Bacterial NadQ (COG4111) is a Nudix-like, ATP-responsive regulator of NAD biosynthesis.

Nicotinamide-adenine dinucleotide (NAD) is centrally important to metabolic reactions that involve redox chemistry. In bacteria, NAD biosynthesis is controlled by different transcription factors, depending on the species. Among the four regulators identified so far, the protein NadQ is reported to act as a repressor of the de novo NAD biosynthetic pathway in proteobacteria. Using comparative genomics, a systematic reconstruction of NadQ regulons in thousands of fully sequenced bacterial genomes has been performed, confirming that NadQ is present in α-proteobacteria and some ß- and γ-proteobacteria, including pathogens like Bordetella pertussis and Neisseria meningitidis, where it likely controls de novo NAD biosynthesis. Through mobility shift assay and mutagenesis, the DNA binding activity of NadQ from Agrobacterium tumefaciens was experimentally validated and determined to be suppressed by ATP. The crystal structures of NadQ in native form and in complex with ATP were determined, indicating that NadQ is a dimer, with each monomer composed of an N-terminal Nudix domain hosting the effector binding site and a C-terminal winged helix-turn-helix domain that binds DNA. Within the dimer, we found one ATP molecule bound, at saturating concentration of the ligand, in keeping with an intrinsic asymmetry of the quaternary structure. Overall, this study provided the basis for depicting a working model of NadQ regulation mechanism.

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.

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.

Ring Separation Highlights the Protein-Folding Mechanism Used by the Phage EL-Encoded Chaperonin.

Chaperonins are ubiquitous, ATP-dependent protein-folding molecular machines that are essential for all forms of life. Bacteriophage φEL encodes its own chaperonin to presumably fold exceedingly large viral proteins via profoundly different nucleotide-binding conformations. Our structural investigations indicate that ATP likely binds to both rings simultaneously and that a misfolded substrate acts as the trigger for ATP hydrolysis. More importantly, the φEL complex dissociates into two single rings resulting from an evolutionarily altered residue in the highly conserved ATP-binding pocket. Conformational changes also more than double the volume of the single-ring internal chamber such that larger viral proteins are accommodated. This is illustrated by the fact that φEL is capable of folding ß-galactosidase, a 116-kDa protein. Collectively, the architecture and protein-folding mechanism of the φEL chaperonin are significantly different from those observed in group I and II chaperonins.

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

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

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.

Genomic reconstruction of transcriptional regulatory networks in lactic acid bacteria.

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

Comparative genomics of CytR, an unusual member of the LacI family of transcription factors.

CytR is a transcription regulator from the LacI family, present in some gamma-proteobacteria including Escherichia coli and known not only for its cellular role, control of transport and utilization of nucleosides, but for a number of unusual structural properties. The present study addressed three related problems: structure of CytR-binding sites and motifs, their evolutionary conservation, and identification of new members of the CytR regulon. While the majority of CytR-binding sites are imperfect inverted repeats situated between binding sites for another transcription factor, CRP, other architectures were observed, in particular, direct repeats. While the similarity between sites for different genes in one genome is rather low, and hence the consensus motif is weak, there is high conservation of orthologous sites in different genomes (mainly in the Enterobacteriales) arguing for the presence of specific CytR-DNA contacts. On larger evolutionary distances candidate CytR sites may migrate but the approximate distance between flanking CRP sites tends to be conserved, which demonstrates that the overall structure of the CRP-CytR-DNA complex is gene-specific. The analysis yielded candidate CytR-binding sites for orthologs of known regulon members in less studied genomes of the Enterobacteriales and Vibrionales and identified a new candidate member of the CytR regulon, encoding a transporter named NupT (YcdZ).

Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile.

The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

Identification of replication origins in prokaryotic genomes.

The availability of hundreds of complete bacterial genomes has created new challenges and simultaneously opportunities for bioinformatics. In the area of statistical analysis of genomic sequences, the studies of nucleotide compositional bias and gene bias between strands and replichores paved way to the development of tools for prediction of bacterial replication origins. Only a few (about 20) origin regions for eubacteria and archaea have been proven experimentally. One reason for that may be that this is now considered as an essentially bioinformatics problem, where predictions are sufficiently reliable not to run labor-intensive experiments, unless specifically needed. Here we describe the main existing approaches to the identification of replication origin (oriC) and termination (terC) loci in prokaryotic chromosomes and characterize a number of computational tools based on various skew types and other types of evidence. We also classify the eubacterial and archaeal chromosomes by predictability of their replication origins using skew plots. Finally, we discuss possible combined approaches to the identification of the oriC sites that may be used to improve the prediction tools, in particular, the analysis of DnaA binding sites using the comparative genomic methods.

Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.