Semen A. Leyn

Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children

Microbiota and infant development Malnutrition in children is a persistent challenge that is not always remedied by improvements in nutrition. This is because a characteristic community of gut microbes seems to mediate some of the pathology. Human gut microbes can be transplanted effectively into germ-free mice to recapitulate their associated phenotypes. Using this model, Blanton et al. found that the microbiota of healthy children relieved the harmful effects on growth caused by the microbiota of malnourished children. In infant mammals, chronic undernutrition results in growth hormone resistance and stunting. In mice, Schwarzer et al. showed that strains of Lactobacillus plantarum in the gut microbiota sustained growth hormone activity via signaling pathways in the liver, thus overcoming growth hormone resistance. Together these studies reveal that specific beneficial microbes could potentially be exploited to resolve undernutrition syndromes. Science, this issue p. 10.1126/science.aad3311, p. 854 Microbes from healthy children protect mice from the detrimental effects of the microbiota of malnourished infants. INTRODUCTION As we come to appreciate how our microbial communities (microbiota) assemble following birth, there is an opportunity to determine how this facet of our developmental biology relates to the healthy or impaired growth of infants and children. Childhood undernutrition is a devastating global health problem whose long-term sequelae, including stunting, neurodevelopmental abnormalities, and immune dysfunction, remain largely refractory to current therapeutic interventions. RATIONALE To test the hypothesis that perturbations in the normal development of the gut microbiota are causally related to undernutrition, we first applied random forests (RF), a machine learning method, to bacterial 16S ribosomal RNA data sets generated from fecal samples that were collected serially from healthy Malawian infants and children during their first 3 postnatal years. Age-discriminatory bacterial taxa were identified with distinctive time-dependent changes in their relative abundances; they were used to construct a sparse RF-derived model describing a program of normal postnatal gut microbiota development that is shared across biologically unrelated individuals. A metric based on this model (microbiota-for-age Z-score) was used to define the state of development (maturation) of fecal microbiota from infants and children with varying degrees of undernutrition. Fecal samples obtained from 6- and 18-month-old children with healthy growth patterns or with varying degrees of undernutrition were transplanted into young germ-free mice that were fed a representative Malawian diet. The recipient animals’ rate of lean body mass gain was characterized by serial quantitative magnetic resonance, their metabolic phenotypes were determined by targeted mass spectrometry, and their femoral bone morphologic features were delineated by microcomputed tomography. RESULTS Undernourished children in the Malawian birth cohort that we studied have immature gut microbiota. Unlike microbiota from healthy children, immature microbiota transmit impaired growth, altered bone morphology, and metabolic abnormalities in the muscle, liver, and brain to recipient gnotobiotic mice. The representation of several age-discriminatory taxa in the transplanted microbiota harbored by recipient animals correlated with their growth rates. Microbiota from 6-month-old infants produced greater effects on growth than did microbiota from 18-month-old children, although in each age bin, the growth effects produced by a healthy donor’s community were greater than those produced by an undernourished donor’s community. Cohousing coprophagic mice shortly after they received microbiota from healthy or severely stunted and underweight 6-month-old infants resulted in the invasion of age- and growth-discriminatory taxa from the former into the latter microbiota in the recipient animals, with associated prevention of growth impairments. Introducing cultured members from this group of invasive species ameliorated growth and metabolic abnormalities in recipients of microbiota from undernourished donors. CONCLUSION These preclinical findings provide evidence that gut microbiota immaturity is causally related to childhood undernutrition. The age- and growth-discriminatory taxa that we identified should help direct studies of the effects of host and environmental factors on gut microbial community development, and they represent therapeutic targets for repairing or preventing gut microbiota immaturity. Preclinical evidence that gut microbiota immaturity is causally related to childhood undernutrition. (A) A model of normal gut microbial community development in Malawian infants and children, based on the relative abundances of 25 bacterial taxa that provide a microbial signature defining the “age,” or state of maturation, of an individual’s (fecal) microbiota. (Hierarchical clusterings of operational taxonomic units are indicated on the left.) (B) Fecal samples from healthy (H) or stunted and underweight (Un) infants and children were transplanted into separate groups of young germ-free mice that were fed a Malawian diet. The immature microbiota of Un donors transmitted impaired growth phenotypes to the mice

RegPrecise 3.0 – A resource for genome-scale exploration of transcriptional regulation in bacteria

BackgroundGenome-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).DescriptionRegPrecise (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.ConclusionsRegPrecise 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 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.

Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

The transcription factor AraR controls utilization of L-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraR-mediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of L-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of L-arabinose.

Control of Proteobacterial Central Carbon Metabolism by the HexR Transcriptional Regulator A CASE STUDY IN SHEWANELLA ONEIDENSIS

Background: Different regulatory strategies are utilized by bacteria to control central carbohydrate metabolism. Results: Transcriptional factor HexR is a global regulator of the central carbohydrate metabolism genes in various groups of proteobacterial genomes. Conclusion: HexR in Shewanella is a repressor/activator of the glycolytic/gluconeogenic genes. Significance: Integration of the comparative genomics and experimental approaches is efficient for reconstruction of transcriptional regulons in bacteria. Bacteria exploit multiple mechanisms for controlling central carbon metabolism (CCM). Thus, a bioinformatic analysis together with some experimental data implicated the HexR transcriptional factor as a global CCM regulator in some lineages of Gammaproteobacteria operating as a functional replacement of the Cra regulator characteristic of Enterobacteriales. In this study, we combined a large scale comparative genomic reconstruction of HexR-controlled regulons in 87 species of Proteobacteria with the detailed experimental analysis of the HexR regulatory network in the Shewanella oneidensis model system. Although nearly all of the HexR-controlled genes are associated with CCM, remarkable variations were revealed in the scale (from 1 to 2 target operons in Enterobacteriales up to 20 operons in Aeromonadales) and gene content of HexR regulons between 11 compared lineages. A predicted 17-bp pseudo-palindrome with a consensus tTGTAATwwwATTACa was confirmed as a HexR-binding motif for 15 target operons (comprising 30 genes) by in vitro binding assays. The negative effect of the key CCM intermediate, 2-keto-3-deoxy-6-phosphogluconate, on the DNA-regulator complex formation was verified. A dual mode of HexR action on various target promoters, repression of genes involved in catabolic pathways and activation of gluconeogenic genes, was for the first time predicted by the bioinformatic analysis and experimentally verified by changed gene expression pattern in S. oneidensis ΔhexR mutant. Phenotypic profiling revealed the inability of this mutant to grow on lactate or pyruvate as a single carbon source. A comparative metabolic flux analysis of wild-type and mutant strains of S. oneidensis using [13C]lactate labeling and GC-MS analysis confirmed the hypothesized HexR role as a master regulator of gluconeogenic flux from pyruvate via the transcriptional activation of phosphoenolpyruvate synthase (PpsA).

Genomic Reconstruction of the Transcriptional Regulatory Network in Bacillus subtilis

The adaptation of microorganisms to their environment is controlled by complex transcriptional regulatory networks (TRNs), which are still only partially understood even for model species. Genome scale annotation of regulatory features of genes and TRN reconstruction are challenging tasks of microbial genomics. We used the knowledge-driven comparative-genomics approach implemented in the RegPredict Web server to infer TRN in the model Gram-positive bacterium Bacillus subtilis and 10 related Bacillales species. For transcription factor (TF) regulons, we combined the available information from the DBTBS database and the literature with bioinformatics tools, allowing inference of TF binding sites (TFBSs), comparative analysis of the genomic context of predicted TFBSs, functional assignment of target genes, and effector prediction. For RNA regulons, we used known RNA regulatory motifs collected in the Rfam database to scan genomes and analyze the genomic context of new RNA sites. The inferred TRN in B. subtilis comprises regulons for 129 TFs and 24 regulatory RNA families. First, we analyzed 66 TF regulons with previously known TFBSs in B. subtilis and projected them to other Bacillales genomes, resulting in refinement of TFBS motifs and identification of novel regulon members. Second, we inferred motifs and described regulons for 28 experimentally studied TFs with previously unknown TFBSs. Third, we discovered novel motifs and reconstructed regulons for 36 previously uncharacterized TFs. The inferred collection of regulons is available in the RegPrecise database (http://regprecise.lbl.gov/) and can be used in genetic experiments, metabolic modeling, and evolutionary analysis.

Comparative genomics of metabolic capacities of regulons controlled by cis-regulatory RNA motifs in bacteria

BackgroundIn silico comparative genomics approaches have been efficiently used for functional prediction and reconstruction of metabolic and regulatory networks. Riboswitches are metabolite-sensing structures often found in bacterial mRNA leaders controlling gene expression on transcriptional or translational levels.An increasing number of riboswitches and other cis-regulatory RNAs have been recently classified into numerous RNA families in the Rfam database. High conservation of these RNA motifs provides a unique advantage for their genomic identification and comparative analysis.ResultsA comparative genomics approach implemented in the RegPredict tool was used for reconstruction and functional annotation of regulons controlled by RNAs from 43 Rfam families in diverse taxonomic groups of Bacteria. The inferred regulons include ~5200 cis-regulatory RNAs and more than 12000 target genes in 255 microbial genomes. All predicted RNA-regulated genes were classified into specific and overall functional categories. Analysis of taxonomic distribution of these categories allowed us to establish major functional preferences for each analyzed cis-regulatory RNA motif family. Overall, most RNA motif regulons showed predictable functional content in accordance with their experimentally established effector ligands. Our results suggest that some RNA motifs (including thiamin pyrophosphate and cobalamin riboswitches that control the cofactor metabolism) are widespread and likely originated from the last common ancestor of all bacteria. However, many more analyzed RNA motifs are restricted to a narrow taxonomic group of bacteria and likely represent more recent evolutionary innovations.ConclusionsThe reconstructed regulatory networks for major known RNA motifs substantially expand the existing knowledge of transcriptional regulation in bacteria. The inferred regulons can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. The obtained genome-wide collection of reference RNA motif regulons is available in the RegPrecise database (http://regprecise.lbl.gov/).

Transcriptional Regulation of Carbohydrate Utilization Pathways in the Bifidobacterium Genus.

Bifidobacteria, which represent common commensals of mammalian gut, are believed to have positive effects on human health. The influence of certain non-digestible carbohydrates (and their use as so-called prebiotics) on growth and metabolic activity of bifidobacteria is of increasing interest; however, mechanisms of transcriptional control of carbohydrate metabolism are poorly understood in these species. We used a comparative genomics approach to reconstruct carbohydrate utilization pathways and transcriptional regulons in 10 Bifidobacterium genomes. Analysis of regulatory gene regions revealed candidate DNA motifs and reconstructed regulons for 268 transcription factors from the LacI, ROK, DeoR, AraC, GntR, and TetR families that form 64 orthologous groups of regulators. Most of the reconstructed regulons are local and control specific catabolic pathways for host- and diet-derived glycans and monosaccharides. Mosaic distributions of many of these local regulators across Bifidobacterium species correlate with distribution of corresponding catabolic pathways. In contrast, the maltose, galactose, sucrose, and fructose regulons, as well as a novel global LacI-family regulator that is predicted to control the central carbohydrate metabolism and arabinose catabolism genes, are universally present in all 10 studied bifidobacteria. A novel group of TetR-family regulators presumably controls the glucoside and galactoside utilization pathways. Paralogs of the ribose repressor RbsR control the pyrimidine nucleoside utilization genes. Multiple paralogs of the maltose regulator MalR co-regulate large sets of genes involved in maltodextrin utilization. The inferred metabolic regulons provide new insights on diverse carbohydrate utilization networks in bifidobacteria that can be employed in metabolic modeling, phenotype prediction and the rational development of novel prebiotics.

Novel inositol catabolic pathway in Thermotoga maritima.

myo-inositol (MI) is a key sugar alcohol component of various metabolites, e.g. phosphatidylinositol-based phospholipids that are abundant in animal and plant cells. The seven-step pathway of MI degradation was previously characterized in various soil bacteria including Bacillus subtilis. Through a combination of bioinformatics and experimental techniques we identified a novel variant of the MI catabolic pathway in the marine hyperthermophilic bacterium Thermotoga maritima. By using in vitro biochemical assays with purified recombinant proteins we characterized four inositol catabolic enzymes encoded in the TM0412-TM0416 chromosomal gene cluster. The novel catabolic pathway in T. maritima starts as the conventional route using the myo-inositol dehydrogenase IolG followed by three novel reactions. The first 2-keto-myo-inositol intermediate is oxidized by another, previously unknown NAD-dependent dehydrogenase TM0412 (named IolM), and a yet unidentified product of this reaction is further hydrolysed by TM0413 (IolN) to form 5-keto-l-gluconate. The fourth step involves epimerization of 5-keto-l-gluconate to d-tagaturonate by TM0416 (IolO). T. maritima is unable to grow on myo-inositol as a single carbon source. The determined in vitro specificity of the InoEFGK (TM0418-TM0421) transporter to myo-inositol-phosphate suggests that the novel pathway in Thermotoga utilizes a phosphorylated derivative of inositol.

N-acetylgalactosamine utilization pathway and regulon in Proteobacteria. Genomic reconstruction and experimental characterization in Shewanella.

Background: Amino sugar utilization pathways are highly variable among different bacteria. Results: The N-acetylgalactosamine utilization pathway and regulon were reconstructed in the genomes of diverse Proteobacteria. Conclusion: In vitro pathway reconstitution confirmed a novel variant of the N-acetylgalactosamine utilization pathway in the Shewanella lineage. Significance: Novel enzymatic activities required for amino sugar utilization were characterized. We used a comparative genomics approach to reconstruct the N-acetyl-d-galactosamine (GalNAc) and galactosamine (GalN) utilization pathways and transcriptional regulons in Proteobacteria. The reconstructed GalNAc/GalN utilization pathways include multiple novel genes with specific functional roles. Most of the pathway variations were attributed to the amino sugar transport, phosphorylation, and deacetylation steps, whereas the downstream catabolic enzymes in the pathway were largely conserved. The predicted GalNAc kinase AgaK, the novel variant of GalNAc-6-phosphate deacetylase AgaAII and the GalN-6-phosphate deaminase AgaS from Shewanella sp. ANA-3 were validated in vitro using individual enzymatic assays and reconstitution of the three-step pathway. By using genetic techniques, we confirmed that AgaS but not AgaI functions as the main GalN-6-P deaminase in the GalNAc/GalN utilization pathway in Escherichia coli. Regulons controlled by AgaR repressors were reconstructed by bioinformatics in most proteobacterial genomes encoding GalNAc pathways. Candidate AgaR-binding motifs share a common sequence with consensus CTTTC that was found in multiple copies and arrangements in regulatory regions of aga genes. This study provides comprehensive insights into the common and distinctive features of the GalNAc/GalN catabolism and its regulation in diverse Proteobacteria.