Evolution of transcriptional regulation of histidine metabolism in Gram-positive bacteria.

BACKGROUND: The histidine metabolism and transport (his) genes are controlled by a variety of RNA-dependent regulatory systems among diverse taxonomic groups of bacteria including T-box riboswitches in Firmicutes and Actinobacteria and RNA attenuators in Proteobacteria. Using a comparative genomic approach, we previously identified a novel DNA-binding transcription factor (named HisR) that controls the histidine metabolism genes in diverse Gram-positive bacteria from the Firmicutes phylum. RESULTS: Here we report the identification of HisR-binding sites within the regulatory regions of the histidine metabolism and transport genes in 395 genomes representing the Bacilli, Clostridia, Negativicutes, and Tissierellia classes of Firmicutes, as well as in 97 other HisR-encoding genomes from the Actinobacteria, Proteobacteria, and Synergistetes phyla. HisR belongs to the TrpR family of transcription factors, and their predicted DNA binding motifs have a similar 20-bp palindromic structure but distinct lineage-specific consensus sequences. The predicted HisR-binding motif was validated in vitro using DNA binding assays with purified protein from the human gut bacterium Ruminococcus gnavus. To fill a knowledge gap in the regulation of histidine metabolism genes in Firmicutes genomes that lack a hisR repressor gene, we systematically searched their upstream regions for potential RNA regulatory elements. As result, we identified 158 T-box riboswitches preceding the histidine biosynthesis and/or transport genes in 129 Firmicutes genomes. Finally, novel candidate RNA attenuators were identified upstream of the histidine biosynthesis operons in six species from the Bacillus cereus group, as well as in five Eubacteriales and six Erysipelotrichales species. CONCLUSIONS: The obtained distribution of the HisR transcription factor and two RNA-mediated regulatory mechanisms for histidine metabolism genes across over 600 species of Firmicutes is discussed from functional and evolutionary points of view.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.

Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Novel Metabolic Pathways and Regulons for Hexuronate Utilization in Proteobacteria.

We used comparative genomics to reconstruct d-galacturonic and d-glucuronic acid catabolic pathways and associated transcriptional regulons involving the tripartite ATP-independent periplasmic (TRAP) family transporters that bind hexuronates in proteobacteria. The reconstructed catabolic network involves novel transcription factors, catabolic enzymes, and transporters for utilization of both hexuronates and aldarates (d-glucarate and meso-galactarate). The reconstructed regulons for a novel GntR family transcription factor, GguR, include the majority of hexuronate/aldarate utilization genes in 47 species from the Burkholderiaceae, Comamonadaceae, Halomonadaceae, and Pseudomonadaceae families. GudR, GulR, and UdhR are additional local regulators of some hexuronate/aldarate utilization genes in some of the above-mentioned organisms. The predicted DNA binding motifs of GguR and GudR regulators from Ralstonia pickettii and Polaromonas were validated by in vitro binding assays. Genes from the GulR- and GguR-controlled loci were differentially expressed in R. pickettii grown on hexuronates and aldarates. By a combination of bioinformatics and experimental techniques we identified a novel variant of the oxidative pathway for hexuronate utilization, including two previously uncharacterized subfamilies of lactone hydrolases (UxuL and UxuF). The genomic context of respective genes and reconstruction of associated pathways suggest that both enzymes catalyze the conversion of d-galactaro- and d-glucaro-1,5-lactones to the ring-opened aldarates. The activities of the purified recombinant enzymes, UxuL and UxuF, from four proteobacterial species were directly confirmed and kinetically characterized. The inferred novel aldarate-specific transporter from the tripartite tricarboxylate transporter (TTT) family transporter TctC was confirmed to bind d-glucarate in vitro This study expands our knowledge of bacterial carbohydrate catabolic pathways by identifying novel families of catabolic enzymes, transcriptional regulators, and transporters.IMPORTANCE Hexuronate catabolic pathways and their transcriptional networks are highly variable among different bacteria. We identified novel transcriptional regulators that control the hexuronate and aldarate utilization genes in four families of proteobacteria. By regulon reconstruction and genome context analysis we identified several novel components of the common hexuronate/aldarate utilization pathways, including novel uptake transporters and catabolic enzymes. Two novel families of lactonases involved in the oxidative pathway of hexuronate catabolism were characterized. Novel transcriptional regulons were validated via in vitro binding assays and gene expression studies with Polaromonas and Ralstonia species. The reconstructed catabolic pathways are interconnected with each other metabolically and coregulated via the GguR regulons in proteobacteria.

The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC.

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the Khalf of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

A novel bifunctional transcriptional regulator of riboflavin metabolism in Archaea.

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide, which are essential coenzymes in all free-living organisms. Riboflavin biosynthesis in many Bacteria but not in Archaea is controlled by FMN-responsive riboswitches. We identified a novel bifunctional riboflavin kinase/regulator (RbkR), which controls riboflavin biosynthesis and transport genes in major lineages of Crenarchaeota, Euryarchaeota and Thaumarchaeota. RbkR proteins are composed of the riboflavin kinase domain and a DNA-binding winged helix-turn-helix-like domain. Using comparative genomics, we predicted RbkR operator sites and reconstructed RbkR regulons in 94 archaeal genomes. While the identified RbkR operators showed significant variability between archaeal lineages, the conserved core of RbkR regulons includes riboflavin biosynthesis genes, known/predicted vitamin uptake transporters and the rbkR gene. The DNA motifs and CTP-dependent riboflavin kinase activity of two RbkR proteins were experimentally validated in vitro. The DNA binding activity of RbkR was stimulated by CTP and suppressed by FMN, a product of riboflavin kinase. The crystallographic structure of RbkR from Thermoplasma acidophilum was determined in complex with CTP and its DNA operator revealing key residues for operator and ligand recognition. Overall, this study contributes to our understanding of metabolic and regulatory networks for vitamin homeostasis in Archaea.

The Nitrogen Regulatory PII Protein (GlnB) and N-Acetylglucosamine 6-Phosphate Epimerase (NanE) Allosterically Activate Glucosamine 6-Phosphate Deaminase (NagB) in Escherichia coli.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli.

The histidine-phosphorylatable phosphocarrier protein (HPr) is an essential component of the sugar-transporting phosphotransferase system (PTS) in many bacteria. Recent interactome findings suggested that HPr interacts with several carbohydrate-metabolizing enzymes, but whether HPr plays a regulatory role was unclear. Here, we provide evidence that HPr interacts with a large number of proteins in Escherichia coli We demonstrate HPr-dependent allosteric regulation of the activities of pyruvate kinase (PykF, but not PykA), phosphofructokinase (PfkB, but not PfkA), glucosamine-6-phosphate deaminase (NagB), and adenylate kinase (Adk). HPr is either phosphorylated on a histidyl residue (HPr-P) or non-phosphorylated (HPr). PykF is activated only by non-phosphorylated HPr, which decreases the PykF Khalf for phosphoenolpyruvate by 10-fold (from 3.5 to 0.36 mm), thus influencing glycolysis. PfkB activation by HPr, but not by HPr-P, resulted from a decrease in the Khalf for fructose-6-P, which likely influences both gluconeogenesis and glycolysis. Moreover, NagB activation by HPr was important for the utilization of amino sugars, and allosteric inhibition of Adk activity by HPr-P, but not by HPr, allows HPr to regulate the cellular energy charge coordinately with glycolysis. These observations suggest that HPr serves as a directly interacting global regulator of carbon and energy metabolism and probably of other physiological processes in enteric bacteria.

A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea.

Thiamine (vitamin B1) is a precursor of thiamine pyrophosphate (TPP), an essential coenzyme in the central metabolism of all living organisms. Bacterial thiamine biosynthesis and salvage genes are controlled at the RNA level by TPP-responsive riboswitches. In Archaea, TPP riboswitches are restricted to the Thermoplasmatales order. Mechanisms of transcriptional control of thiamine genes in other archaeal lineages remain unknown. Using the comparative genomics approach, we identified a novel family of transcriptional regulators (named ThiR) controlling thiamine biosynthesis and transport genes in diverse lineages in the Crenarchaeota phylum as well as in the Halobacteria and Thermococci classes of the Euryarchaeota ThiR regulators are composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which is similar to the archaeal thiamine phosphate synthase ThiN. By using comparative genomics, we predicted ThiR-binding DNA motifs and reconstructed ThiR regulons in 67 genomes representing all above-mentioned lineages. The predicted ThiR-binding motifs are characterized by palindromic symmetry with several distinct lineage-specific consensus sequences. In addition to thiamine biosynthesis genes, the reconstructed ThiR regulons include various transporters for thiamine and its precursors. Bioinformatics predictions were experimentally validated by in vitro DNA-binding assays with the recombinant ThiR protein from the hyperthermophilic archaeon Metallosphaera yellowstonensis MK1. Thiamine phosphate and, to some extent, TPP and hydroxyethylthiazole phosphate were required for the binding of ThiR to its DNA targets, suggesting that ThiR is derepressed by limitation of thiamine phosphates. The thiamine phosphate-binding residues previously identified in ThiN are highly conserved in ThiR regulators, suggesting a conserved mechanism for effector recognition. IMPORTANCE: Thiamine pyrophosphate is a cofactor for many essential enzymes for glucose and energy metabolism. Thiamine or vitamin B1 biosynthesis and its transcriptional regulation in Archaea are poorly understood. We applied the comparative genomics approach to identify a novel family of regulators for the transcriptional control of thiamine metabolism genes in Archaea and reconstructed the respective regulons. The predicted ThiR regulons in archaeal genomes control the majority of thiamine biosynthesis genes. The reconstructed regulon content suggests that numerous uptake transporters for thiamine and/or its precursors are encoded in archaeal genomes. The ThiR regulon was experimentally validated by DNA-binding assays with Metallosphaera spp. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in diverse lineages of Archaea.

Mycobacterial nicotinate mononucleotide adenylyltransferase: structure, mechanism, and implications for drug discovery.

Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (Ki ∼ 25 µM) and antimycobacterial activity (MIC80) ∼ 40-80 µM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.

Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota.

UNLABELLED: Autotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylum Crenarchaeota. Aerobic members of the order Sulfolobales utilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobic Thermoproteales use the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways in Archaea is limited. We applied a comparative genomics approach to predict novel autotrophic regulons in the Crenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in the Sulfolobales (HHC box) and Thermoproteales (DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in all Sulfolobales genomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed by in vitro binding assays with the recombinant HhcR protein from Metallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the order Thermoproteales. DhcR in Thermoproteus neutrophilus (Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data in Metallosphaera and Thermoproteus spp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in the Crenarchaeota. IMPORTANCE: Little is known about transcriptional regulation of carbon dioxide fixation pathways in Archaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages of Archaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages of Crenarchaeota and to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays in Metallosphaera spp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways in Archaea.

Quinolinate salvage and insights for targeting NAD biosynthesis in group A streptococci.

The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen. The predicted niacin auxotrophy was experimentally verified, as well as the essential role of the nicotinamidase PncA in the utilization of nicotinamide (Nm). PncA is dispensable in the presence of nicotinate (Na), ruling it out as a viable antibacterial target. The function of the "orphan" NadC enzyme, which is uniquely present in all GAS species despite the absence of other genes of NAD de novo synthesis, was elucidated. Indeed, the quinolinate (Qa) phosphoribosyltransferase activity of NadC from S. pyogenes allows the organism to sustain growth when Qa is present as a sole pyridine precursor. Finally, the redundancy of functional upstream salvage pathways in GAS species narrows the choice of potential drug targets to the two indispensable downstream enzymes of NAD synthesis, nicotinate adenylyltransferase (NadD family) and NAD synthetase (NadE family). Biochemical characterization of NadD confirmed its functional role in S. pyogenes, and its potential as an antibacterial target was supported by inhibition studies with previously identified class I inhibitors of the NadD enzyme family. One of these inhibitors efficiently inhibited S. pyogenes NadD (sp.NadD) in vitro (50% inhibitory concentration [IC(50)], 15 µM), exhibiting a noncompetitive mechanism with a K(i) of 8 µM.

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.

E. coli allantoinase is activated by the downstream metabolic enzyme, glycerate kinase, and stabilizes the putative allantoin transporter by direct binding.

Allantoin is a good source of ammonium for many organisms, and in Escherichia coli it is utilized under anaerobic conditions. We provide evidence that allantoinase (AllB) is allosterically activated by direct binding of the allantoin catabolic enzyme, glycerate 2-kinase (GlxK) in the presence of glyoxylate. Glyoxylate is known to be an effector of the AllR repressor which regulates the allantoin utilization operons in E. coli. AllB has low affinity for allantoin, but its activation by GlxK leads to increased affinity for its substrate. We also show that the predicted allantoin transporter YbbW (re-named AllW) has allantoin specificity and the protein-protein interaction with AllB. Our results show that the AllB-dependent allantoin degradative pathway is subject to previously unrecognized regulatory mechanisms involving direct protein-protein interactions.

CyuR is a Dual Regulator for L-Cysteine Dependent Antimicrobial Resistance in Escherichia coli.

Hydrogen sulfide (H 2 S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress. This mitigation of oxidative stress was suggested to be an important survival mechanism to achieve antimicrobial resistance (AMR) in many pathogenic bacteria. CyuR (known as DecR or YbaO) is a recently characterized Cys-dependent transcription regulator, responsible for the activation of the cyuAP operon and generation of hydrogen sulfide from Cys. Despite its potential importance, the regulatory network of CyuR remains poorly understood. In this study, we investigated the roles of the CyuR regulon in a Cys-dependent AMR mechanism in E. coli strains. We found: 1) Cys metabolism has a significant role in AMR and its effect is conserved in many E. coli strains, including clinical isolates; 2) CyuR negatively controls the expression of mdlAB encoding a transporter that exports antibiotics such as cefazolin and vancomycin; 3) CyuR binds to a DNA sequence motif 'GAAwAAATTGTxGxxATTTsyCC' in the absence of Cys, confirmed by an in vitro binding assay; and 4) CyuR may regulate 25 additional genes as suggested by in silico motif scanning and transcriptome sequencing. Collectively, our findings expanded the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys.

Diversity and versatility of the Thermotoga maritima sugar kinome.

Sugar phosphorylation is an indispensable committed step in a large variety of sugar catabolic pathways, which are major suppliers of carbon and energy in heterotrophic species. Specialized sugar kinases that are indispensable for most of these pathways can be utilized as signature enzymes for the reconstruction of carbohydrate utilization machinery from microbial genomic and metagenomic data. Sugar kinases occur in several structurally distinct families with various partially overlapping as well as yet unknown substrate specificities that often cannot be accurately assigned by homology-based techniques. A subsystems-based metabolic reconstruction combined with the analysis of genome context and followed by experimental testing of predicted gene functions is a powerful approach of functional gene annotation. Here we applied this integrated approach for functional mapping of all sugar kinases constituting an extensive and diverse sugar kinome in the thermophilic bacterium Thermotoga maritima. Substrate preferences of 14 kinases mainly from the FGGY and PfkB families were inferred by bioinformatics analysis and biochemically characterized by screening with a panel of 45 different carbohydrates. Most of the analyzed enzymes displayed narrow substrate preferences corresponding to their predicted physiological roles in their respective catabolic pathways. The observed consistency supports the choice of kinases as signature enzymes for genomics-based identification and reconstruction of sugar utilization pathways. Use of the integrated genomic and experimental approach greatly speeds up the identification of the biochemical function of unknown proteins and improves the quality of reconstructed pathways.

Tagaturonate-fructuronate epimerase UxaE, a novel enzyme in the hexuronate catabolic network in Thermotoga maritima.

Thermotoga maritima is a marine hyperthermophilic microorganism that degrades a wide range of simple and complex carbohydrates including pectin and produces fermentative hydrogen at high yield. Galacturonate and glucuronate, two abundant hexuronic acids in pectin and xylan, respectively, are catabolized via committed metabolic pathways to supply carbon and energy for a variety of microorganisms. By a combination of bioinformatics and experimental techniques we identified a novel enzyme family (named UxaE) catalysing a previously unknown reaction in the hexuronic acid catabolic pathway, epimerization of tagaturonate to fructuronate. The enzymatic activity of the purified recombinant tagaturonate epimerase from T. maritima was directly confirmed and kinetically characterized. Its function was also confirmed by genetic complementation of the growth of the Escherichia coli uxaB knockout mutant strain on galacturonate. An inferred novel galacturonate to mannonate catabolic pathway in T. maritima was reconstituted in vitro using a mixture of recombinant purified enzymes UxaE, UxaC and UxuB. Members of the newly identified UxaE family were identified in ~50 phylogenetically diverse heterotrophic bacteria from aquatic and soil environments. The genomic context of respective genes and reconstruction of associated pathways suggest that UxaE enzymatic and biological function remains conserved in all of these species.

A systems approach discovers the role and characteristics of seven LysR type transcription factors in Escherichia coli.

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YcaN, YbhD, YgfI, YiaU, YneJ. We show that: (1) YbdO (tentatively re-named CitR) regulation has an effect on bacterial growth at low pH with citrate supplementation. CitR is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); (2) YgfI (tentatively re-named DhfA) activates the dhaKLM operon that encodes the phosphotransferase system, DhfA is involved in formate, glycerol and dihydroxyacetone utilization; (3) YiaU (tentatively re-named LpsR) regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase and resistance to oxacillin microaerobically; (4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen/energy sources; and (5) YbhD and YcaN regulate adjacent y-genes on the genome. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

A roadmap for the functional annotation of protein families: a community perspective.

Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3-4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward.

Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus.

BACKGROUND: Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. RESULTS: To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR), numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation) and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp). CONCLUSIONS: We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S. oneidensis MR-1. Analysis of correlations in gene expression patterns helps to interpret the reconstructed regulatory network. The inferred regulatory interactions will provide an additional regulatory constrains for an integrated model of metabolism and regulation in S. oneidensis MR-1.