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Genome sequencing of Pediococcus acidilactici (NRCC1), a novel isolate from dromedary camel (Camelus dromedarius) rumen fluid

Genome sequencing of Pediococcus acidilactici (NRCC1), a novel isolate from dromedary camel... The lactic acid bacterium Pediococcus acidilactici has recently been reported to help in treating constipation, diarrhea, relieving stress, and enhancing growth rate and immune response in humans, birds, fishes, and small animals. In the present study, we sequenced and analyzed the whole genome of P. acidilactici NRCC1, a novel isolate from rumen fluid of dromedary camel (Camelus dromedarius). The genome of P. acidilactici NRCC1 was assembled into 60 contigs, comprising 1,785,679 bp and 42.5% GC content. The 1705 CDS were predicted and annotated using the RAST server. The genome encodes numerous enzymes for utilization of different carbohydrates. It also harbors genes for antibiotic biosynthesis and many others which might confer probiotic properties. The comparative genome analysis with P. acidilactici DSM 20284 revealed some unique features in P. acidilactici NRCC1. Thus, the genome sequencing of P. acidilactici NRCC1 has opened up new horizons for further research in animal probiotics and feed supplements. . . . . Keywords Camel Genome analysis Pediococcus acidilactici Probiotic Rumen microflora Introduction camel comprises only three chambers (C1, C2, and C3), while in true ruminants, four chambers are present (Fowler 2010). The dromedary camel (Camelus dromedarius), a unique ani- Chamber C1 is a large anaerobic fermentation chamber anal- mal highly adapted to the desert ecosystem, can digest a range ogous to the rumen in function and harbors a distinct micro- of plant materials, including low-quality shrubs and trees. This bial community that enables the camel to digest, ferment, and ability can be attributed to the extensive microbial population extract the nutrients efficiently from plant lignocellulosic ma- in the forestomach comprising bacteria, archaea, fungi, and terial (Kay and Maloiy 1989). Studies suggest that the camel protozoa (Bhatt et al. 2013). The digestive anatomy and phys- rumen microbiome is structurally similar but compositionally iology of the dromedary camel is different from that of true distinct from other ruminants (Gharechahi et al. 2015;Dande ruminants like cattle, sheep, and goat. The forestomach of et al. 2015). At the phylum level, Firmicutes comprises the second largest group, accounting for about 31% of the total bacterial population in camel rumen (Gharechahi et al. 2015). Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-017-1320-0) contains supplementary Pediococcus acidilactici is a homofermentative, Gram- material, which is available to authorized users. positive, nonmotile, catalase-negative facultative anaerobe be- longing to phylum, Firmicutes; class, Bacilli; order, * Rakesh Ranjan Lactobacillales; and family, Lactobacillaceae. It can grow in rakesh_ranjan3@rediffmail.com a wide range of pH, temperature, and osmotic pressure and can colonize the digestive tract of humans and animals ICAR—National Research Centre on Camel, Jorbeer, (Klaenhammer 1993). Besides, it is also present in fermented Bikaner, Rajasthan 334001, India vegetables, fermented dairy products, and meat. They possess Department of Animal Biotechnology, College of Veterinary Science several beneficial health effects, helpful in treating constipa- and Animal Husbandry, Anand Agricultural University (AAU), tion, diarrhea, relieving stress, and enhancing growth rate and Anand, Gujarat 388001, India immune response in birds, fishes, and small animals Department of Microbiology and Biogas Research, Gujarat (Ferguson et al. 2010). Some strains isolated from food and Vidyapith, Sadra, Ahmedabad, Gujarat 382320, India 104 Ann Microbiol (2018) 68:103–110 the human gastrointestinal system have recently been tested as Research Centre on Equines, Hisar, Haryana, India with ac- a probiotic supplement and found to prevent colonization of cession no. VTCCRM0000259B. pathogens such as Shigella spp., Salmonella spp., Clostridium difficile,and Escherichia coli in the small intestine (Feng et al. Biochemical analysis for utilization of different sugars 2016). Pediococcus acidilactici produces distinct pediocins that are active against a broad spectrum of Gram-positive bac- The API 20A test kit (bioMérieux, La-Balme-les-Grottes, teria (Cintas et al. 1995). To our knowledge, this is the first France) was used to test the carbon utilization profile of study documenting the isolation of P. acidilactici from camel P. acidilactici NRCC1, as per the manufacturer’sinstructions. rumen fluid and its genetic analysis. The draft genome se- Strict anaerobic conditions were maintained using gas phase quence of P. acidilactici NRCC1 was analyzed with particular N :CO (80:20) and the glovebox was maintained in anaero- 2 2 reference to its probiotic potential and functional characteris- bic conditions with N :H :CO (80:10:10) during the incuba- 2 2 2 tics were compared with P. acidilactici DSM 20284. tion. The results of the biochemical tests were later compared with genome features for the presence of genes coding specif- ically for sugar metabolism. Materials and methods Genomic DNA isolation and sequencing Rumen fluid collection A pure culture with uniform colony characteristics and mor- phology was processed for DNA isolation. The genomic DNA A rumen fluid sample was collected from an 8-year-old was isolated using a commercial DNA isolation kit (GenElute healthy male dromedary camel (Camelus dromedarius)using Bacterial Genomic DNA Kit, NA2110; Sigma-Aldrich) and a rumen fluid extraction unit designed for camels as described the concentration was measured using a NanoDrop elsewhere (Bhatt et al. 2013). The first 200-mL sample obtain- Spectrophotometer ND1000 (Thermo Scientific, USA). The ed was discarded to avoid contamination with saliva. whole genome was sequenced using Ion Torrent PGM. The Thereafter, rumen contents (50 mL) was collected and filtered library was prepared using the Ion Plus Fragment Library Kit through four layers of autoclaved gauze, and the filtered ru- by following the manufacturer’s instructions. In brief, the ge- men fluid was used for microbial culture and isolation. The nomic DNA was fragmented and adaptors were ligated. The animal was maintained under an intensive system of manage- desired size of library fragments was selected using the E-Gel ment and fed daily with guar (Cyamopsis tetragonoloba)meal SizeSelect kit (Thermo Fisher Scientific, USA), subjected to (mixture of 30–33% hull, 27–30% endosperm, and 43–47% emulsion polymerase chain reaction (PCR), and sequencing germ) and groundnut (Arachis hypogaea) haulms. Ethical by Ion PGM using the 318 Chip. standards and guidelines as recommended by the Committee for the Purpose of Control and Supervision of Experiments on Genome assembly and annotation Animals (CPCSEA), Govt. of India and as approved by the Institute Animal Ethics Committee were followed throughout PRINSEQ (Schmieder and Edwards 2011) was used for qual- during animal handling for sample collection. ity filtering, where sequences with mean quality score < 20 and sequences shorter than 40 bp were filtered out. The re- Microbial culture and isolation maining good-quality reads were mapped to P. acidilactici DSM 20284 using GS Reference Mapper (Newbler) v2.3. The rumen fluid was processed for culturing as described by The assembled genome was uploaded to the RAST server Mah and Smith (2009). Briefly, the samples was cultured on for feature prediction and annotation. The whole genome shot- the modified Mah et al. (1978) medium containing starch 1 g, gun sequence has been deposited in DDBJ/EMBL/GenBank yeast extract 0.25 g, K HPo 0.04 g, MgCl 0.01 g, MgCl under the accession number LQNQ00000000 and the version 2 4 2 2 0.01 g, NaHCo 0.05 g, cysteine hydrochloride 0.01 g, pep- described in this paper is version LQNQ01000000. tone 0.25 g, mineral solution 5 mL, vitamin solution 0.5 mL, The reference genome of P. acidilactici strain DSM 20284 and distilled water 100 mL. The culture was incubated anaer- was processed with the same pipeline (RAST). CGView obically at 38 °C for 48 to 72 h. A mixture of nitrogen and Server (Grant and Stothard 2008) was used to show the phys- carbon dioxide (80:20) was purged continuously during the ical map of the genome. Further, we also calculated the aver- incubationtomaintainanaerobic conditions. Repeated age nucleotide identity (ANI) between these two genomes subculturing was done on the same media until pure culture using the ANI calculator (Goris et al. 2007)and the was obtained. The isolated pure culture was submitted to the Orthologous Average Nucleotide Identity Tool (OAT) (Lee rumen microbes repository of the National Centre for et al. 2016). Benchmarking Universal Single-Copy Orthologs Veterinary Type Culture Collection, ICAR-National (BUSCO) v2 (Simão et al. 2015) was used to check the Ann Microbiol (2018) 68:103–110 105 completeness of the assembled genome. tRNAscan-SE (Lowe content. The genome of P. acidilactici NRCC1 was sequenced and Eddy 1997) was used to identify tRNAs in the genome. with 198X coverage. The genome features of P. acidilactici NRCC1 are presented in Table 1 and Fig. 1 shows the physical Metabolic features map of the genome. At the phyla level (Firmicutes), BUSCO revealed 226 com- The carbohydate-active enzyme profiles of P. acidilactici plete and single-copy BUSCOs, which correspond to 97.5% NRCC1 and reference strain DSM 20284 were compared completeness, while at the order level, a total of 428 single- using the Carbohydrate-Active Enzymes (CAZy) database copy BUSCOs were present, corresponding to 96.6% com- (Lombard et al. 2014). A Pfam-based sequence annotation pleteness of the genome (Supplementary Table 1). tRNAscan- of the predicted amino acids gene sequences of both the ge- SE identified a total of 52 tRNAs in the P. acidilactici genome, nomes was performed using the CAZymes Analysis Toolkit encoding for 20 different amino acids (Supplementary Table 2). (Park et al. 2010) with minimum E value 1E-5. Enzymes and pathways information was retrieved using KEGG analysis. Functional annotation antiSMASH (Medema et al. 2011) was used to identify sec- ondary metabolite biosynthesis gene clusters. RAST annotation predicated a total of 1705 CDS (Supplementary Table 3) and, of these, 1013 were functionally Phylogenetic analysis classified with subsystems (Fig. 2a and Supplementary Table 4). A large proportion of the CDS were classified into the carbohy- A BLASTn search of the assembled genome was carried out drate and protein metabolism. A total of 172 CDS of against 16S ribosomal RNA sequences of bacteria and archaea P. acidilactici NRCC1 was found to participate in the carbohy- to identify the organism. The 16S rRNA gene sequence of drate metabolism. The feature was further classified into enzyme P. acidilactici was submitted to the NCBI with accession no. coding for subcategories, such as the central carbohydrate met- KU504251. For phylogenetic analysis, all of the 16S rRNA abolic pathways (43), monosaccharides (54), di- and oligosac- gene sequences of P. acidilactici with size > 1.5Kb were charides (37), fermentation (13), amino sugars (8), organic acids downloaded from the NCBI. Sequences were aligned using (6), sugar alcohols (6), one-carbon metabolism (4), and carbo- MAAFT (Katoh et al. 2012) and poorly aligned regions were hydrates, no subcategory (1). The total number of CDS partici- removed using the Gblocks server (Talavera and Castresana pating in the carbohydrate metabolism in P. acidilactici DSM 2007). Thereafter, the neighbor joining (NJ) tree was con- 20284 was 183, with an almost similar pattern of enzyme coding structed using MEGA (Molecular Evolutionary Genetics for subcategories like central carbohydrate metabolic pathways Analysis) version 7.0 (Kumar et al. 2016). The evolutionary (43), monosaccharides (54), di- and oligosaccharides (38), fer- distances were computed using the maximum composite like- mentation (13), amino sugars (10), organic acids (14), sugar lihood method and are in the units of the number of base alcohols (6), one-carbon metabolism (4), and carbohydrates, no substitutions per site. subcategory (1). Likewise, the protein metabolism profiles of the Table 1 General features of the Pediococcus acidilactici NRCC1 genome Results Feature Value Colonies of P. acidilactici NRCC1 appeared as small, round, milky-white, and opaque, with irregular margins and smooth Total bases 1,785,679 Total contigs 60 moist surface. The organism after Gram staining appeared as Gram-positive, long, single rods. Maximum contig length (bp) 188,345 Mean contig length (bp) 29,761 Biochemical properties and relative enzyme encoding Minimum contig length (bp) 295 CDS in the genome GC content (%) 42.5 Coverage 198X After removing low-quality sequences (Phred score < 20 and N50 78,213 minimum length 40 bp), a total of 1,951,776 sequences N75 50,987 (493,599,611 bp) were subjected to mapping against the N90 17,876 P. acidilactici DSM 20284 (RefSeq ID NZ_CP015206.1) ge- N95 7995 nome using Newbler v2.6. From these, a total of 1,374,981 CDS 1705 sequences corresponding to 353,510,036 bases were mapped tRNAs 52 against the reference genome with 60 consensus contigs. The CAZy domains 335 genome comprises of 1,785,679 bases with 42.5% GC 106 Ann Microbiol (2018) 68:103–110 Fig. 1 Circular genome map of Pediococcus acidilactici NRCC1 two strains were similar, with the only difference being in the Metabolic features lower number of CDS present in NRCC1 for protein biosynthe- sis (120 vs. 150). There were 23 unique features in P. acidilactici From the predicted amino acid sequences in the P. acidilactici NRCC1 in comparison to P. acidilactici DSM 20284 when all genome, 335 putative sequences harbor domains for the CAZy annotations of RAST were considered (Fig. 2b). Moreover, the family of enzymes (Supplementary Table 5). Comparative anal- ANI was 99.13% and 99.18% using the ANI calculator ysis of the carbohydrate-active enzyme profile of P. acidilactici (Supplementary Fig. 1) and OAT v0.93, respectively. NRCC1 revealed the presence of an equal number of enzymes in Fig. 2 a The COG functional classification of the P. acidilactici NRCC1 NRCC1 genomic features with reference strain P. acidilactici DSM genome. The number of genes associated with different functional 20284 categories is shown in parentheses. b The comparison of P. acidilactici Ann Microbiol (2018) 68:103–110 107 the carbohydrate esterase (CE) class, but a slightly lower number glycerol, esculin, mannose, rhamnose, trehalose, and xylose. of enzymes in the glycoside hydrolases (GH) and glycosyltrans- Genome analysis confirmed the presence of genes coding for ferases (GT) classes found in the DSM 20284 strain (Fig. 3). A enzymes involved in the metabolic pathway for utilizing these total of 712 different Pfam domains are present in the genome. sugars (Table 2). Gene sequences for metabolism of sugars Further, we identified a total of 11 secondary metabolite biosyn- like mannitol, melezitose, esculin, gelatin, and raffinose were thesis gene clusters in the genome (Supplementary Fig. 2)using absent, which corroborated the negative results as obtained in the antiSMASH database. One of them is the fusaricidin biosyn- the API 20A test kits and roll tubes. thesis gene cluster (25% of genes showed identity), which is a peptide antibiotic (Supplementary Fig. 3). KEGG analysis Phylogenetic analysis showed that 1045 (61.3%) of 1705 CDS were annotated, where genetic information processing was at the top, followed by en- BLASTn search against bacterial and archaeal 16S rRNA da- vironmental information processing, carbohydrate metabolism, tabases revealed that contig number 58, which is of 1783 bp, unclassified, and others (Supplementary Fig. 4 and showed 99% similarity with the complete 16S rRNA gene Supplementary Table 6). With KEGG, 29 hits for peptidases sequence of P. acidilactici strain DSM 20284 (1569 bp), with (ko01002) were identified where serine, metallo, and cysteine query position 66–1634 bp. As shown in Fig. 4,the isolate peptidases were higher in count (Supplementary Table 7). A total P. acidilactici NRCC1 is more closely related to the of 73 KOs were assigned to the biosynthesis of antibiotics. The P. acidilactici strain SM1 (KX688797.1), which was isolated pathway for anaerobic metabolism of carbohydrate was from fermented moong in Gujarat, India. The second close highlighted, where the pathway showed the formation of pyru- neighbor is P. acidilactici strain JFP1 (KM062019.1), which vate mostly via glycolysis (Supplementary Fig. 5)and subse- was isolated from traditional food in Jeju, South Korea. quent anaerobic fermentation. Enzymes related to pyruvate (Supplementary Fig. 6), butanoate (Supplementary Fig. 7a), and propanoate metabolism (Supplementary Fig. 7b) were also Discussion identified. The overall carbon metabolism pathway in P. acidilactici NRCC1 is shown in Supplementary Fig. 8. The rumen (C1 chamber) of the dromedary camel is inhibited by a high density of resident microorganisms, including bac- teria, protozoa, archaea, and fungi, which play a vital role in Biochemical analysis for utilization of different sugars degradation and digestion of the ingested plant materials. In fact, the camel is more efficient in the digestion of the fiber of Biochemical tests for the fermentation profile using API 20A range plants, fodder, and grasses than other domestic rumi- kits and roll tubes showed positive results for the fermentation nants (Holzapfel et al. 2009). of nine sugars, including arabinose, galactose, glucose, Pediococcus acidilactici is an important lactic acid bacteria used as starter cultures in meat, vegetable, and dairy fermen- tation, causing characteristic flavor changes, improving hy- gienic quality, and extending the shelf life of products. A range of bacteriocins (pediocins) have been identified from P. acidilactici strains, which, in conjunction with organic acids (such as lactic and acetic acids), result in antagonistic proper- ties against a range of Gram-positive and Gram-negative bac- teria (Ferguson et al. 2010). Some strains of the organism, like P. acidilactici MA18/5M isolated in France from natural- pasture Gramineae, are commercially available probiotics widely used in swine, poultry, aquaculture feeds, and human dietary supplements. In the present study, we sequenced and assembled the genome of P. acidilactici NRCC1 which was isolated from camel rumen fluid. The organism was able to utilize various sugars as the carbon source. This is likely be- Fig. 3 Comparison of the carbohydrate-active enzyme profiles of cause several fibrolytic rumen bacteria hydrolyze complex P. acidilactici NRCC1 and P. acidilactici DSM 20284. GH stands for glycoside hydrolases (hydrolysis and/or rearrangement of glycosidic plant fibers and release the monomers which are subsequently bonds), GT for glycosyltransferases (formation of glycosidic bonds), PL utilized by other rumen bacteria. Not only the biochemical for polysaccharide lyase, CE for carbohydrate esterase (nonhydrolytic tests data but genome analysis also showed the presence of cleavage of glycosidic bonds), CBM for carbohydrate-binding module CDS encoding enzymes needed for the utilization of various (adhesion to carbohydrates), and AA for auxiliary activities (redox enzymes that act in conjunction with CAZymes) sugars. Moreover, the presence of tRNAs for all the amino 108 Ann Microbiol (2018) 68:103–110 Table 2 Results of sugar fermentation tests vis-à-vis coding sequences for enzymes involved in utilizing various sugars as the carbon source, present in P. acidilactici NRCC1 S. no. Sugar API 20A result Roll tubes CDS for enzyme(s) involved in carbon source utilization 1. Arabinose + Not included L-ribulose-5-phosphate 4-epimerase, ribulokinase, L-arabinose isomerase 2. Galactose Not included + 6-Phosphofructokinase, galactosamine-6-phosphate isomerase 3. Glucose + Not included Glucose-6-phosphate 1-dehydrogenase, glucose 1 dehydrogenase 4. Glycerol + Not included 2-Hydroxy-3-oxopropionate reductase, pyruvate kinase, glycerate kinase 5. Esculin + Beta-glucosidase 6. Mannose + Not included Alpha-mannosidase, phosphomannomutase, mannose-6-phosphate isomerase 7. Rhamnose + Not included Rhamnulose-1-phosphate aldolase, rhamnulokinase, L-rhamnose isomerase, alpha-L-rhamnosidase 8. Trehalose + Not included Trehalose-6-phosphate hydrolase, trehalose-specific IIB component 9. Xylose + Not included Xylulose-5-phosphate phosphoketolase acids suggest that P. acidilactici can synthesize all the amino on the presence of universal single-copy orthologous genes, acids on its own. the genome of P. acidilactici was found to be ~ 97% complete. BUSCO is widely used to access the assembly and com- This study revealed that the genome size and GC content of pleteness of the assembled genome (Simão et al. 2015). Based P. acidilactici NRCC1 and P. acidilactici DSM 20284 are Fig. 4 Phylogenetic tree based on 16S rRNA sequences of P. acidilactici Ann Microbiol (2018) 68:103–110 109 similar and within the normal range reported in the literature Conclusion for most of the P. acidilactici strains (Holzapfel et al. 2009; Barreau et al. 2012). The carbohydrate-active enzyme profiles This is the first study reporting the isolation and genomic of these two strains were also similar, though a lower number sequencing of Pediococcus acidilactici NRCC1 from drome- of enzymes in glycoside hydrolases and glycosyltransferases dary camel rumen. The genomic analysis of the organism re- were recorded. This may be perhaps due to the larger amount vealed that it can utilize different carbohydrates, biosynthesize of genomic data available for the reference strain. The antibiotics, and have several unique features suggesting its CAZyme analysis profile further supports the results revealed possible use in probiotics and feed supplements in animals. by the RAST CDS prediction that the organism possesses the Acknowledgements The authors are thankful to Ankit T Hinsu and Ravi potential to digest various plant polysaccharides. Shah, College of Veterinary Science and Animal Husbandry, Anand Different sequences coding for lactose uptake, adherence, Agricultural University (AAU), Anand, Gujarat for extending their tech- resistance to oxidative stress, and folate synthesis were present, nical support in the sequencing and Kashinath, Senior Technical Officer, suggesting the probiotic potential of P. acidilactici NRCC1 ICAR-NRCC, Bikaner for the help in rumen fluid collection. (Prajapati et al. 2013). Resistance to acid and oxidative stress Funding This work is supported by a grant from ICAR-National Centre are important criteria for sustained survival of the organism in for Veterinary Type Culture Collection, National Research Centre on the gastrointestinal tract. Moreover, the presence of antibiotic Equines, Hisar, Haryana, India under Veterinary Type Culture genes as revealed by KEGG analysis as well as the presence of Collection-Rumen Microbe project. the fusaricidin biosynthesis gene cluster also suggests a probiotic property. Fusaricidin is a peptide antibiotic isolated from several Compliance with ethical standards soil bacteria effective against both fungi and Gram-positive bac- Conflict of interest None. teria (Choi et al. 2008;Yuet al. 2012). The fusaricidin-like gene cluster might be responsible for the biosynthesis of pediocins, which are bacteriocins produced by several lactic acid bacteria References (Papagianni 2003). Pediocins from pediococci have been isolat- ed and characterized (Porto et al. 2017). It has been observed that Abd El-Tawab MM, Youssef IM, Bakr HA, Fthenakis GC, Giadinis ND probiotic bacteria can modulate the rumen microbiota and im- (2016) Role of probiotics in nutrition and health of small ruminants. prove health and productivity (Qadis et al. 2014; Uyeno et al. Pol J Vet Sci 19:893–906. https://doi.org/10.1515/pjvs-2016-0114 2015; Abd El-Tawab et al. 2016). Barreau G, Tompkins TA, de Carvalho VG (2012) Draft genome se- Phylogenetically, Pediococcus and Lactobacillus form a quence of probiotic strain Pediococcus acidilactici MA18/5M. J Bacteriol 194:901. https://doi.org/10.1128/JB.06563-11 supercluster that can be divided into two subclusters; all spe- Bhatt VD, Dande SS, Patil NV, Joshi CG (2013) Molecular analysis of the cies of Pediococcus fall within the Lactobacillus casei– bacterial microbiome in the forestomach fluid from the dromedary Pediococcus subcluster. Pediococci are morphologically dis- camel (Camelus dromedarius). 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Genome sequencing of Pediococcus acidilactici (NRCC1), a novel isolate from dromedary camel (Camelus dromedarius) rumen fluid

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Springer Journals
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Copyright © 2017 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-017-1320-0
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Abstract

The lactic acid bacterium Pediococcus acidilactici has recently been reported to help in treating constipation, diarrhea, relieving stress, and enhancing growth rate and immune response in humans, birds, fishes, and small animals. In the present study, we sequenced and analyzed the whole genome of P. acidilactici NRCC1, a novel isolate from rumen fluid of dromedary camel (Camelus dromedarius). The genome of P. acidilactici NRCC1 was assembled into 60 contigs, comprising 1,785,679 bp and 42.5% GC content. The 1705 CDS were predicted and annotated using the RAST server. The genome encodes numerous enzymes for utilization of different carbohydrates. It also harbors genes for antibiotic biosynthesis and many others which might confer probiotic properties. The comparative genome analysis with P. acidilactici DSM 20284 revealed some unique features in P. acidilactici NRCC1. Thus, the genome sequencing of P. acidilactici NRCC1 has opened up new horizons for further research in animal probiotics and feed supplements. . . . . Keywords Camel Genome analysis Pediococcus acidilactici Probiotic Rumen microflora Introduction camel comprises only three chambers (C1, C2, and C3), while in true ruminants, four chambers are present (Fowler 2010). The dromedary camel (Camelus dromedarius), a unique ani- Chamber C1 is a large anaerobic fermentation chamber anal- mal highly adapted to the desert ecosystem, can digest a range ogous to the rumen in function and harbors a distinct micro- of plant materials, including low-quality shrubs and trees. This bial community that enables the camel to digest, ferment, and ability can be attributed to the extensive microbial population extract the nutrients efficiently from plant lignocellulosic ma- in the forestomach comprising bacteria, archaea, fungi, and terial (Kay and Maloiy 1989). Studies suggest that the camel protozoa (Bhatt et al. 2013). The digestive anatomy and phys- rumen microbiome is structurally similar but compositionally iology of the dromedary camel is different from that of true distinct from other ruminants (Gharechahi et al. 2015;Dande ruminants like cattle, sheep, and goat. The forestomach of et al. 2015). At the phylum level, Firmicutes comprises the second largest group, accounting for about 31% of the total bacterial population in camel rumen (Gharechahi et al. 2015). Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-017-1320-0) contains supplementary Pediococcus acidilactici is a homofermentative, Gram- material, which is available to authorized users. positive, nonmotile, catalase-negative facultative anaerobe be- longing to phylum, Firmicutes; class, Bacilli; order, * Rakesh Ranjan Lactobacillales; and family, Lactobacillaceae. It can grow in rakesh_ranjan3@rediffmail.com a wide range of pH, temperature, and osmotic pressure and can colonize the digestive tract of humans and animals ICAR—National Research Centre on Camel, Jorbeer, (Klaenhammer 1993). Besides, it is also present in fermented Bikaner, Rajasthan 334001, India vegetables, fermented dairy products, and meat. They possess Department of Animal Biotechnology, College of Veterinary Science several beneficial health effects, helpful in treating constipa- and Animal Husbandry, Anand Agricultural University (AAU), tion, diarrhea, relieving stress, and enhancing growth rate and Anand, Gujarat 388001, India immune response in birds, fishes, and small animals Department of Microbiology and Biogas Research, Gujarat (Ferguson et al. 2010). Some strains isolated from food and Vidyapith, Sadra, Ahmedabad, Gujarat 382320, India 104 Ann Microbiol (2018) 68:103–110 the human gastrointestinal system have recently been tested as Research Centre on Equines, Hisar, Haryana, India with ac- a probiotic supplement and found to prevent colonization of cession no. VTCCRM0000259B. pathogens such as Shigella spp., Salmonella spp., Clostridium difficile,and Escherichia coli in the small intestine (Feng et al. Biochemical analysis for utilization of different sugars 2016). Pediococcus acidilactici produces distinct pediocins that are active against a broad spectrum of Gram-positive bac- The API 20A test kit (bioMérieux, La-Balme-les-Grottes, teria (Cintas et al. 1995). To our knowledge, this is the first France) was used to test the carbon utilization profile of study documenting the isolation of P. acidilactici from camel P. acidilactici NRCC1, as per the manufacturer’sinstructions. rumen fluid and its genetic analysis. The draft genome se- Strict anaerobic conditions were maintained using gas phase quence of P. acidilactici NRCC1 was analyzed with particular N :CO (80:20) and the glovebox was maintained in anaero- 2 2 reference to its probiotic potential and functional characteris- bic conditions with N :H :CO (80:10:10) during the incuba- 2 2 2 tics were compared with P. acidilactici DSM 20284. tion. The results of the biochemical tests were later compared with genome features for the presence of genes coding specif- ically for sugar metabolism. Materials and methods Genomic DNA isolation and sequencing Rumen fluid collection A pure culture with uniform colony characteristics and mor- phology was processed for DNA isolation. The genomic DNA A rumen fluid sample was collected from an 8-year-old was isolated using a commercial DNA isolation kit (GenElute healthy male dromedary camel (Camelus dromedarius)using Bacterial Genomic DNA Kit, NA2110; Sigma-Aldrich) and a rumen fluid extraction unit designed for camels as described the concentration was measured using a NanoDrop elsewhere (Bhatt et al. 2013). The first 200-mL sample obtain- Spectrophotometer ND1000 (Thermo Scientific, USA). The ed was discarded to avoid contamination with saliva. whole genome was sequenced using Ion Torrent PGM. The Thereafter, rumen contents (50 mL) was collected and filtered library was prepared using the Ion Plus Fragment Library Kit through four layers of autoclaved gauze, and the filtered ru- by following the manufacturer’s instructions. In brief, the ge- men fluid was used for microbial culture and isolation. The nomic DNA was fragmented and adaptors were ligated. The animal was maintained under an intensive system of manage- desired size of library fragments was selected using the E-Gel ment and fed daily with guar (Cyamopsis tetragonoloba)meal SizeSelect kit (Thermo Fisher Scientific, USA), subjected to (mixture of 30–33% hull, 27–30% endosperm, and 43–47% emulsion polymerase chain reaction (PCR), and sequencing germ) and groundnut (Arachis hypogaea) haulms. Ethical by Ion PGM using the 318 Chip. standards and guidelines as recommended by the Committee for the Purpose of Control and Supervision of Experiments on Genome assembly and annotation Animals (CPCSEA), Govt. of India and as approved by the Institute Animal Ethics Committee were followed throughout PRINSEQ (Schmieder and Edwards 2011) was used for qual- during animal handling for sample collection. ity filtering, where sequences with mean quality score < 20 and sequences shorter than 40 bp were filtered out. The re- Microbial culture and isolation maining good-quality reads were mapped to P. acidilactici DSM 20284 using GS Reference Mapper (Newbler) v2.3. The rumen fluid was processed for culturing as described by The assembled genome was uploaded to the RAST server Mah and Smith (2009). Briefly, the samples was cultured on for feature prediction and annotation. The whole genome shot- the modified Mah et al. (1978) medium containing starch 1 g, gun sequence has been deposited in DDBJ/EMBL/GenBank yeast extract 0.25 g, K HPo 0.04 g, MgCl 0.01 g, MgCl under the accession number LQNQ00000000 and the version 2 4 2 2 0.01 g, NaHCo 0.05 g, cysteine hydrochloride 0.01 g, pep- described in this paper is version LQNQ01000000. tone 0.25 g, mineral solution 5 mL, vitamin solution 0.5 mL, The reference genome of P. acidilactici strain DSM 20284 and distilled water 100 mL. The culture was incubated anaer- was processed with the same pipeline (RAST). CGView obically at 38 °C for 48 to 72 h. A mixture of nitrogen and Server (Grant and Stothard 2008) was used to show the phys- carbon dioxide (80:20) was purged continuously during the ical map of the genome. Further, we also calculated the aver- incubationtomaintainanaerobic conditions. Repeated age nucleotide identity (ANI) between these two genomes subculturing was done on the same media until pure culture using the ANI calculator (Goris et al. 2007)and the was obtained. The isolated pure culture was submitted to the Orthologous Average Nucleotide Identity Tool (OAT) (Lee rumen microbes repository of the National Centre for et al. 2016). Benchmarking Universal Single-Copy Orthologs Veterinary Type Culture Collection, ICAR-National (BUSCO) v2 (Simão et al. 2015) was used to check the Ann Microbiol (2018) 68:103–110 105 completeness of the assembled genome. tRNAscan-SE (Lowe content. The genome of P. acidilactici NRCC1 was sequenced and Eddy 1997) was used to identify tRNAs in the genome. with 198X coverage. The genome features of P. acidilactici NRCC1 are presented in Table 1 and Fig. 1 shows the physical Metabolic features map of the genome. At the phyla level (Firmicutes), BUSCO revealed 226 com- The carbohydate-active enzyme profiles of P. acidilactici plete and single-copy BUSCOs, which correspond to 97.5% NRCC1 and reference strain DSM 20284 were compared completeness, while at the order level, a total of 428 single- using the Carbohydrate-Active Enzymes (CAZy) database copy BUSCOs were present, corresponding to 96.6% com- (Lombard et al. 2014). A Pfam-based sequence annotation pleteness of the genome (Supplementary Table 1). tRNAscan- of the predicted amino acids gene sequences of both the ge- SE identified a total of 52 tRNAs in the P. acidilactici genome, nomes was performed using the CAZymes Analysis Toolkit encoding for 20 different amino acids (Supplementary Table 2). (Park et al. 2010) with minimum E value 1E-5. Enzymes and pathways information was retrieved using KEGG analysis. Functional annotation antiSMASH (Medema et al. 2011) was used to identify sec- ondary metabolite biosynthesis gene clusters. RAST annotation predicated a total of 1705 CDS (Supplementary Table 3) and, of these, 1013 were functionally Phylogenetic analysis classified with subsystems (Fig. 2a and Supplementary Table 4). A large proportion of the CDS were classified into the carbohy- A BLASTn search of the assembled genome was carried out drate and protein metabolism. A total of 172 CDS of against 16S ribosomal RNA sequences of bacteria and archaea P. acidilactici NRCC1 was found to participate in the carbohy- to identify the organism. The 16S rRNA gene sequence of drate metabolism. The feature was further classified into enzyme P. acidilactici was submitted to the NCBI with accession no. coding for subcategories, such as the central carbohydrate met- KU504251. For phylogenetic analysis, all of the 16S rRNA abolic pathways (43), monosaccharides (54), di- and oligosac- gene sequences of P. acidilactici with size > 1.5Kb were charides (37), fermentation (13), amino sugars (8), organic acids downloaded from the NCBI. Sequences were aligned using (6), sugar alcohols (6), one-carbon metabolism (4), and carbo- MAAFT (Katoh et al. 2012) and poorly aligned regions were hydrates, no subcategory (1). The total number of CDS partici- removed using the Gblocks server (Talavera and Castresana pating in the carbohydrate metabolism in P. acidilactici DSM 2007). Thereafter, the neighbor joining (NJ) tree was con- 20284 was 183, with an almost similar pattern of enzyme coding structed using MEGA (Molecular Evolutionary Genetics for subcategories like central carbohydrate metabolic pathways Analysis) version 7.0 (Kumar et al. 2016). The evolutionary (43), monosaccharides (54), di- and oligosaccharides (38), fer- distances were computed using the maximum composite like- mentation (13), amino sugars (10), organic acids (14), sugar lihood method and are in the units of the number of base alcohols (6), one-carbon metabolism (4), and carbohydrates, no substitutions per site. subcategory (1). Likewise, the protein metabolism profiles of the Table 1 General features of the Pediococcus acidilactici NRCC1 genome Results Feature Value Colonies of P. acidilactici NRCC1 appeared as small, round, milky-white, and opaque, with irregular margins and smooth Total bases 1,785,679 Total contigs 60 moist surface. The organism after Gram staining appeared as Gram-positive, long, single rods. Maximum contig length (bp) 188,345 Mean contig length (bp) 29,761 Biochemical properties and relative enzyme encoding Minimum contig length (bp) 295 CDS in the genome GC content (%) 42.5 Coverage 198X After removing low-quality sequences (Phred score < 20 and N50 78,213 minimum length 40 bp), a total of 1,951,776 sequences N75 50,987 (493,599,611 bp) were subjected to mapping against the N90 17,876 P. acidilactici DSM 20284 (RefSeq ID NZ_CP015206.1) ge- N95 7995 nome using Newbler v2.6. From these, a total of 1,374,981 CDS 1705 sequences corresponding to 353,510,036 bases were mapped tRNAs 52 against the reference genome with 60 consensus contigs. The CAZy domains 335 genome comprises of 1,785,679 bases with 42.5% GC 106 Ann Microbiol (2018) 68:103–110 Fig. 1 Circular genome map of Pediococcus acidilactici NRCC1 two strains were similar, with the only difference being in the Metabolic features lower number of CDS present in NRCC1 for protein biosynthe- sis (120 vs. 150). There were 23 unique features in P. acidilactici From the predicted amino acid sequences in the P. acidilactici NRCC1 in comparison to P. acidilactici DSM 20284 when all genome, 335 putative sequences harbor domains for the CAZy annotations of RAST were considered (Fig. 2b). Moreover, the family of enzymes (Supplementary Table 5). Comparative anal- ANI was 99.13% and 99.18% using the ANI calculator ysis of the carbohydrate-active enzyme profile of P. acidilactici (Supplementary Fig. 1) and OAT v0.93, respectively. NRCC1 revealed the presence of an equal number of enzymes in Fig. 2 a The COG functional classification of the P. acidilactici NRCC1 NRCC1 genomic features with reference strain P. acidilactici DSM genome. The number of genes associated with different functional 20284 categories is shown in parentheses. b The comparison of P. acidilactici Ann Microbiol (2018) 68:103–110 107 the carbohydrate esterase (CE) class, but a slightly lower number glycerol, esculin, mannose, rhamnose, trehalose, and xylose. of enzymes in the glycoside hydrolases (GH) and glycosyltrans- Genome analysis confirmed the presence of genes coding for ferases (GT) classes found in the DSM 20284 strain (Fig. 3). A enzymes involved in the metabolic pathway for utilizing these total of 712 different Pfam domains are present in the genome. sugars (Table 2). Gene sequences for metabolism of sugars Further, we identified a total of 11 secondary metabolite biosyn- like mannitol, melezitose, esculin, gelatin, and raffinose were thesis gene clusters in the genome (Supplementary Fig. 2)using absent, which corroborated the negative results as obtained in the antiSMASH database. One of them is the fusaricidin biosyn- the API 20A test kits and roll tubes. thesis gene cluster (25% of genes showed identity), which is a peptide antibiotic (Supplementary Fig. 3). KEGG analysis Phylogenetic analysis showed that 1045 (61.3%) of 1705 CDS were annotated, where genetic information processing was at the top, followed by en- BLASTn search against bacterial and archaeal 16S rRNA da- vironmental information processing, carbohydrate metabolism, tabases revealed that contig number 58, which is of 1783 bp, unclassified, and others (Supplementary Fig. 4 and showed 99% similarity with the complete 16S rRNA gene Supplementary Table 6). With KEGG, 29 hits for peptidases sequence of P. acidilactici strain DSM 20284 (1569 bp), with (ko01002) were identified where serine, metallo, and cysteine query position 66–1634 bp. As shown in Fig. 4,the isolate peptidases were higher in count (Supplementary Table 7). A total P. acidilactici NRCC1 is more closely related to the of 73 KOs were assigned to the biosynthesis of antibiotics. The P. acidilactici strain SM1 (KX688797.1), which was isolated pathway for anaerobic metabolism of carbohydrate was from fermented moong in Gujarat, India. The second close highlighted, where the pathway showed the formation of pyru- neighbor is P. acidilactici strain JFP1 (KM062019.1), which vate mostly via glycolysis (Supplementary Fig. 5)and subse- was isolated from traditional food in Jeju, South Korea. quent anaerobic fermentation. Enzymes related to pyruvate (Supplementary Fig. 6), butanoate (Supplementary Fig. 7a), and propanoate metabolism (Supplementary Fig. 7b) were also Discussion identified. The overall carbon metabolism pathway in P. acidilactici NRCC1 is shown in Supplementary Fig. 8. The rumen (C1 chamber) of the dromedary camel is inhibited by a high density of resident microorganisms, including bac- teria, protozoa, archaea, and fungi, which play a vital role in Biochemical analysis for utilization of different sugars degradation and digestion of the ingested plant materials. In fact, the camel is more efficient in the digestion of the fiber of Biochemical tests for the fermentation profile using API 20A range plants, fodder, and grasses than other domestic rumi- kits and roll tubes showed positive results for the fermentation nants (Holzapfel et al. 2009). of nine sugars, including arabinose, galactose, glucose, Pediococcus acidilactici is an important lactic acid bacteria used as starter cultures in meat, vegetable, and dairy fermen- tation, causing characteristic flavor changes, improving hy- gienic quality, and extending the shelf life of products. A range of bacteriocins (pediocins) have been identified from P. acidilactici strains, which, in conjunction with organic acids (such as lactic and acetic acids), result in antagonistic proper- ties against a range of Gram-positive and Gram-negative bac- teria (Ferguson et al. 2010). Some strains of the organism, like P. acidilactici MA18/5M isolated in France from natural- pasture Gramineae, are commercially available probiotics widely used in swine, poultry, aquaculture feeds, and human dietary supplements. In the present study, we sequenced and assembled the genome of P. acidilactici NRCC1 which was isolated from camel rumen fluid. The organism was able to utilize various sugars as the carbon source. This is likely be- Fig. 3 Comparison of the carbohydrate-active enzyme profiles of cause several fibrolytic rumen bacteria hydrolyze complex P. acidilactici NRCC1 and P. acidilactici DSM 20284. GH stands for glycoside hydrolases (hydrolysis and/or rearrangement of glycosidic plant fibers and release the monomers which are subsequently bonds), GT for glycosyltransferases (formation of glycosidic bonds), PL utilized by other rumen bacteria. Not only the biochemical for polysaccharide lyase, CE for carbohydrate esterase (nonhydrolytic tests data but genome analysis also showed the presence of cleavage of glycosidic bonds), CBM for carbohydrate-binding module CDS encoding enzymes needed for the utilization of various (adhesion to carbohydrates), and AA for auxiliary activities (redox enzymes that act in conjunction with CAZymes) sugars. Moreover, the presence of tRNAs for all the amino 108 Ann Microbiol (2018) 68:103–110 Table 2 Results of sugar fermentation tests vis-à-vis coding sequences for enzymes involved in utilizing various sugars as the carbon source, present in P. acidilactici NRCC1 S. no. Sugar API 20A result Roll tubes CDS for enzyme(s) involved in carbon source utilization 1. Arabinose + Not included L-ribulose-5-phosphate 4-epimerase, ribulokinase, L-arabinose isomerase 2. Galactose Not included + 6-Phosphofructokinase, galactosamine-6-phosphate isomerase 3. Glucose + Not included Glucose-6-phosphate 1-dehydrogenase, glucose 1 dehydrogenase 4. Glycerol + Not included 2-Hydroxy-3-oxopropionate reductase, pyruvate kinase, glycerate kinase 5. Esculin + Beta-glucosidase 6. Mannose + Not included Alpha-mannosidase, phosphomannomutase, mannose-6-phosphate isomerase 7. Rhamnose + Not included Rhamnulose-1-phosphate aldolase, rhamnulokinase, L-rhamnose isomerase, alpha-L-rhamnosidase 8. Trehalose + Not included Trehalose-6-phosphate hydrolase, trehalose-specific IIB component 9. Xylose + Not included Xylulose-5-phosphate phosphoketolase acids suggest that P. acidilactici can synthesize all the amino on the presence of universal single-copy orthologous genes, acids on its own. the genome of P. acidilactici was found to be ~ 97% complete. BUSCO is widely used to access the assembly and com- This study revealed that the genome size and GC content of pleteness of the assembled genome (Simão et al. 2015). Based P. acidilactici NRCC1 and P. acidilactici DSM 20284 are Fig. 4 Phylogenetic tree based on 16S rRNA sequences of P. acidilactici Ann Microbiol (2018) 68:103–110 109 similar and within the normal range reported in the literature Conclusion for most of the P. acidilactici strains (Holzapfel et al. 2009; Barreau et al. 2012). The carbohydrate-active enzyme profiles This is the first study reporting the isolation and genomic of these two strains were also similar, though a lower number sequencing of Pediococcus acidilactici NRCC1 from drome- of enzymes in glycoside hydrolases and glycosyltransferases dary camel rumen. The genomic analysis of the organism re- were recorded. This may be perhaps due to the larger amount vealed that it can utilize different carbohydrates, biosynthesize of genomic data available for the reference strain. The antibiotics, and have several unique features suggesting its CAZyme analysis profile further supports the results revealed possible use in probiotics and feed supplements in animals. by the RAST CDS prediction that the organism possesses the Acknowledgements The authors are thankful to Ankit T Hinsu and Ravi potential to digest various plant polysaccharides. Shah, College of Veterinary Science and Animal Husbandry, Anand Different sequences coding for lactose uptake, adherence, Agricultural University (AAU), Anand, Gujarat for extending their tech- resistance to oxidative stress, and folate synthesis were present, nical support in the sequencing and Kashinath, Senior Technical Officer, suggesting the probiotic potential of P. acidilactici NRCC1 ICAR-NRCC, Bikaner for the help in rumen fluid collection. (Prajapati et al. 2013). Resistance to acid and oxidative stress Funding This work is supported by a grant from ICAR-National Centre are important criteria for sustained survival of the organism in for Veterinary Type Culture Collection, National Research Centre on the gastrointestinal tract. Moreover, the presence of antibiotic Equines, Hisar, Haryana, India under Veterinary Type Culture genes as revealed by KEGG analysis as well as the presence of Collection-Rumen Microbe project. the fusaricidin biosynthesis gene cluster also suggests a probiotic property. Fusaricidin is a peptide antibiotic isolated from several Compliance with ethical standards soil bacteria effective against both fungi and Gram-positive bac- Conflict of interest None. teria (Choi et al. 2008;Yuet al. 2012). The fusaricidin-like gene cluster might be responsible for the biosynthesis of pediocins, which are bacteriocins produced by several lactic acid bacteria References (Papagianni 2003). Pediocins from pediococci have been isolat- ed and characterized (Porto et al. 2017). It has been observed that Abd El-Tawab MM, Youssef IM, Bakr HA, Fthenakis GC, Giadinis ND probiotic bacteria can modulate the rumen microbiota and im- (2016) Role of probiotics in nutrition and health of small ruminants. prove health and productivity (Qadis et al. 2014; Uyeno et al. Pol J Vet Sci 19:893–906. https://doi.org/10.1515/pjvs-2016-0114 2015; Abd El-Tawab et al. 2016). Barreau G, Tompkins TA, de Carvalho VG (2012) Draft genome se- Phylogenetically, Pediococcus and Lactobacillus form a quence of probiotic strain Pediococcus acidilactici MA18/5M. J Bacteriol 194:901. https://doi.org/10.1128/JB.06563-11 supercluster that can be divided into two subclusters; all spe- Bhatt VD, Dande SS, Patil NV, Joshi CG (2013) Molecular analysis of the cies of Pediococcus fall within the Lactobacillus casei– bacterial microbiome in the forestomach fluid from the dromedary Pediococcus subcluster. Pediococci are morphologically dis- camel (Camelus dromedarius). Mol Biol Rep 40:3363–3371 tinct from lactobacilli (rods). Pediococci are only acidophilic, Choi SK,ParkSY, KimR,Lee CH,Kim JF,ParkSH(2008) homofermentative, lactic acid bacteria that divide alternatively Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681. Biochem Biophys Res in two perpendicular directions to form tetrads (Simpson and Commun 365:89–95. https://doi.org/10.1016/j.bbrc.2007.10.147 Taguchi 1995). Phylogenetic analysis of P. acidilactici Cintas LM, Rodriguez JM, Fernandez MF, Sletten K, Nes IF, Hernandez NRCC1 based on the full-length 16S rRNA gene sequence PE, Holo H (1995) Isolation and characterization of pediocin L50, a revealed that it is closely related to the P. acidilactici strain new bacteriocin from Pediococcus acidilactici with a broad inhibi- SM1 (KX688797.1) and P. acidilactici strain JFP1 tory spectrum. 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Journal

Annals of MicrobiologySpringer Journals

Published: Dec 28, 2017

References