Open Access Research2004RodionovetVolume al. 5, Issue 11, Article R90 Reconstruction of regulatory and metabolic pathways in comment metal-reducing δ-proteobacteria Dmitry A Rodionov*, Inna Dubchak†, Adam Arkin‡§¶, Eric Alm‡ and Mikhail S Gelfand*¥ Addresses: *Institute for Information Transmission Problems, Russian Academy of Sciences, Bolshoi Karetny per. 19, Moscow 127994, Russia. †Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. ‡Physical Biosciences Division, Lawrence Berkeley § ¶ National Laboratory, Berkeley, CA 94720, USA. Howard Hughes Medical Institute, Berkeley, CA 94720, USA. University of California, reviews Berkeley, CA 94720, USA. ¥State Scientific Center GosniiGenetika, 1st Dorozhny pr. 1, Moscow 117545, Russia. Correspondence: Dmitry A Rodionov. E-mail: [email protected] Published: 22 October 2004 Received: 2 July 2004 Revised: 20 September 2004 Genome Biology 2004, 5:R90 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 reports © 2004 Rodionov et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Reconstruction<p>Apathways study suggests of the of regulatory geneticthat phylogenetically and and regulatory metabolic diverse factors pathways delta-proteobacteria in several in metal-reducing biosynthesis, have delta-proteobacteriametal homologous ion homeostasis, regulatory stress components.</p> response, and energy metabolism deposited research Abstract Background: Relatively 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 research refereed 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. interactions 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. information Background Bdellovibrio, which prey on other bacteria [1]. In this study, The delta subdivision of proteobacteria is a very diverse group we focus on anaerobic metal-reducing δ-proteobacteria, of Gram-negative microorganisms that include aerobic gen- seven representatives of which have been sequenced recently, era Myxococcus with complex developmental lifestyles and providing an opportunity for comparative genomic analysis. 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 Desul- onas palmitatis has been sequenced, resulting in a large fovibrio and Desulfotalea species, are metabolically and eco- number of small scaffolds, the identity of which (acetoxidans logically versatile prokaryotes often characterized by their or palmitatis) has not been determined, and we refer to this ability to reduce sulfate to sulfide [2]. They can be found in sequence set simply as Desulfuromonas. Though draft-qual- aquatic habitats or waterlogged soils containing abundant ity sequence can make it difficult to assert with confidence the organic material and sufficient levels of sulfate, and play a key absence of any particular gene, we have included these role in the global sulfur and carbon cycles [1]. Industrial inter- genomes in our study because they do provide insight as to est in sulfate reducers has focused on their role in corrosion the presence or absence of entire pathways, they can be com- of metal equipment and the souring of petroleum reservoirs, pared to the related finished genome of G. sulfurreducens, while their ability to reduce toxic heavy metals has drawn and because complete genome sequence is not necessary for attention from researchers interested in exploiting this ability the methodology we use to detect regulatory sequences. for bioremediation. Psychrophilic sulfate-reducing Desulfo- talea psychrophila has been isolated from permanently cold In this comprehensive study, we identify a large number of arctic marine sediments [3]. In contrast to sulfate-reducing regulatory elements in these δ-proteobacteria. Some of the bacteria, the genera Geobacter and Desulfuromonas com- corresponding regulons are highly conserved among various prise dissimilative metal-reducing bacteria, which cannot bacteria (for example, riboswitches, BirA, CIRCE), whereas reduce sulfate, but include representatives that require sulfur others are specific only for δ-proteobacteria. We also present as a respiratory electron acceptor for oxidation of acetate to the reconstruction of a number of biosynthetic pathways and carbon dioxide [4]. These bacteria are an important compo- systems for metal-ion homeostasis and stress response in nent of the subsurface biota that oxidizes organic compounds, these bacteria. The most important result of this study is hydrogen or sulfur with the reduction of insoluble Fe(III) identification of a novel regulon involved in sulfate reduction oxides [5], and have also been implicated in corrosion and and energy metabolism in sulfate-reducing bacteria, which is toxic metal reduction. most probably controlled by a regulator from the CRP/FNR family. Knowledge of transcriptional regulatory networks is essential for understanding cellular processes in bacteria. However, experimental data about regulation of gene expression in δ- Results proteobacteria are very limited. Different approaches could The results are organized under four main headings for con- be used for identification of co-regulated genes (regulons). venience. In the first, we analyze a number of specific regu- Transcriptional profiling using DNA microarrays allows one lons for biosynthesis of various amino acids and cofactors in to compare the expression levels of thousands of genes in dif- δ-proteobacteria. Most of them are controlled by RNA regula- ferent experimental conditions, and is a valuable tool for dis- tory elements, or riboswitches, that are highly conserved secting bacterial adaptation to various environments. across bacteria [18]. In the next section we describe several Computational approaches, on the other hand, provide an regulons for the uptake and homeostasis of transition metal opportunity to describe regulons in poorly characterized ions that are necessary for growth. These regulons operate by genomes. Comparison of upstream sequences of genes can, in transcription factors that are homologous to factors in principle, identify co-regulated genes. From large-scale stud- Escherichia coli, but are predicted to recognize entirely dif- ies [6-9] and analyses of individual regulatory systems [10- ferent DNA signals. We then describe two stress-response 14] it is clear that the comparative analysis of binding sites for regulons: heat-shock regulons (σ32 and HrcA/CIRCE), which transcriptional regulators is a powerful approach to the func- operate by regulatory elements conserved in diverse bacteria, tional annotation of bacterial genomes. Additional tech- and newly identified peroxide stress response regulons that niques used in genome context analysis, such as are quite diverse and conserved only in closely related spe- chromosomal gene clustering, protein fusions and co-occur- cies. Finally, we present a completely new global regulon in rence profiles, in combination with metabolic reconstruction, metal-reducing δ-proteobacteria, which includes various allow the inference of functional coupling between genes and genes involved in energy metabolism and sulfate reduction. the prediction of gene function [15]. Biosynthesis and transport of vitamins and amino acids Recent completion of finished and draft quality genome Biotin sequences for δ-proteobacteria provides an opportunity for Biotin (vitamin H) is an essential cofactor for numerous comparative analysis of transcriptional regulation and meta- biotin-dependent carboxylases in a variety of microorgan- bolic pathways in these bacteria. The finished genomes isms [19]. The strict control of biotin biosynthesis is mediated include sulfate-reducing