Genome-scale metabolic reconstruction of 7,302 human microorganisms for personalized medicine.

The human microbiome influences the efficacy and safety of a wide variety of commonly prescribed drugs. Designing precision medicine approaches that incorporate microbial metabolism would require strain- and molecule-resolved, scalable computational modeling. Here, we extend our previous resource of genome-scale metabolic reconstructions of human gut microorganisms with a greatly expanded version. AGORA2 (assembly of gut organisms through reconstruction and analysis, version 2) accounts for 7,302 strains, includes strain-resolved drug degradation and biotransformation capabilities for 98 drugs, and was extensively curated based on comparative genomics and literature searches. The microbial reconstructions performed very well against three independently assembled experimental datasets with an accuracy of 0.72 to 0.84, surpassing other reconstruction resources and predicted known microbial drug transformations with an accuracy of 0.81. We demonstrate that AGORA2 enables personalized, strain-resolved modeling by predicting the drug conversion potential of the gut microbiomes from 616 patients with colorectal cancer and controls, which greatly varied between individuals and correlated with age, sex, body mass index and disease stages. AGORA2 serves as a knowledge base for the human microbiome and paves the way to personalized, predictive analysis of host-microbiome metabolic interactions.

Comparative Genomic Analysis Reveals Novel Microcompartment-Associated Metabolic Pathways in the Human Gut Microbiome.

Bacterial microcompartments are self-assembling subcellular structures surrounded by a semipermeable protein shell and found only in bacteria, but not archaea or eukaryotes. The general functions of the bacterial microcompartments are to concentrate enzymes, metabolites, and cofactors for multistep pathways; maintain the cofactor ratio; protect the cell from toxic metabolic intermediates; and protect the encapsulated pathway from unwanted side reactions. The bacterial microcompartments were suggested to play a significant role in organisms of the human gut microbiome, especially for various pathogens. Here, we used a comparative genomics approach to analyze the bacterial microcompartments in 646 individual genomes of organisms commonly found in the human gut microbiome. The bacterial microcompartments were found in 150 (23.2%) analyzed genomes. These microcompartments include previously known ones for the utilization of ethanolamine, 1,2-propanediol, choline, and fucose/rhamnose. Moreover, we reconstructed two novel pathways associated with the bacterial microcompartments. These pathways are catabolic pathways for the utilization of 1-amino-2-propanol/1-amino-2-propanone and xanthine. Remarkably, the xanthine utilization pathway does not demonstrate similarity to previously known microcompartment-associated pathways. Thus, we describe a novel type of bacterial microcompartment.

Systematic assessment of secondary bile acid metabolism in gut microbes reveals distinct metabolic capabilities in inflammatory bowel disease.

BACKGROUND: The human gut microbiome performs important functions in human health and disease. A classic example for host-gut microbial co-metabolism is host biosynthesis of primary bile acids and their subsequent deconjugation and transformation by the gut microbiome. To understand these system-level host-microbe interactions, a mechanistic, multi-scale computational systems biology approach that integrates the different types of omic data is needed. Here, we use a systematic workflow to computationally model bile acid metabolism in gut microbes and microbial communities. RESULTS: Therefore, we first performed a comparative genomic analysis of bile acid deconjugation and biotransformation pathways in 693 human gut microbial genomes and expanded 232 curated genome-scale microbial metabolic reconstructions with the corresponding reactions (available at https://vmh.life ). We then predicted the bile acid biotransformation potential of each microbe and in combination with other microbes. We found that each microbe could produce maximally six of the 13 secondary bile acids in silico, while microbial pairs could produce up to 12 bile acids, suggesting bile acid biotransformation being a microbial community task. To investigate the metabolic potential of a given microbiome, publicly available metagenomics data from healthy Western individuals, as well as inflammatory bowel disease patients and healthy controls, were mapped onto the genomes of the reconstructed strains. We constructed for each individual a large-scale personalized microbial community model that takes into account strain-level abundances. Using flux balance analysis, we found considerable variation in the potential to deconjugate and transform primary bile acids between the gut microbiomes of healthy individuals. Moreover, the microbiomes of pediatric inflammatory bowel disease patients were significantly depleted in their bile acid production potential compared with that of controls. The contributions of each strain to overall bile acid production potential across individuals were found to be distinct between inflammatory bowel disease patients and controls. Finally, bottlenecks limiting secondary bile acid production potential were identified in each microbiome model. CONCLUSIONS: This large-scale modeling approach provides a novel way of analyzing metagenomics data to accelerate our understanding of the metabolic interactions between the host and gut microbiomes in health and diseases states. Our models and tools are freely available to the scientific community.

The Virtual Metabolic Human database: integrating human and gut microbiome metabolism with nutrition and disease.

A multitude of factors contribute to complex diseases and can be measured with 'omics' methods. Databases facilitate data interpretation for underlying mechanisms. Here, we describe the Virtual Metabolic Human (VMH, www.vmh.life) database encapsulating current knowledge of human metabolism within five interlinked resources 'Human metabolism', 'Gut microbiome', 'Disease', 'Nutrition', and 'ReconMaps'. The VMH captures 5180 unique metabolites, 17 730 unique reactions, 3695 human genes, 255 Mendelian diseases, 818 microbes, 632 685 microbial genes and 8790 food items. The VMH's unique features are (i) the hosting of the metabolic reconstructions of human and gut microbes amenable for metabolic modeling; (ii) seven human metabolic maps for data visualization; (iii) a nutrition designer; (iv) a user-friendly webpage and application-programming interface to access its content; (v) user feedback option for community engagement and (vi) the connection of its entities to 57 other web resources. The VMH represents a novel, interdisciplinary database for data interpretation and hypothesis generation to the biomedical community.

Comparative Genomic Analysis of the Human Gut Microbiome Reveals a Broad Distribution of Metabolic Pathways for the Degradation of Host-Synthetized Mucin Glycans and Utilization of Mucin-Derived Monosaccharides.

The colonic mucus layer is a dynamic and complex structure formed by secreted and transmembrane mucins, which are high-molecular-weight and heavily glycosylated proteins. Colonic mucus consists of a loose outer layer and a dense epithelium-attached layer. The outer layer is inhabited by various representatives of the human gut microbiota (HGM). Glycans of the colonic mucus can be used by the HGM as a source of carbon and energy when dietary fibers are not sufficiently available. Both commensals and pathogens can utilize mucin glycans. Commensals are mostly involved in the cleavage of glycans, while pathogens mostly utilize monosaccharides released by commensals. This HGM-derived degradation of the mucus layer increases pathogen susceptibility and causes many other health disorders. Here, we analyzed 397 individual HGM genomes to identify pathways for the cleavage of host-synthetized mucin glycans to monosaccharides as well as for the catabolism of the derived monosaccharides. Our key results are as follows: (i) Genes for the cleavage of mucin glycans were found in 86% of the analyzed genomes, which significantly higher than a previous estimation. (ii) Genes for the catabolism of derived monosaccharides were found in 89% of the analyzed genomes. (iii) Comparative genomic analysis identified four alternative forms of the monosaccharide-catabolizing enzymes and four alternative forms of monosaccharide transporters. (iv) Eighty-five percent of the analyzed genomes may be involved in potential feeding pathways for the monosaccharides derived from cleaved mucin glycans. (v) The analyzed genomes demonstrated different abilities to degrade known mucin glycans. Generally, the ability to degrade at least one type of mucin glycan was predicted for 81% of the analyzed genomes. (vi) Eighty-two percent of the analyzed genomes can form mutualistic pairs that are able to degrade mucin glycans and are not degradable by any of the paired organisms alone. Taken together, these findings provide further insight into the inter-microbial communications of the HGM as well as into host-HGM interactions.

Generation of genome-scale metabolic reconstructions for 773 members of the human gut microbiota.

Genome-scale metabolic models derived from human gut metagenomic data can be used as a framework to elucidate how microbial communities modulate human metabolism and health. We present AGORA (assembly of gut organisms through reconstruction and analysis), a resource of genome-scale metabolic reconstructions semi-automatically generated for 773 human gut bacteria. Using this resource, we identified a defined growth medium for Bacteroides caccae ATCC 34185. We also showed that interactions among modeled species depend on both the metabolic potential of each species and the nutrients available. AGORA reconstructions can integrate either metagenomic or 16S rRNA sequencing data sets to infer the metabolic diversity of microbial communities. AGORA reconstructions could provide a starting point for the generation of high-quality, manually curated metabolic reconstructions. AGORA is fully compatible with Recon 2, a comprehensive metabolic reconstruction of human metabolism, which will facilitate studies of host-microbiome interactions.

Nitrogen Fixation and Molecular Oxygen: Comparative Genomic Reconstruction of Transcription Regulation in Alphaproteobacteria.

Biological nitrogen fixation plays a crucial role in the nitrogen cycle. An ability to fix atmospheric nitrogen, reducing it to ammonium, was described for multiple species of Bacteria and Archaea. The transcriptional regulatory network for nitrogen fixation was extensively studied in several representatives of the class Alphaproteobacteria. This regulatory network includes the activator of nitrogen fixation NifA, working in tandem with the alternative sigma-factor RpoN as well as oxygen-responsive regulatory systems, one-component regulators FnrN/FixK and two-component system FixLJ. Here we used a comparative genomics approach for in silico study of the transcriptional regulatory network in 50 genomes of Alphaproteobacteria. We extended the known regulons and proposed the scenario for the evolution of the nitrogen fixation transcriptional network. The reconstructed network substantially expands the existing knowledge of transcriptional regulation in nitrogen-fixing microorganisms and can be used for genetic experiments, metabolic reconstruction, and evolutionary analysis.

Genomic Analysis of the Human Gut Microbiome Suggests Novel Enzymes Involved in Quinone Biosynthesis.

Ubiquinone and menaquinone are membrane lipid-soluble carriers of electrons that are essential for cellular respiration. Eukaryotic cells can synthesize ubiquinone but not menaquinone, whereas prokaryotes can synthesize both quinones. So far, most of the human gut microbiome (HGM) studies have been based on metagenomic analysis. Here, we applied an analysis of individual HGM genomes to the identification of ubiquinone and menaquinone biosynthetic pathways. In our opinion, the shift from metagenomics to analysis of individual genomes is a pivotal milestone in investigation of bacterial communities, including the HGM. The key results of this study are as follows. (i) The distribution of the canonical pathways in the HGM genomes was consistent with previous reports and with the distribution of the quinone-dependent reductases for electron acceptors. (ii) The comparative genomics analysis identified four alternative forms of the previously known enzymes for quinone biosynthesis. (iii) Genes for the previously unknown part of the futalosine pathway were identified, and the corresponding biochemical reactions were proposed. We discuss the remaining gaps in the menaquinone and ubiquinone pathways in some of the microbes, which indicate the existence of further alternate genes or routes. Together, these findings provide further insight into the biosynthesis of quinones in bacteria and the physiology of the HGM.

Comparative genomics and evolution of transcriptional regulons in Proteobacteria.

Comparative genomics approaches are broadly used for analysis of transcriptional regulation in bacterial genomes. In this work, we identified binding sites and reconstructed regulons for 33 orthologous groups of transcription factors (TFs) in 196 reference genomes from 21 taxonomic groups of Proteobacteria. Overall, we predict over 10 600 TF binding sites and identified more than 15 600 target genes for 1896 TFs constituting the studied orthologous groups of regulators. These include a set of orthologues for 21 metabolism-associated TFs from Escherichia coli and/or Shewanella that are conserved in five or more taxonomic groups and several additional TFs that represent non-orthologous substitutions of the metabolic regulators in some lineages of Proteobacteria. By comparing gene contents of the reconstructed regulons, we identified the core, taxonomy-specific and genome-specific TF regulon members and classified them by their metabolic functions. Detailed analysis of ArgR, TyrR, TrpR, HutC, HypR and other amino-acid-specific regulons demonstrated remarkable differences in regulatory strategies used by various lineages of Proteobacteria. The obtained genomic collection of in silico reconstructed TF regulons contains a large number of new regulatory interactions that await future experimental validation. The collection provides a framework for future evolutionary studies of transcriptional regulatory networks in Bacteria. It can be also used for functional annotation of putative metabolic transporters and enzymes that are abundant in the reconstructed regulons.

Two novel regulators of N-acetyl-galactosamine utilization pathway and distinct roles in bacterial infections.

Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d-galactosamine (GalN)/N-acetyl-d-galactosamine (GalNAc) catabolism pathway in bacterial pathogenesis. Here, we report the genomic reconstruction of GalN/GalNAc utilization pathway in Streptococci and the diversified aga regulons. We delineated two new paralogous AgaR regulators for the GalN/GalNAc catabolism pathway. The electrophoretic mobility shift assays experiment demonstrated that AgaR2 (AgaR1) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and RNA-seq suggested that AgaR2 (not AgaR1) can effectively repress the transcription of the target genes. Removal of agaR2 (not agaR1) from Streptococcus suis 05ZYH33 augments significantly the abilities of both adherence to Hep-2 cells and anti-phagocytosis against RAW264.7 macrophage. As anticipated, the dysfunction in AgaR2-mediated regulation of S. suis impairs its pathogenicity in experimental models of both mice and piglets. Our finding discovered two novel regulators specific for GalN/GalNAc catabolism and assigned them distinct roles into bacterial infections. To the best of our knowledge, it might represent a first paradigm that links the GalN/GalNAc catabolism pathway to bacterial pathogenesis.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins.

Systematic genomic analysis reveals the complementary aerobic and anaerobic respiration capacities of the human gut microbiota.

Because of the specific anatomical and physiological properties of the human intestine, a specific oxygen gradient builds up within this organ that influences the intestinal microbiota. The intestinal microbiome has been intensively studied in recent years, and certain respiratory substrates used by gut inhabiting microbes have been shown to play a crucial role in human health. Unfortunately, a systematic analysis has not been previously performed to determine the respiratory capabilities of human gut microbes (HGM). Here, we analyzed the distribution of aerobic and anaerobic respiratory reductases in 254 HGM genomes. In addition to the annotation of known enzymes, we also predicted a novel microaerobic reductase and novel thiosulfate reductase. Based on this comprehensive assessment of respiratory reductases in the HGM, we proposed a number of exchange pathways among different bacteria involved in the reduction of various nitrogen oxides. The results significantly expanded our knowledge of HGM metabolism and interactions in bacterial communities.

Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in Clostridium acetobutylicum.

Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD(+) ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD(+) concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD(+) ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD(+) homeostasis in clostridia.

Comparative genomics and evolution of regulons of the LacI-family transcription factors.

DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study the function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons, we tentatively assigned functional roles and predicted candidate effectors for 78 and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, as well as the three novel regulators-GluR, GapR, and PckR-that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of the LacI protein family. The obtained genomic collection of in silico reconstructed LacI-TF regulons in bacteria is available in the RegPrecise database (http://regprecise.lbl.gov). It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacterial lineages.

Polysaccharides utilization in human gut bacterium Bacteroides thetaiotaomicron: comparative genomics reconstruction of metabolic and regulatory networks.

BACKGROUND: Bacteroides thetaiotaomicron, a predominant member of the human gut microbiota, is characterized by its ability to utilize a wide variety of polysaccharides using the extensive saccharolytic machinery that is controlled by an expanded repertoire of transcription factors (TFs). The availability of genomic sequences for multiple Bacteroides species opens an opportunity for their comparative analysis to enable characterization of their metabolic and regulatory networks. RESULTS: A comparative genomics approach was applied for the reconstruction and functional annotation of the carbohydrate utilization regulatory networks in 11 Bacteroides genomes. Bioinformatics analysis of promoter regions revealed putative DNA-binding motifs and regulons for 31 orthologous TFs in the Bacteroides. Among the analyzed TFs there are 4 SusR-like regulators, 16 AraC-like hybrid two-component systems (HTCSs), and 11 regulators from other families. Novel DNA motifs of HTCSs and SusR-like regulators in the Bacteroides have the common structure of direct repeats with a long spacer between two conserved sites. CONCLUSIONS: The inferred regulatory network in B. thetaiotaomicron contains 308 genes encoding polysaccharide and sugar catabolic enzymes, carbohydrate-binding and transport systems, and TFs. The analyzed TFs control pathways for utilization of host and dietary glycans to monosaccharides and their further interconversions to intermediates of the central metabolism. The reconstructed regulatory network allowed us to suggest and refine specific functional assignments for sugar catabolic enzymes and transporters, providing a substantial improvement to the existing metabolic models for B. thetaiotaomicron. The obtained collection of reconstructed TF regulons is available in the RegPrecise database (http://regprecise.lbl.gov).

RegPrecise 3.0--a resource for genome-scale exploration of transcriptional regulation in bacteria.

BACKGROUND: Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in prokaryotes is one of the critical tasks of modern genomics. Bacteria from different taxonomic groups, whose lifestyles and natural environments are substantially different, possess highly diverged transcriptional regulatory networks. The comparative genomics approaches are useful for in silico reconstruction of bacterial regulons and networks operated by both transcription factors (TFs) and RNA regulatory elements (riboswitches). DESCRIPTION: RegPrecise (http://regprecise.lbl.gov) is a web resource for collection, visualization and analysis of transcriptional regulons reconstructed by comparative genomics. We significantly expanded a reference collection of manually curated regulons we introduced earlier. RegPrecise 3.0 provides access to inferred regulatory interactions organized by phylogenetic, structural and functional properties. Taxonomy-specific collections include 781 TF regulogs inferred in more than 160 genomes representing 14 taxonomic groups of Bacteria. TF-specific collections include regulogs for a selected subset of 40 TFs reconstructed across more than 30 taxonomic lineages. Novel collections of regulons operated by RNA regulatory elements (riboswitches) include near 400 regulogs inferred in 24 bacterial lineages. RegPrecise 3.0 provides four classifications of the reference regulons implemented as controlled vocabularies: 55 TF protein families; 43 RNA motif families; ~150 biological processes or metabolic pathways; and ~200 effectors or environmental signals. Genome-wide visualization of regulatory networks and metabolic pathways covered by the reference regulons are available for all studied genomes. A separate section of RegPrecise 3.0 contains draft regulatory networks in 640 genomes obtained by an conservative propagation of the reference regulons to closely related genomes. CONCLUSIONS: RegPrecise 3.0 gives access to the transcriptional regulons reconstructed in bacterial genomes. Analytical capabilities include exploration of: regulon content, structure and function; TF binding site motifs; conservation and variations in genome-wide regulatory networks across all taxonomic groups of Bacteria. RegPrecise 3.0 was selected as a core resource on transcriptional regulation of the Department of Energy Systems Biology Knowledgebase, an emerging software and data environment designed to enable researchers to collaboratively generate, test and share new hypotheses about gene and protein functions, perform large-scale analyses, and model interactions in microbes, plants, and their communities.

Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima.

Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

Genomic reconstruction of transcriptional regulatory networks in lactic acid bacteria.

BACKGROUND: Genome scale annotation of regulatory interactions and reconstruction of regulatory networks are the crucial problems in bacterial genomics. The Lactobacillales order of bacteria collates various microorganisms having a large economic impact, including both human and animal pathogens and strains used in the food industry. Nonetheless, no systematic genome-wide analysis of transcriptional regulation has been previously made for this taxonomic group. RESULTS: A comparative genomics approach was used for reconstruction of transcriptional regulatory networks in 30 selected genomes of lactic acid bacteria. The inferred networks comprise regulons for 102 orthologous transcription factors (TFs), including 47 novel regulons for previously uncharacterized TFs. Numerous differences between regulatory networks of the Streptococcaceae and Lactobacillaceae groups were described on several levels. The two groups are characterized by substantially different sets of TFs encoded in their genomes. Content of the inferred regulons and structure of their cognate TF binding motifs differ for many orthologous TFs between the two groups. Multiple cases of non-orthologous displacements of TFs that control specific metabolic pathways were reported. CONCLUSIONS: The reconstructed regulatory networks substantially expand the existing knowledge of transcriptional regulation in lactic acid bacteria. In each of 30 studied genomes the obtained regulatory network contains on average 36 TFs and 250 target genes that are mostly involved in carbohydrate metabolism, stress response, metal homeostasis and amino acids biosynthesis. The inferred networks can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. All reconstructed regulons are captured within the Streptococcaceae and Lactobacillaceae collections in the RegPrecise database (http://regprecise.lbl.gov).

Temporal regulation of gene expression of the Escherichia coli bacteriophage phiEco32.

Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase (RNAP): gp79, a novel protein, and gp36, a distant homolog of σ(70) family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during infection. Host transcription shutoff and three distinct bacteriophage temporal gene classes (early, middle, and late) were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by a host RNAP holoenzyme containing the σ(70) factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by an RNAP holoenzyme containing gp36. These promoters are characterized by a single-consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by σ(70) holoenzyme, activated transcription by the gp36 holoenzyme in vitro.

Transcriptional regulation of central carbon and energy metabolism in bacteria by redox-responsive repressor Rex.

Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD(+) levels in gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD(+) balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.