_A3360 Electron transport protein SCO1/SenC (210 aa) functional similarity between RegB, PrrB, and RegO, the Rru_A3359 Cobyrinate a,c‑ diamide synthase/hydrogenobyrinic acid latter shows poor sequence homology with the former a,c‑ diamide synthase (Glutamine‑hydroly (460 aa) two proteins. Indeed, homology searches using either Rru_A3366 Hydrogenobyrinic acid a,c‑ diamide cobaltochelatase (1240 aa) the nucleotide or amino acid sequences of RegB/PrrB, showed the absence of homologous genes and/or pro- teins in Rsp. rubrum. In addition to the sensor kinase, the second component of this two-component signal results are shown in (Fig. 5) and the predicted functional transduction system is the response regulator. For the partners are listed in Table 5. Screening of the Rsp. rubrum RegB/PrrB sensor proteins, the cognate response regu- proteome for potential interaction partners predicts that lators RegA/PrrA were identified in Rb. capsulatus three CheY chemotaxis response regulators are strong can- and Rb. sphaeroides, respectively. Both components didates for direct interaction with RegO. Other interesting form two divergent transcriptional units in most pho- predictions include potential interactions with the cobala- totrophic bacterial species investigated (see Fig. 1). min biosynthetic genes and the scoI homologue senC, all In Rsp. rubrum, none of the regO neighboring genes existing in the vicinity of regO. is predicted to encode a candidate response regulator. The general features of the system, however, seem to be maintained in Rsp. rubrum: the transduction of an Discussion environmental signal, such as anaerobiosis, involves a In this study, a novel regulatory gene, regO, is described phosphorylation cascade. This signal is initiated when and shown to be clearly involved in the positive regu- the sensor kinase undergoes autophosphorylation at lation of photosynthesis gene expression in response to a conserved histidine residue, forming a high-energy removal of oxygen from cultures of the wild-type strain state phosphate that can subsequently be transferred to Rsp. rubrum S1. Disruption of regO by interposon a conserved aspartate residue on the cognate response mutagenesis produced the mutant strain Rsp. rubrum regulator. The highly conserved histidine residue (H277) REGO , which is unable to grow phototrophically at low in RegO suggests that this protein maintains the phos- light intensities and could only be grown either anaero- phorylation feature of other sensor kinases. Although bically in the dark in the presence of DMSO (an exter- not investigated in this study, many sensor kinases nal electron acceptor) or aerobically in the dark, albeit also exhibit phosphatase activity with the capability of very slowly. Under the microscope, the mutant strain removing phosphate. This activity has been shown to possessed elongated dark cells, reminiscent of fts muta- be affected by the extent of the gene disruption during tions in bacterial cell division machinery proteins (fts = the construction of mutant strains. Thus, Mosley et al. filamentous temperature sensitive). Sequence compari - (Mosley et al. 1995) found that a RegB mutant with a sons indicated that regO is another member of the sen- simple point mutation has a significantly lower level of sor kinase family of prokaryotic sensory transduction aerobic expression than does a putative knockout muta- factors known to regulate a number of diverse cellular tion in RegB. They explained that the first mutant has processes. In this respect, regO seems to be a func- possibly lost the kinase activity but retained the phos- tional homologue of regB in Rb. capsulatus and prrB in phatase activity, whereas the second mutant has lost Rb. sphaeroides, which were identified earlier (Mosley both the kinase and phosphatase activities. Similarly, et al. 1995; Eraso and Kaplan 1994). Interestingly, each dephosphorylation has been shown to play a role in of the three members of the histidine kinase family is the oxygen regulation of expression regulated by FixL physically adjacent to another gene, designated senC in Rhizobium meliloti (Lois et al. 1993). Although a in both Rsp. rubrum and Rb. capsulatus and prrC in RegA response-regulator-homologue was not identified Rb. sphaeroides. The senC gene bears strong similarity Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 10 of 12 Fig. 6 Domain structure of RegB in Rhodobacter capsulatus, generated by SMART (http:// smart. embl‑ heide lberg. de/ smart/ show_ motifs. pl). RegB has a histidin kinase (HisKA) domain and HATPase_c domain. No PAS domains in this study, a network analysis using the programme Gong et al. 1998). ArcB, was shown to detect changes STRING 8.2 predicted two potential CheY-like pro- in aerobic or anaerobic culture conditions by monitor- teins as possible candidates (Fig. 5). CheY is known as ing cell redox poise, through direct interaction with the the response regulator of the chemotaxis sensory pro- cytoplasmically localized respiratory chain (Jung et al. tein CheA (Thakor et al. 2011; Hirschman et al. 2001). 2008). A Kyte-Doolittle hydropathy plot of the deduced The predicted cytosolic nature of RegO (see “Results” RegO polypeptide sequence revealed the lack of any section) suggests a possible crosstalk between RegO putative transmembrane domains, and therefore RegO and other response regulators that are likely to obtain appears to be a cytosolic protein (Fig. 4). the phosphate from RegO. Screening of the genome of The presence of several tetrapyrrole-encoding genes Rsp. rubrum for potential response regulators revealed near regO encourages us to hypothesize that a heme the presence of more than 200 potential proteins pos- group is a possible “sensing” ligand of the predicted PAS sessing DNA binding domains, characteristic for this domains. Further, both PAS-domain duplication and the type of interaction (data not shown). As described in predicted interaction with multiple response regulators Results, amino acid sequence analysis of RegO using suggest that the described cytosolic sensor RegO might BLAST, SMART, or Prosite engines predicted two Per- respond to multiple signals. Arnt-Sim (PAS) domains at the N-terminus, a middle Conflict of interest histidine kinase domain, and a histidine ATP kinase at The authors declare no conflict of interest the C-terminus (Fig. 2) (Table 4). Each of the predicted PAS domains has a photoactivated adenylyl cyclase Authors’ contributions (PAC) at its C-terminus. The latter motifs are sug - Conceptualization: K. A. Methodology: K.A. Investigation: M. M. Writing original gested to contribute to the PAS fold (Taylor and Zhulin draft: M.M. Writing review and editing: K. A. Supervision: K.A. All authors read and approved the final manuscript. 1999). One major difference between RegO and other sensor kinases that respond to changes in oxygen lev- Funding els, e.g., the RegB (PrrB) system in most purple bacte- Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge rial species investigated (Masuda et al. 1999; Sebastien Bank (EKB). The authors declare that no funds, grants, or other support were et al. 2010), and FixL in Rhizobium meliloti and ArcB received during the preparation of this manuscript. in E. coli, is that these kinases lack the N-terminus PAS/ Availability of data and materials PAC domain (Fig. 6). Instead, these proteins exhibit The data sets generated during and/or analyzed during the current study are membrane-spanning domains. However, mutational available from the corresponding author on reasonable request. studies show that apparently the membrane-spanning and periplasmic regions of these kinases do not func- Declarations tion as receptors but act as anchors to the membrane, Ethics approval and consent to participate which are required for optimal in vivo activity (Lois Not applicable et al. 1993). On the other hand, FixL was found to inter- act directly with oxygen via a cytoplasmic heme moiety Consent for publication Not applicable (Gilles-Gonzalez et al. 1991). However, FixL proteins from Bradyrhizobium japonicum (Anthamatten and Competing interests Hennecke 1991; Lois et al. 1993) do not appear to have The authors have no relevant financial or non‑financial interests to disclose. any transmembrane region and apparently are soluble cytoplasmic proteins. Both oxygen sensing and kinase Received: 7 June 2022 Accepted: 20 December 2022 activities appear to be similar in membrane-bound and soluble FixL proteins (Gilles-Gonzalez et al. 1991; M ansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 11 of 12 References xanthus. Mol Microbiol 23:1–7. https:// doi. org/ 10. 1111/j. 1365‑ 2958. 2008. Ann Hirschman, Marina Boukhvalova, Ricaele VanBruggen, Alan J. Wolfe and 06238.x. The RCS (2001) No titleactive site mutations in CheA, the signal‑transducing Jung WS, Jung YR, Oh DB et al (2008) Characterization of the Arc two‑ com‑ protein kinase of the chemotaxis system in Escherichia coli. Biochemistry ponent signal transduction system of the capnophilic rumen bacterium 40:13876–13887 Mannheimia succiniciproducens. FEMS Microbiol Lett 284:109–119. Anthamatten D, Hennecke H (1991) The regulatory status of the fixL ‑ and https:// doi. org/ 10. 1111/j. 1574‑ 6968. 2008. 01187.x fixJ‑like genes in Bradyrhizobium japonicum may be different from that Key J, Moffat K (2005) Crystal structures of deoxy and CO ‑bound bjFixLH reveal in Rhizobium meliloti. MGG Mol Gen Genet 225:38–48. https:// doi. org/ 10. details of ligand recognition and signaling. Biochemistry 44:4627–4635. 1007/ BF002 82640https:// doi. org/ 10. 1021/ bi047 942r Armitage JAP (1997) Behavioural responses of bacteria to light and oxygen. Kořený L, Oborník M, Horáková E et al (2021) The convoluted history of haem Arch Microbiol 168:249–261. https:// doi. org/ 10. 1007/ s0020 30050 496 biosynthesis. Biol Rev 44. https:// doi. org/ 10. 1111/ brv. 12794 Buelow DR, Raivio TL (2010) Three (and more) component regulatory sys‑ Lee Y T, Wang MC (2019) The bacterivore’s solution: fight and flight to promote tems ‑ Auxiliary regulators of bacterial histidine kinases. Mol Microbiol survival. Dev Cell 49:7–9. https:// doi. org/ 10. 1016/j. devcel. 2019. 03. 021 75:547–566. https:// doi. org/ 10. 1111/j. 1365‑ 2958. 2009. 06982.x Lois AF, Weinstein M, Ditta GS, Helinski DR (1993) Autophosphorylation and Casino P, Rubio V, Marina A (2009) Structural insight into partner specificity phosphatase activities of the oxygen‑sensing protein FixL of Rhizobium and phosphoryl transfer in two‑ component signal transduction. Cell meliloti are coordinately regulated by oxygen. J Biol Chem 268:4370– 139:325–336. https:// doi. org/ 10. 1016/j. cell. 2009. 08. 032 4375. https:// doi. org/ 10. 1016/ s0021‑ 9258(18) 53619‑x Creager‑Allen RL, Silversmith RE, Bourret RB (2013) A link between dimeriza‑ Mann TH, Shapiro L (2018) Integration of cell cycle signals by multi‑PAS tion and autophosphorylation of the response regulator PhoB. J Biol domain kinases. Proc Natl Acad Sci U S A 115:E7166–E7173. https:// doi. Chem 288:21755–21769. https:// doi. org/ 10. 1074/ jbc. M113. 471763org/ 10. 1073/ pnas. 18085 43115 Desai SK, Kenney LJ (2017) To ∼P or Not to ∼P? Non‑ canonical activation Martínez‑Argudo I, Salinas P, Maldonado R, Contreras A (2002) Domain by two‑ component response regulators. Mol Microbiol 103:203–213. interactions on the ntr signal transduction pathway: Two‑hybrid analysis https:// doi. org/ 10. 1111/ mmi. 13532 of mutant and truncated derivatives of histidine kinase NtrB. J Bacteriol Eraso JM, Jung HR, Zeng X et al (2008) Role of the global transcriptional regu‑ 184:200–206. https:// doi. org/ 10. 1128/ JB. 184.1. 200‑ 206. 2002 lator PrrA in Rhodobacter sphaeroides 2.4.1: Combined transcriptome Mascher T, Helmann JD, Unden G (2006) Stimulus Perception in Bacterial and proteome analysis. J Bacteriol 190:4831–4848. https:// doi. org/ 10. Signal‑ Transducing Histidine Kinases. Microbiol Mol Biol Rev 70:910–938. 1128/ JB. 00301‑ 08https:// doi. org/ 10. 1128/ mmbr. 00020‑ 06 Eraso JM, Kaplan S (1994) prrA, a putative response regulator involved in oxygen Masuda S, Matsumoto Y, Nagashima KVP et al (1999) Structural and functional regulation of photosynthesis gene expression in Rhodobacter sphaeroides. analyses of photosynthetic regulatory genes regA and regB from J Bacteriol 176:32–43. https:// doi. org/ 10. 1128/ jb. 176.1. 3243.‑ 1994 Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter Flory JE, Donohue TJ (1997) Transcriptional central of several aerobically capsulatus. J Bacteriol 181:4205–4215. https:// doi. org/ 10. 1128/ jb. 181. 14. induced cytochrome structural genes in Rhodobacter sphaeroides. 4205‑ 4215. 1999 Microbiology 143:3101–3110. https:// doi. org/ 10. 1099/ 00221 Mitrophanov AY, Groisman EA (2008) Signal integration in bacterial two‑ 287‑ 143‑ 10‑ 3101 component regulatory systems. Genes Dev 22:2601–2611. https:// doi. Francis VI, Porter SL (2019) Multikinase networks: two‑ component signaling org/ 10. 1101/ gad. 17003 08 networks integrating multiple stimuli. Annu Rev Microbiol 73:199–223. Möglich A, Ayers RA, Moffat K (2009) Structure and signaling mechanism https:// doi. org/ 10. 1146/ annur ev‑ micro‑ 020518‑ 115846 of Per‑ARNT ‑Sim domains. Structure 17:1282–1294. https:// doi. org/ 10. Francis VI, Waters EM, Finton‑ James SE et al (2018) Multiple communica‑1016/j. str. 2009. 08. 011 tion mechanisms between sensor kinases are crucial for virulence in Mosley CS, Suzuki JY, Bauer CE (1995) Erratum: Identification and molecular Pseudomonas aeruginosa. Nat Commun 9. https:// doi. org/ 10. 1038/ genetic characterization of a sensor kinase responsible for coordinately s41467‑ 018‑ 04640‑8 regulating light harvesting and reaction center gene expression in Galperin MY (2006) Structural classification of bacterial response regulators: response to anaerobiosis (Journal of Bacteriology 176:24 (7569)). J Bacte‑ Diversity of output domains and domain combinations. J Bacteriol riol 177:3359. https:// doi. org/ 10. 1128/ jb. 177. 11. 3359‑ 3359. 1995 188:4169–4182. https:// doi. org/ 10. 1128/ JB. 01887‑ 05 Neumann B, Pospiech A, Schairer HU (1992) Rapid isolation of genomic DNA Ghosh R, Hardmeyer A, Thoenen I, Bachofen R (1994) Optimization of the Sis‑ from Gram‑negative bacteria. Trends Genet 8:332–333. https:// doi. org/ 10. trom culture medium for large‑scale batch cultivation of Rhodospirillum 1016/ 0168‑ 9525(92) 90269‑A rubrum under semiaerobic conditions with maximal yield of photosyn‑ Nijhoff M, Publishers J, Hague T (1984) 77 © 1984. 77–87 thetic membranes. Appl Environ Microbiol 60:1698–1700. https:// doi. org/ Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable 10. 1128/ aem. 60.5. 1698‑ 1700. 1994 DNA fragment. Gene 29:303–313. https:// doi. org/ 10. 1111/j. 1442‑ 200X. Gilles‑ Gonzalez MA, Ditta GS, Helinski DR (1991) A haemoprotein with kinase 1992. tb009 31.x activity encoded by the oxygen sensor of Rhizobium meliloti. Nature Sebastien Z, Keran L, Bauer CE (2010) The tetrapyrrole biosynthetic path‑ 350:170–172. https:// doi. org/ 10. 1038/ 35017 0a0 way and its regulation in Rhodobacter capsulatus. Adv Exp Med Biol Gong W, Hao B, Mansy SS et al (1998) Structure of a biological oxygen sensor: 13(675):229–250. https:// doi. org/ 10. 1007/ 978‑1‑ 4419‑ 1528‑3 A new mechanism for heme‑ driven signal transduction. Proc Natl Acad Simon R, Priefer U, Pühler A (1983) A broad host range mobiloization system Sci U S A 95:15177–15182. https:// doi. org/ 10. 1073/ pnas. 95. 26. 15177 for invivo genetic‑ engineering ‑ transposon mutagenasis in gram nega‑ Grammel H, Gilles ED, Ghosh R (2003) Microaerophilic cooperation of reduc‑ tive bacteria. Bio‑ Technology. 1(9):784–791 tive and oxidative pathways allows maximal photosynthetic mem‑ Singh R, Rao V, Shakila H et al (2003) Disruption of mptpB impairs the ability brane biosynthesis in Rhodospirillum rubrum. Appl Environ Microbiol of Mycobacterium tuberculosis to survive in guinea pigs. Mol Microbiol 69:6577–6586. https:// doi. org/ 10. 1128/ AEM. 69. 11. 6577‑ 6586. 2003 50:751–762. https:// doi. org/ 10. 1046/j. 1365‑ 2958. 2003. 03712.x Happ HN, Braatsch S, Broschek V et al (2005) Light‑ dependent regulation of Skerker JM, Perchuk BS, Siryaporn A et al (2008) Rewiring the specificity of photosynthesis genes in Rhodobacter sphaeroides 2.4.1 is coordinately two‑ component signal transduction systems. Cell 133:1043–1054. controlled by photosynthetic electron transport via the PrrBA two‑ com‑https:// doi. org/ 10. 1016/j. cell. 2008. 04. 040 ponent system and the photoreceptor AppA. Mol Microbiol 58:903–914. Smith CA, Suzuki JY, Bauer CE (1996) Cloning and characterization of the https:// doi. org/ 10. 1111/j. 1365‑ 2958. 2005. 04882.x chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by Hoch JA, Varughese KI (2001) Keeping signals straight in phosphorelay signal complementation of a bacteriochlorophyll biosynthesis mutant of transduction. J Bacteriol 183:4941–4949. https:// doi. org/ 10. 1128/ JB. 183. Rhodobacter capsulatus. Plant Mol Biol 30:1307–1314. https:// doi. org/ 10. 17. 4941‑ 4949. 20011007/ BF000 19561 Isaacson D, Mueller JL, JCN and SS (2006) The receiver domain of FrzE, a CheA‑ Stein BJ, Fiebig A, Crosson S (2020) Feedback control of a two‑ component CheY fusion protein, regulates the CheA histidine kinase activity and signaling system by an Fe‑S‑binding receiver domain. MBio 11. https:// downstream signaling to the A‑ and S‑motility systems of Myxococcus doi. org/ 10. 1128/ mBio. 03383‑ 19 Mansour and Abou‑Aisha Annals of Microbiology (2023) 73:5 Page 12 of 12 Stock AM, Robinson VL, Goudreau PN (2000) Two‑ component signal transduc‑ tion. Annu Rev Biochem 69:183–215. https:// doi. org/ 10. 1146/ annur ev. bioch em. 69.1. 183 Stock JB, Ninfa AJ, Stock AM (1989) Protein phosphorylation and regulation of adaptive responses in bacteria Szurmant H, White RA, Hoch JA (2007) Sensor complexes regulating two‑ com‑ ponent signal transduction. Curr Opin Struct Biol 17:706–715. https:// doi. org/ 10. 1016/j. sbi. 2007. 08. 019 Taylor BL (2007) Aer on the inside looking out: paradigm for a PAS‑HAMP role in sensing oxygen, redox and energy. Mol Microbiol 65:1415–1424. https:// doi. org/ 10. 1111/j. 1365‑ 2958. 2007. 05889.x Taylor BL, Zhulin IB (1999) PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63:479–506. https:// doi. org/ 10. 1128/ mmbr. 63.2. 479‑ 506. 1999 Thakor H, Nicholas S, Porter IM et al (2011) Identification of an anchor residue for CheA‑ CheY interactions in the chemotaxis system of Escherichia coli. J Bacteriol 193:3894–3903. https:// doi. org/ 10. 1128/ JB. 00426‑ 11 West AH, Stock AM (2001) Histidine kinases and response regulator proteins in two‑ component signaling systems. Trends Biochem Sci 26:369–376. https:// doi. org/ 10. 1016/ S0968‑ 0004(01) 01852‑7 Yang C, Tang D (2000) Patient‑specific carotid plaque progression simulation. C Model Eng Sci 1:119–131. https:// doi. org/ 10. 1016/j. biote chadv. 2011. 08. 021. Secre ted Zeilstra‑Ryalls JH (2009) Regulation of the tetrapyrrole biosynthetic pathway, pp 777–798. https:// doi. org/ 10. 1007/ 978‑1‑ 4020‑ 8815‑5_ 39 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Journal
Annals of Microbiology – Springer Journals
Published: Jan 19, 2023
Keywords: Sensor histidine kinase; Rhodosprillum rubrum; Tetrapyrrole biosynthesis; Two-component systems