Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

Whole Genome Mining and Characterization of a New Probiotic Strain Levilactobacillus brevis ILSH3 from Handia: An Ethnic Fermented Beverage of Odisha, India.

Isolation and characterization of probiotics from traditional fermented food have contributed many beneficial strains to the field of health and nutritional sciences. Handia, a traditional fermented alcoholic beverage popular in different parts of Odisha, was our source of isolation. This study characterizes one such potential bacteria, Levilactobacillus brevis ILSH3 (H3) isolated from Handia. The investigation for the probiotic attributes as per ICMR-DBT guidelines qualified the checkpoint assays like acid and bile tolerance, bile salt hydrolase activity, antimicrobial properties, and pathogen exclusion ability. The whole genome sequence of H3 (2,460,966 bp in size with GC content of 45.62%) was subjected to comparative genome analysis for its taxonomic identification and validation of probiotic potential. Various genes pertaining to its probiotic potential were identified in the genome and it showed zero matches against any pathogenic families. Metabolite profiling of cell-free supernatant using liquid chromatography-mass spectrometry revealed the presence of essential amino acids, short-chain fatty acids, antimicrobial molecules, immunomodulatory molecules, and flavor/aroma-enhancing compounds. Immunomodulatory property investigation using Bioplex and qRT-PCR showed a reduction in the levels of pro-inflammatory cytokines in L. brevis ILSH3-treated Caco-2 cells. Collectively, the results demonstrate that this Handia-origin bacteria Levilactobacillus brevis ILSH3 possesses desirable attributes of a probiotic, which is now open for nutritional and health biologists to explore. This new probiotic strain may show promising results when utilized in healthcare or functional foods.

Identification of conserved genomic signatures specific to Bifidobacterium species colonising the human gut.

Bifidobacterium species are known for their ability to inhabit various habitats and are often regarded as the first colonisers of the human gut. In the present work, we have used comparative genomics to identify conserved genomic signatures specific to Bifidobacterium species associated with the human gut. Our approach discovered five genomic signatures with varying lengths and confidence. Among the predicted five signatures, a 1790 bp multi-drug resistance (MDR) signature was found to be remarkably specific to only those species that can colonise the human gut. The signature codes for a membrane transport protein belonging to the major facilitator superfamily (MFS) generally involved in MDR. Phylogenetic analyses of the MDR signature suggest a lineage-specific evolution of the MDR signature in bifidobacteria colonising the human gut. Functional annotation led to the discovery of two conserved domains in the protein; a catalytic MFS domain involved in the efflux of drugs and toxins, and a regulatory cystathionine-ß-synthase (CBS) domain that can interact with adenosyl-carriers. Molecular docking simulation performed with the modelled tertiary structure of the MDR signature revealed the putative functional role of the covalently linked domains. The MFS domain displayed a high affinity towards various protein synthesis inhibitor antibiotics and human bile acids, whereas the C-terminally linked CBS domain exhibited favourable binding with molecular structures of ATP and AMP. Therefore, we believe that the predicted signature represents a niche-specific survival trait involved in bile and antibiotic resistance, imparting an adaptive advantage to the Bifidobacterium species colonising the human gut. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03492-4.

Decoding the roles of extremophilic microbes in the anaerobic environments: Past, Present, and Future.

The genome of an organism is directly or indirectly correlated with its environment. Consequently, different microbes have evolved to survive and sustain themselves in a variety of environments, including unusual anaerobic environments. It is believed that their genetic material could have played an important role in the early evolution of their existence in the past. Presently, out of the uncountable number of microbes found in different ecosystems we have been able to discover only one percent of the total communities. A large majority of the microbial populations exists in the most unusual and extreme environments. For instance, many anaerobic bacteria are found in the gastrointestinal tract of humans, soil, and hydrothermal vents. The recent advancements in Metagenomics and Next Generation Sequencing technologies have improved the understanding of their roles in these environments. Presently, anaerobic bacteria are used in various industries associated with biofuels, fermentation, production of enzymes, vaccines, vitamins, and dairy products. This broad applicability brings focus to the significant contribution of their genomes in these functions. Although the anaerobic microbes have become an irreplaceable component of our lives, a major and important section of such anaerobic microbes still remain unexplored. Therefore, it can be said that unlocking the role of the microbial genomes of the anaerobes can be a noteworthy discovery not just for mankind but for the entire biosystem as well.

Gut microbiota alternation under the intestinal epithelium-specific knockout of mouse Piga gene.

Crosstalk between the gut microbiota and intestinal epithelium shapes the gut environment and profoundly influences the intestinal immune homeostasis. Glycosylphosphatidylinositol anchored proteins (GPI - APs) contribute to a variety of gut-associated immune functions, including microbial surveillance and defense, and epithelial cell polarity. Properly polarised epithelial cells are essential for the establishment of the barrier function of gut epithelia. The Piga gene is one among seven genes that encode for an enzyme which is involved in the first step of GPI-anchor biosynthesis. This is the first study reporting a knockout of the intestinal epithelial cell-specific Piga gene (Piga-/-) and its association with the gut microbiota in mice using a whole metagenome shotgun-based sequencing approach. An overall reduced microbiota diversity has been observed in the Piga-/- group as compared to the control group (ANOVA p = 0.34). The taxonomic biomarkers, namely: Gammaproteobacteria (class), Enterobacterales (order), Enterobacteriaceae (family), Escherichia (genus), Proteus (genus) and Escherichia coli (species), increased more in the Piga-/- mice as compared to in the control group. Further, the pathogenic E. coli strains, namely E. coli O157:H7 str. EDL 933 (EHEC), E. coli CFT073 (UPEC) and E. coli 536 (UPEC), were found in the Piga-/- mice which also harbored virulence factor transporters. In addition, the taxa responsible for short chain fatty acid production were decreased in the Piga-/- group. The Piga-/- mice gut harbored an increased number of microbial functions responsible for the survival of pathogens in the inflamed gut environment. Our observations clearly indicate that the Piga-/- mice gut might have an overall enhancement in pathogenic behaviour and reduced capabilities beneficial to health.

Shotgun metagenomic sequencing revealed the prebiotic potential of a grain-based diet in mice.

In the present study, we elucidated the effect of grain-based (GB) diet containing both soluble and insoluble fibers and purified ingredients-based (PIB) diet containing only insoluble fiber, namely cellulose on mice gut microbiome using whole shotgun based metagenomic sequencing. Although the fiber content in both diet types is the same (5%) the presence of soluble fiber only in the GB diet differentiates it from the PIB diet. The taxonomic analysis of sequenced reads reveals a significantly higher enrichment of probiotic Lactobacilli in the GB group as compared to the PIB group. Further, the enhancement of energy expensive cellular processes namely, cell cycle control, cell division, chromosome partitioning, and transcription is observed in the GB group which could be due to the metabolization of the soluble fiber for faster energy production. In contrast, a higher abundance of cellulolytic bacterial community namely, the members of family Lachnospiraceae and Ruminococcaceae and the metabolism functions are found in the PIB group. The PIB group shows a significant increase in host-derived oligosaccharide metabolism functions indicating that they might first target the host-derived oligosaccharides and self-stored glycogen in addition to utilising the available cellulose. In addition to the beneficial microbial community variations, both the groups also exhibited an increased abundance of opportunistic pathobionts which could be due to an overall low amount of fiber in the diet. Furthermore, backtracing analysis identified probiotic members of Lactobacillus, viz., L. crispatus ST1, L. fermentum CECT 5716, L. gasseri ATCC 33323, L. johnsonii NCC 533 and L. reuteri 100-23 in the GB group, while Bilophila wadsworthia 3_1_6, Desulfovibrio piger ATCC 29098, Clostridium symbiosum WAL-14163, and Ruminococcaceae bacterium D16 in the PIB group. These data suggest that Lactobacilli, a probiotic community of microorganisms, are the predominant functional contributors in the gut of GB diet-fed mice, whereas pathobionts too coexisted with commensals in the gut microbiome of the PIB group. Thus at 5% fiber, GB modifies the gut microbial ecology more effectively than PIB and the inclusion of soluble fiber in the GB diet may be one of the primary factors responsible for this impact.

Impact of dietary fructooligosaccharides (FOS) on murine gut microbiota and intestinal IgA secretion.

Fructooligosaccharides (FOS) are considered as prebiotics and are well known for their health-promoting properties, including antitumor, allergy-preventive, and infection-protective effects. They exert these effects by modulating the gut microbial composition and dynamics. In the present study, we performed a comparative whole metagenome shotgun sequencing analysis (WMGS) to elucidate the gut microbiota and secretary Immunoglobulin A (SIgA) dynamics as a result of 5% (w/w) FOS supplementation over a period of 7 days (fecal samples were collected every day). A number of taxa including Bacteroides, Lactobacillus, Roseburia, Clostridia, Faecalibaculum, and Enterorhabdus were found to be modulated with SIgA production in the murine gut. The process of SIgA production from FOS metabolization was found to be carried out via the production of short-chain fatty acids in the gut. Species of Bacteroides and Roseburia; namely, B. caccae, B. finegoldii, B. ovatus, B. thetaiotamicron, and Roseburia intestinalis, respectively, are predominantly responsible for FOS metabolization in the murine gut. The abundances of these bacterial species and their corresponding functions involved in FOS metabolization decreased over time even though these prebiotics were administered continuously for seven days. This suggests that there is a decrease in FOS metabolization over time. In addition, the present analysis suggests that the administration of FOS may help to reduce the pathogenic bacteria from the gut via SIgA production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03116-3.

In silico functional and evolutionary analyses of rubber oxygenases (RoxA and RoxB).

The study presents an in silico identification of poly (cis-1,4-isoprene) cleaving enzymes, viz., RoxA and RoxB in bacteria, followed by their functional and evolutionary exploration using comparative genomics. The orthologs of these proteins were found to be restricted to Gram-negative beta-, gamma-, and delta-proteobacteria. Toward the evolutionary propagation, the RoxA and RoxB genes were predicted to have evolved via a common interclass route of horizontal gene transfer in the phylum Proteobacteria (delta → gamma → beta). Besides, recombination, mutation, and gene conversion were also detected in both the genes leading to their diversification. Further, the differential selective pressure is predicted to be operating on entire RoxA and RoxB genes such that the former is diversifying further, whereas the latter is evolving to reduce its genetic diversity. However, the structurally and functionally important sites/residues of these genes were found to be preventing changes implying their evolutionary conservation. Further, the phylogenetic analysis demonstrated a sharp split between the RoxA and RoxB orthologs and indicated the emergence of their variant as another type of putative rubber oxygenase (RoxC) in the class Gammaproteobacteria. A detailed in silico analysis of the signature motifs and residues of Rox sequences exhibited important differences as well as similarities among the RoxA, RoxB, and putative RoxC sequences. Although RoxC appears to be a hybrid of RoxA and RoxB, the signature motifs and residues of RoxC are more similar to RoxB.

Comparative gut microbiome analysis of the Prakriti and Sasang systems reveals functional level similarities in constitutionally similar classes.

The traditional medicinal systems (TMS) of India (Prakriti) and Korea (Sasang) classify human individuals based on their constitution determined by the physiological and psychological traits of individuals. Similarities in the constitutions are already found between the classes of Prakriti (Vata, Pitta, and Kapha) and Sasang (TE: Taeeumin, SE: Soeumin, and SY: Soyangin) systems. Gut health is an important aspect of this constitution based classification in TMS. To determine the role of gut microbes in such classifications, we have analyzed the gut microbiome (taxa and imputed functions) in the constitutionally similar Prakriti and Sasang classes. An enrichment of Bacteroides and Prevotella enterotypes is observed in the Sasang and Prakriti samples, respectively. The impact of the constitution is found to be more prominent with respect to the taxa and predicted-functions within the Prakriti classes. Gut microbiome functional-level similarities are found to correlate well with the host phenotypes of the constitutionally similar Prakriti and Sasang classes. An enrichment of carbohydrate and amino-acid metabolism is observed in the Vata and SE classes which may be responsible for meeting with their high energy demands and lean phenotype. The Pitta and SY classes exhibit the high capacity to metabolize toxins. An enrichment of functions responsible for predisposition to obesity and high drug metabolism is observed in the Kapha and TE classes. The contribution of gut adaptive functions is found to correlate with the constitution-based classification in both Prakriti and Sasang systems. The TE class harboured the highest number of biofilm-forming and stress-tolerant microbes thus exhibiting the maximum tolerance of environmental stress. Similarities in the gut microbiota and the resulting disease predisposition patterns are found to exist between the constitutionally matching Prakriti and Sasang classes.

Association of colitis with gut-microbiota dysbiosis in clathrin adapter AP-1B knockout mice.

Inflammatory bowel disease results from alterations in the immune system and intestinal microbiota. The role of intestinal epithelial cells (IECs) in maintaining gut homeostasis is well known and its perturbation often causes gastrointestinal disorders including IBD. The epithelial specific adaptor protein (AP)-1B is involved in the establishment of the polarity of IECs. Deficiency of the AP-1B µ subunit (Ap1m2-/-) leads to the development of chronic colitis in mice. However, how this deficiency affects the gut microbes and its potential functions remains elusive. To gain insights into the gut microbiome of Ap1m2-/- mice having the colitis phenotype, we undertook shotgun metagenomic sequencing analysis of knockout mice. We found important links to the microbial features involved in altering various physiological pathways, including carbohydrate metabolism, nutrient transportation, oxidative stress, and bacterial pathogenesis (cell motility). In addition, an increased abundance of sulfur-reducing and lactate-producing bacteria has been observed which may aggravate the colitis condition.

The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum.

In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [13C]glucose (99% [1-13C]glucose or 99% [1,2-13C]glucose or 40% [13C6]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum The 13C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes.IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3, 4). This work provided evidence from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood.

Functional signature analysis of extreme Prakriti endophenotypes in gut microbiome of western Indian rural population.

Ayurveda is practiced in India from ancient times and stratifies the individuals based on their Prakriti constitution. Advancements in modern science have led to the association of Prakriti with molecular, biochemical, genomic and other entities. We have recently explored the gut microbiome composition and microbial signatures in healthy extreme Prakriti endo-phenotypes. However, their functional potentials are still lacking. The present study includes 63 females (29 Vata, 11 Pitta, and 23 Kapha) and 50 males (13 Vata, 18 Pitta, and 19 Kapha) samples. The predictive functional profiling and organism level functional traits of the human gut microbiome have been carried out in Prakriti groups using imputed metagenomic approach. A higher functional level redundancy is found than the taxonomy across the Prakriti groups, however the dominant taxa contributing to the functional profiles are found to be different. A high number of functional signatures specific to the Prakriti groups were identified in female datasets. Some of the functional signatures were found to be gender specific. For example, a higher abundance of microbes contributing potential pathogenic and stress tolerance related functions was found in Kapha in female and Pitta in male. The functional signatures correlated well with phenotypes and disease predisposition of Prakriti groups.

Exploration of Survival Traits, Probiotic Determinants, Host Interactions, and Functional Evolution of Bifidobacterial Genomes Using Comparative Genomics.

Members of the genus Bifidobacterium are found in a wide-range of habitats and are used as important probiotics. Thus, exploration of their functional traits at the genus level is of utmost significance. Besides, this genus has been demonstrated to exhibit an open pan-genome based on the limited number of genomes used in earlier studies. However, the number of genomes is a crucial factor for pan-genome calculations. We have analyzed the pan-genome of a comparatively larger dataset of 215 members of the genus Bifidobacterium belonging to different habitats, which revealed an open nature. The pan-genome for the 56 probiotic and human-gut strains of this genus, was also found to be open. The accessory- and unique-components of this pan-genome were found to be under the operation of Darwinian selection pressure. Further, their genome-size variation was predicted to be attributed to the abundance of certain functions carried by genomic islands, which are facilitated by insertion elements and prophages. In silico functional and host-microbe interaction analyses of their core-genome revealed significant genomic factors for niche-specific adaptations and probiotic traits. The core survival traits include stress tolerance, biofilm formation, nutrient transport, and Sec-secretion system, whereas the core probiotic traits are imparted by the factors involved in carbohydrate- and protein-metabolism and host-immunomodulations.

Correction: Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

[This corrects the article DOI: 10.1371/journal.pone.0197448.].

Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

The pathway of rubber (poly [cis-1,4-isoprene]) catabolism is well documented for Gram-positive rubber degraders but only little information exists for Gram-negative species. The first documented potent rubber degrading Gram-negative strain is Xanthomonas sp. strain 35Y that uses extracellular rubber oxygenases for the initial cleavage of the polyisoprene molecule. However, neither the exact phylogenetic position of Xanthomonas sp. strain 35Y nor the catabolic pathway of the primary polyisoprene cleavage products have been investigated. In this contribution, we started to address both these issues by a comprehensive taxonomic characterization and by the analysis of the draft genome sequence of strain 35Y. Evaluation of the 16S rRNA gene sequence pointed to a borderline taxonomic position of strain 35Y as a novel species of the genus Steroidobacter. Further, substantial differences in the genotypic properties of strain 35Y and the members of the genus Steroidobacter, including average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH), resolved the taxonomic position of strain 35Y and suggested its positioning as a novel species of the genus Steroidobacter. This was further confirmed by comparative analysis of physiological and biochemical features of strain 35Y with other members of the genus Steroidobacter. Thus, we conclude that strain 35Y represents a novel species of the genus Steroidobacter, for which we propose the designation Steroidobacter cummioxidans sp. nov., strain 35YT. A comprehensive analysis of the draft genome of S. cummioxidans strain 35Y revealed similarities but also substantial differences to rubber degrading Gram-positive counterparts. In particular, the putative transporters for the uptake of polyisoprene cleavage products differ from Gram-positive rubber degrading species. The draft genome sequence of S. cummioxidans strain 35Y will be useful for researchers to experimentally verify the predicted similarities and differences in the pathways of polyisoprene catabolism in Gram-positive and Gram-negative rubber degrading species.

Lactobacillus fermentum FTDC 8312 combats hypercholesterolemia via alteration of gut microbiota.

In this study, hypercholesterolemic mice fed with Lactobacillus fermentum FTDC 8312 after a seven-week feeding trial showed a reduction in serum total cholesterol (TC) levels, accompanied by a decrease in serum low-density lipoprotein cholesterol (LDL-C) levels, an increase in serum high-density lipoprotein cholesterol (HDL-C) levels, and a decreased ratio of apoB100:apoA1 when compared to those fed with control or a type strain, L. fermentum JCM 1173. These have contributed to a decrease in atherogenic indices (TC/HDL-C) of mice on the FTDC 8312 diet. Serum triglyceride (TG) levels of mice fed with FTDC 8312 and JCM 1173 were comparable to those of the controls. A decreased ratio of cholesterol and phospholipids (C/P) was also observed for mice fed with FTDC 8312, leading to a decreased number of spur red blood cells (RBC) formation in mice. Additionally, there was an increase in fecal TC, TG, and total bile acid levels in mice on FTDC 8312 diet compared to those with JCM 1173 and controls. The administration of FTDC 8312 also altered the gut microbiota population such as an increase in the members of genera Akkermansia and Oscillospira, affecting lipid metabolism and fecal bile excretion in the mice. Overall, we demonstrated that FTDC 8312 exerted a cholesterol lowering effect that may be attributed to gut microbiota modulation.

Comparative Genomics of Herpesviridae Family to Look for Potential Signatures of Human Infecting Strains.

Herpesviridae family is one of the significant viral families which comprises major pathogens of a wide range of hosts. This family includes at least eight species of viruses which are known to infect humans. This family has evolved 180-220 million years ago and the present study highlights that it is still evolving and more genes can be added to the repertoire of this family. In addition, its core-genome includes important viral proteins including glycoprotein B and helicase. Most of the infections caused by human herpesviruses have no definitive cure; thus, search for new therapeutic strategies is necessary. The present study finds core-genome of human herpesviruses that differs from that of Herpesviridae family and nonhuman herpes strains of this family and might be a putative target for vaccine development. The phylogenetic reconstruction based upon the protein sequences of core gene set of Herpesviridae family reveals the sharp splits of its different subfamilies and supports the hypothesis of coevolution of viruses with their hosts. In addition, data mining for cis-elements in the genomes of human herpesviruses results in the prediction of numerous regulatory elements which can be used for regulating the expression of viral based vectors implicated in gene therapies.

Functional assignment of metagenomic data: challenges and applications.

Metagenomic sequencing provides a unique opportunity to explore earth's limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely 'In a given environment, among the microbes present, what are they doing, and how are they doing it?' Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.

Fast and accurate taxonomic assignments of metagenomic sequences using MetaBin.

Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp). To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp) with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.

Complete genome sequences of rat and mouse segmented filamentous bacteria, a potent inducer of th17 cell differentiation.

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E. coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.