Mikhail S. Gelfand

Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes

Using comparative analysis of genes, operons, and regulatory elements, we describe the cobalamin (vitamin B12) biosynthetic pathway in available prokaryotic genomes. Here we found a highly conserved RNA secondary structure, the regulatory B12 element, which is widely distributed in the upstream regions of cobalamin biosynthetic/transport genes in eubacteria. In addition, the binding signal (CBL-box) for a hypothetical B12 regulator was identified in some archaea. A search for B12 elements and CBL-boxes and positional analysis identified a large number of new candidate B12-regulated genes in various prokaryotes. Among newly assigned functions associated with the cobalamin biosynthesis, there are several new types of cobalt transporters, ChlI and ChlD subunits of the CobN-dependent cobaltochelatase complex, cobalt reductase BluB, adenosyltransferase PduO, several new proteins linked to the lower ligand assembly pathway, l-threonine kinase PduX, and a large number of other hypothetical proteins. Most missing genes detected within the cobalamin biosynthetic pathways of various bacteria were identified as nonorthologous substitutes. The variable parts of the cobalamin metabolism appear to be the cobalt transport and insertion, the CobG/CbiG- and CobF/CbiD-catalyzed reactions, and the lower ligand synthesis pathway. The most interesting result of analysis of B12 elements is that B12-independent isozymes of the methionine synthase and ribonucleotide reductase are regulated by B12 elements in bacteria that have both B12-dependent and B12-independent isozymes. Moreover, B12 regulons of various bacteria are thought to include enzymes from known B12-dependent or alternative pathways.

Comparative Genomics of Thiamin Biosynthesis in Procaryotes NEW GENES AND REGULATORY MECHANISMS

Vitamin B1 in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria. Using comparative analysis of genes, operons, and regulatory elements, we describe the thiamin biosynthetic pathway in available bacterial genomes. The previously detected thiamin-regulatory element,thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9736–9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea. Search for THIelements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters, in various prokaryotic organisms. In particular, we assign the thiamin transporter function to yuaJ in theBacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes. By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated regulation based on formation of alternative RNA structures involving theTHI element. Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites. Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea, and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis.

Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins.

Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group. Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for γ-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor. In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis). Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci. Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process. Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins. The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs. We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins. Thus we predict a mechanism for maintaining zinc availability for essential enzymes.

Increase of functional diversity by alternative splicing

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.

Comparative and Functional Genomic Analysis of Prokaryotic Nickel and Cobalt Uptake Transporters: Evidence for a Novel Group of ATP-Binding Cassette Transporters

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B(12) riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B(12) biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.

A Novel Class of Modular Transporters for Vitamins in Prokaryotes

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.

Reconstruction of regulatory and metabolic pathways in metal-reducing δ-proteobacteria

BackgroundRelatively little is known about the genetic basis for the unique physiology of metal-reducing genera in the delta subgroup of the proteobacteria. The recent availability of complete finished or draft-quality genome sequences for seven representatives allowed us to investigate the genetic and regulatory factors in a number of key pathways involved in the biosynthesis of building blocks and cofactors, metal-ion homeostasis, stress response, and energy metabolism using a combination of regulatory sequence detection and analysis of genomic context.ResultsIn the genomes of δ-proteobacteria, we identified candidate binding sites for four regulators of known specificity (BirA, CooA, HrcA, sigma-32), four types of metabolite-binding riboswitches (RFN-, THI-, B12-elements and S-box), and new binding sites for the FUR, ModE, NikR, PerR, and ZUR transcription factors, as well as for the previously uncharacterized factors HcpR and LysX. After reconstruction of the corresponding metabolic pathways and regulatory interactions, we identified possible functions for a large number of previously uncharacterized genes covering a wide range of cellular functions.ConclusionsPhylogenetically diverse δ-proteobacteria appear to have homologous regulatory components. This study for the first time demonstrates the adaptability of the comparative genomic approach to de novo reconstruction of a regulatory network in a poorly studied taxonomic group of bacteria. Recent efforts in large-scale functional genomic characterization of Desulfovibrio species will provide a unique opportunity to test and expand our predictions.

A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes

This work was partially supported by grants from the Russian State ‘Human Genome’ program and the Russian Fund of Basic Research under grant 99-04-48347. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org.

RegPrecise: a database of curated genomic inferences of transcriptional regulatory interactions in prokaryotes.

The RegPrecise database (http://regprecise.lbl.gov) was developed for capturing, visualization and analysis of predicted transcription factor regulons in prokaryotes that were reconstructed and manually curated by utilizing the comparative genomic approach. A significant number of high-quality inferences of transcriptional regulatory interactions have been already accumulated for diverse taxonomic groups of bacteria. The reconstructed regulons include transcription factors, their cognate DNA motifs and regulated genes/operons linked to the candidate transcription factor binding sites. The RegPrecise allows for browsing the regulon collections for: (i) conservation of DNA binding sites and regulated genes for a particular regulon across diverse taxonomic lineages; (ii) sets of regulons for a family of transcription factors; (iii) repertoire of regulons in a particular taxonomic group of species; (iv) regulons associated with a metabolic pathway or a biological process in various genomes. The initial release of the database includes ∼11 500 candidate binding sites for ∼400 orthologous groups of transcription factors from over 350 prokaryotic genomes. Majority of these data are represented by genome-wide regulon reconstructions in Shewanella and Streptococcus genera and a large-scale prediction of regulons for the LacI family of transcription factors. Another section in the database represents the results of accurate regulon propagation to the closely related genomes.

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.