Reconstruction Of Regulatory And Metabolic Pathways In Metal-Reducing delta-Proteobacteria

Background: Relatively little is known about the genetic basis for the unique physiology of metalreducing 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. Results: In 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. Conclusions: Phylogenetically 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. Background The delta subdivision of proteobacteria is a very diverse group of Gram-negative microorganisms that include aerobic genera Myxococcus with complex developmental lifestyles and Bdellovibrio, which prey on other bacteria [1]. In this study, we focus on anaerobic metal-reducing δ-proteobacteria, seven representatives of which have been sequenced recently, providing an opportunity for comparative genomic analysis. Published: 22 October 2004 Genome Biology 2004, 5:R90 Received: 2 July 2004 Revised: 20 September 2004 Accepted: 30 September 2004 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2004/5/11/R90 Genome Biology 2004, 5:R90 R90.2 Genome Biology 2004, Volume 5, Issue 11, Article R90 Rodionov et al. http://genomebiology.com/2004/5/11/R90 Within this group, sulfate-reducing bacteria, including Desulfovibrio and Desulfotalea species, are metabolically and ecologically versatile prokaryotes often characterized by their ability to reduce sulfate to sulfide [2]. They can be found in aquatic habitats or waterlogged soils containing abundant organic material and sufficient levels of sulfate, and play a key role in the global sulfur and carbon cycles [1]. Industrial interest in sulfate reducers has focused on their role in corrosion of metal equipment and the souring of petroleum reservoirs, while their ability to reduce toxic heavy metals has drawn attention from researchers interested in exploiting this ability for bioremediation. Psychrophilic sulfate-reducing Desulfotalea psychrophila has been isolated from permanently cold arctic marine sediments [3]. In contrast to sulfate-reducing bacteria, the genera Geobacter and Desulfuromonas comprise dissimilative metal-reducing bacteria, which cannot reduce sulfate, but include representatives that require sulfur as a respiratory electron acceptor for oxidation of acetate to carbon dioxide [4]. These bacteria are an important component of the subsurface biota that oxidizes organic compounds, hydrogen or sulfur with the reduction of insoluble Fe(III) oxides [5], and have also been implicated in corrosion and toxic metal reduction. Knowledge of transcriptional regulatory networks is essential for understanding cellular processes in bacteria. However, experimental data about regulation of gene expression in δproteobacteria are very limited. Different approaches could be used for identification of co-regulated genes (regulons). Transcriptional profiling using DNA microarrays allows one to compare the expression levels of thousands of genes in different experimental conditions, and is a valuable tool for dissecting bacterial adaptation to various environments. Computational approaches, on the other hand, provide an opportunity to describe regulons in poorly characterized genomes. Comparison of upstream sequences of genes can, in principle, identify co-regulated genes. From large-scale studies [6-9] and analyses of individual regulatory systems [1014] it is clear that the comparative analysis of binding sites for transcriptional regulators is a powerful approach to the functional annotation of bacterial genomes. Additional techniques used in genome context analysis, such as chromosomal gene clustering, protein fusions and co-occurrence profiles, in combination with metabolic reconstruction, allow the inference of functional coupling between genes and the prediction of gene function [15]. Recent completion of finished and draft quality genome sequences for δ-proteobacteria provides an opportunity for comparative analysis of transcriptional regulation and metabolic pathways in these bacteria. The finished genomes include sulfate-reducing Desulfovibrio vulgaris [16], D. desulfuricans G20, and Desulfotalea psychrophila, as well as the sulfur-reducing G. sulfurreducens [17], while the G. metallireducens genome has been completed to draft quality. A mixture of Desulfuromonas acetoxidans and Desulfuromonas palmitatis has been sequenced, resulting in a large number of small scaffolds, the identity of which (acetoxidans or palmitatis) has not been determined, and we refer to this sequence set simply as Desulfuromonas. Though draft-quality sequence can make it difficult to assert with confidence the absence of any particular gene, we have included these genomes in our study because they do provide insight as to the presence or absence of entire pathways, they can be compared to the related finished genome of G. sulfurreducens, and because complete genome sequence is not necessary for the methodology we use to detect regulatory sequences. In this comprehensive study, we identify a large number of regulatory elements in these δ-proteobacteria. Some of the corresponding regulons are highly conserved among various bacteria (for example, riboswitches, BirA, CIRCE), whereas others are specific only for δ-proteobacteria. We also present the reconstruction of a number of biosynthetic pathways and systems for metal-ion homeostasis and stress response in these bacteria. The most important result of this study is identification of a novel regulon involved in sulfate reduction and energy metabolism in sulfate-reducing bacteria, which is most probably controlled by a regulator from the CRP/FNR family. Results The results are organized under four main headings for convenience. In the first, we analyze a number of specific regulons for biosynthesis of various amino acids and cofactors in δ-proteobacteria. Most of them are controlled by RNA regulatory elements, or riboswitches, that are highly conserved across bacteria [18]. In the next section we describe several regulons for the uptake and homeostasis of transition metal ions that are necessary for growth. These regulons operate by transcription factors that are homologous to factors in Escherichia coli, but are predicted to recognize entirely different DNA signals. We then describe two stress-response regulons: heat-shock regulons (σ32 and HrcA/CIRCE), which operate by regulatory elements conserved in diverse bacteria, and newly identified peroxide stress response regulons that are quite diverse and conserved only in closely related species. Finally, we present a completely new global regulon in metal-reducing δ-proteobacteria, which includes various genes involved in energy metabolism and sulfate reduction. Biosynthesis and transport of vitamins and amino acids Biotin Biotin (vitamin H) is an essential cofactor for numerous biotin-dependent carboxylases in a variety of microorganisms [19]. The strict control of biotin biosynthesis is mediated by the bifunctional BirA protein, which acts both as a biotinprotein ligase and a transcriptional repressor of the biotin operon. The consensus binding signal of BirA is a palindromic sequence TTGTAAACC-[N14/15]-GGTTTACAA [20]. Consistent with the presence of the biotin repressor BirA, all bacteria Genome Biology 2004, 5:R90 http://genomebiology.com/2004/5/11/R90 Genome Biology 2004, Volume 5, Issue 11, Article R90 Rodionov et al. R90.3