Andrey A. Mironov

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.

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.

RegPredict: an integrated system for regulon inference in prokaryotes by comparative genomics approach

RegPredict web server is designed to provide comparative genomics tools for reconstruction and analysis of microbial regulons using comparative genomics approach. The server allows the user to rapidly generate reference sets of regulons and regulatory motif profiles in a group of prokaryotic genomes. The new concept of a cluster of co-regulated orthologous operons allows the user to distribute the analysis of large regulons and to perform the comparative analysis of multiple clusters independently. Two major workflows currently implemented in RegPredict are: (i) regulon reconstruction for a known regulatory motif and (ii) ab initio inference of a novel regulon using several scenarios for the generation of starting gene sets. RegPredict provides a comprehensive collection of manually curated positional weight matrices of regulatory motifs. It is based on genomic sequences, ortholog and operon predictions from the MicrobesOnline. An interactive web interface of RegPredict integrates and presents diverse genomic and functional information about the candidate regulon members from several web resources. RegPredict is freely accessible at http://regpredict.lbl.gov.

A Gibbs sampler for identification of symmetrically structured, spaced DNA motifs with improved estimation of the signal length

MOTIVATION Transcription regulatory protein factors often bind DNA as homo-dimers or hetero-dimers. Thus they recognize structured DNA motifs that are inverted or direct repeats or spaced motif pairs. However, these motifs are often difficult to identify owing to their high divergence. The motif structure included explicitly into the motif recognition algorithm improves recognition efficiency for highly divergent motifs as well as estimation of motif geometric parameters. RESULT We present a modification of the Gibbs sampling motif extraction algorithm, SeSiMCMC (Sequence Similarities by Markov Chain Monte Carlo), which finds structured motifs of these types, as well as non-structured motifs, in a set of unaligned DNA sequences. It employs improved estimators of motif and spacer lengths. The probability that a sequence does not contain any motif is accounted for in a rigorous Bayesian manner. We have applied the algorithm to a set of upstream regions of genes from two Escherichia coli regulons involved in respiration. We have demonstrated that accounting for a symmetric motif structure allows the algorithm to identify weak motifs more accurately. In the examples studied, ArcA binding sites were demonstrated to have the structure of a direct spaced repeat, whereas NarP binding sites exhibited the palindromic structure. AVAILABILITY The WWW interface of the program, its FreeBSD (4.0) and Windows 32 console executables are available at http://bioinform.genetika.ru/SeSiMCMC

Automated selection of positions determining functional specificity of proteins by comparative analysis of orthologous groups in protein families

The increasing volume of genomic data opens new possibilities for analysis of protein function. We introduce a method for automated selection of residues that determine the functional specificity of proteins with a common general function (the specificity‐determining positions [SDP] prediction method). Such residues are assumed to be conserved within groups of orthologs (that may be assumed to have the same specificity) and to vary between paralogs. Thus, considering a multiple sequence alignment of a protein family divided into orthologous groups, one can select positions where the distribution of amino acids correlates with this division. Unlike previously published techniques, the introduced method directly takes into account nonuniformity of amino acid substitution frequencies. In addition, it does not require setting arbitrary thresholds. Instead, a formal procedure for threshold selection using the Bernoulli estimator is implemented. We tested the SDP prediction method on the LacI family of bacterial transcription factors and a sample of bacterial water and glycerol transporters belonging to the major intrinsic protein (MIP) family. In both cases, the comparison with available experimental and structural data strongly supported our predictions.

RegTransBase—a database of regulatory sequences and interactions in a wide range of prokaryotic genomes

RegTransBase is a manually curated database of regulatory interactions in prokaryotes that captures the knowledge in public scientific literature using a controlled vocabulary. Although several databases describing interactions between regulatory proteins and their binding sites are already being maintained, they either focus mostly on the model organisms Escherichia coli and Bacillus subtilis or are entirely computationally derived. RegTransBase describes a large number of regulatory interactions reported in many organisms and contains the following types of experimental data: the activation or repression of transcription by an identified direct regulator, determining the transcriptional regulatory function of a protein (or RNA) directly binding to DNA (RNA), mapping or prediction of a binding site for a regulatory protein and characterization of regulatory mutations. Currently, RegTransBase content is derived from about 3000 relevant articles describing over 7000 experiments in relation to 128 microbes. It contains data on the regulation of about 7500 genes and evidence for 6500 interactions with 650 regulators. RegTransBase also contains manually created position weight matrices (PWM) that can be used to identify candidate regulatory sites in over 60 species. RegTransBase is available at .

Conservation of the binding site for the arginine repressor in all bacterial lineages

BackgroundThe arginine repressor ArgR/AhrC is a transcription factor universally conserved in bacterial genomes. Its recognition signal (the ARG box), a weak palindrome, is also conserved between genomes, despite a very low degree of similarity between individual sites within a genome. Thus, the arginine repressor is different from two other universal transcription factors - HrcA, whose recognition signal is very strongly conserved both within and between genomes, and LexA/DinR, whose signal is strongly conserved within, but not between, genomes. The arginine regulon is well studied in Escherichia coli and to some extent in Bacillus subtilis and some other genomes. Here, we apply the comparative genomic approach to the prediction of the ArgR-binding sites in all completely sequenced bacterial genomes.ResultsOrthologs of ArgR/AhrC were identified in the complete genomes of E. coli, Haemophilus influenzae, Vibrio choleras, B. subtilis, Mycobacterium tuberculosis, Thermotoga maritima, Chlamydia pneumoniae and Deinococcus radiodurans. Candidate arginine repressor binding sites were identified upstream of arginine transport and metabolism genes.ConclusionsWe found that the ArgR/AhrC recognition signal is conserved in all genomes that contain genes encoding orthologous transcription factors of this family. All genomes studied except M. tuberculosis contain ABC transport cassettes (related to the Art system of E. coli) belonging to the candidate arginine regulons.

Software for analysis of bacterial genomes

GenomeExplorer is a program for comparative analysis of regulation in prokaryotic genomes. The program has options for signal search, comparison of gene samples, search for paralogs and orthologs, iterative construction of signal profiles. The program has a convenient graphic interface, allowing for navigation in the annotation window, in the genome map, and in the table of gene similarities. The use of the system clipboard allows one to export the results of analysis into Word and Excel, and to call external programs via the Internet.