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.

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.

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.

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.

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.

Identification of a transcription factor, PunR, that regulates the purine and purine nucleoside transporter punC in E. coli.

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii.

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

A Riboflavin Transporter in Bdellovibrio exovorous JSS.

The ImpX transporters of the drug/metabolite transporter superfamily were first proposed to transport riboflavin (RF; vitamin B2) based on findings of a cis-regulatory RNA element responding to flavin mononucleotide (an FMN riboswitch). Bdellovibrio exovorous JSS has a homolog belonging to this superfamily. It has 10 TMSs and shows 30% identity to the previously characterized ImpX transporter from Fusobacterium nucleatum. However, the ImpX homolog is not regulated by an FMN-riboswitch. In order to test the putative function of the ImpX homolog from B. exovorous (BexImpX), we cloned and heterologously expressed its gene. We used functional complementation, growth inhibition experiments, direct uptake experiments and inhibition studies, suggesting a high degree of specificity for RF uptake. The EC50 for growth with RF was estimated to be in the range 0.5-1 µM, estimated from the half-maximal RF concentration supporting the growth of a RF auxotrophic Escherichia coli strain, but the Khalf for RF uptake was 20 µM. Transport experiments suggested that the energy source is the proton motive force but that NaCl stimulates uptake. Thus, members of the ImpX family members are capable of RF uptake, not only in RF prototrophic species such as F.  nucleatum, but also in the B2 auxotrophic species, B. exovorous.

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.

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.

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.

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.

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.

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.

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.

Genomic distribution of B-vitamin auxotrophy and uptake transporters in environmental bacteria from the Chloroflexi phylum.

Bacteria from the Chloroflexi phylum are dominant members of phototrophic microbial mat communities in terrestrial thermal environments. Vitamins of B group are key intermediates (precursors) in the biosynthesis of indispensable enzyme cofactors driving numerous metabolic processes in all forms of life. A genomics-based reconstruction and comparative analysis of respective biosynthetic and salvage pathways and riboswitch regulons in over 20 representative Chloroflexi genomes revealed a widespread auxotrophy for some of the vitamins. The most prominent predicted phenotypic signature, auxotrophy for vitamins B1 and B7 was experimentally confirmed for the best studied model organism Chloroflexus aurantiacus. These observations along with identified candidate genes for the respective uptake transporters pointed to B vitamin cross-feeding as an important aspect of syntrophic metabolism in microbial communities. Inferred specificities of homologous substrate-binding components of ABC transporters for vitamins B1 (ThiY) and B2 (RibY) were verified by thermofluorescent shift approach. A functional activity of the thiamine-specific transporter ThiXYZ from C. aurantiacus was experimentally verified by genetic complementation in E. coli. Expanding the integrative approach, which was applied here for a comprehensive analysis of B-vitamin metabolism in Chloroflexi would allow reconstruction of metabolic interdependencies in microbial communities.

Metabolic and bactericidal effects of targeted suppression of NadD and NadE enzymes in mycobacteria.

UNLABELLED: Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by (13)C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE: To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.