Comparative genomic reconstruction of transcriptional networks controlling central metabolism in the Shewanella genus

Dmitry A Rodionov 0 Pavel S Novichkov Elena D Stavrovskaya Irina A Rodionova 0 Xiaoqing Li 0 Marat D Kazanov 0 Dmitry A Ravcheev 0 Anna V Gerasimova Alexey E Kazakov Galina Yu Kovaleva Elizabeth A Permina Olga N Laikova Ross Overbeek Margaret F Romine James K Fredrickson Adam P Arkin Inna Dubchak Andrei L Osterman 0 Mikhail S Gelfand 0 Sanford-Burnham Medical Research Institute , La Jolla, California , USA Background: Genome-scale prediction of gene regulation and reconstruction of transcriptional regulatory networks in bacteria is one of the critical tasks of modern genomics. The Shewanella genus is comprised of metabolically versatile gamma-proteobacteria, whose lifestyles and natural environments are substantially different from Escherichia coli and other model bacterial species. The comparative genomics approaches and computational identification of regulatory sites are useful for the in silico reconstruction of transcriptional regulatory networks in bacteria. Results: To explore conservation and variations in the Shewanella transcriptional networks we analyzed the repertoire of transcription factors and performed genomics-based reconstruction and comparative analysis of regulons in 16 Shewanella genomes. The inferred regulatory network includes 82 transcription factors and their DNA binding sites, 8 riboswitches and 6 translational attenuators. Forty five regulons were newly inferred from the genome context analysis, whereas others were propagated from previously characterized regulons in the Enterobacteria and Pseudomonas spp.. Multiple variations in regulatory strategies between the Shewanella spp. and E. coli include regulon contraction and expansion (as in the case of PdhR, HexR, FadR), numerous cases of recruiting non-orthologous regulators to control equivalent pathways (e.g. PsrA for fatty acid degradation) and, conversely, orthologous regulators to control distinct pathways (e.g. TyrR, ArgR, Crp). Conclusions: We tentatively defined the first reference collection of ~100 transcriptional regulons in 16 Shewanella genomes. The resulting regulatory network contains ~600 regulated genes per genome that are mostly involved in metabolism of carbohydrates, amino acids, fatty acids, vitamins, metals, and stress responses. Several reconstructed regulons including NagR for N-acetylglucosamine catabolism were experimentally validated in S. oneidensis MR-1. Analysis of correlations in gene expression patterns helps to interpret the reconstructed regulatory network. The inferred regulatory interactions will provide an additional regulatory constrains for an integrated model of metabolism and regulation in S. oneidensis MR-1. - Background Fine-tuned regulation of gene expression in response to extracellular and intracellular signals is a key mechanism for successful adaptation of microorganisms to changing environmental conditions. Activation and repression of gene expression in bacteria is usually mediated by DNA-binding transcription factors (TFs) that specifically recognize TF-binding sites (TFBSs) in upstream regions of target genes, and also by various regulatory RNA structures including cis-acting metabolite-sensing riboswitches and attenuators encoded in the leader regions of target genes. Genes and operons directly co-regulated by the same TF or by an RNA structure are considered to belong to a regulon. All regulons taken together form the transcriptional regulatory network (TRN) of the cell. TFs form more than 50 different protein families and constitute around 5-10% of all genes in an average bacterial genome, and their respective regulons cover a substantial fraction of bacterial TRNs [1]. Traditional experimental methods for the analysis of transcriptional gene regulation and characterization of TFBSs provided a foundation for the current understanding of regulatory interactions [2]. However, taken alone, they are limited in productivity (the scale) and feasibility (often restricted to a few model organisms). High-throughput transcriptome approaches opens new opportunities for measuring the expression of thousands of genes in a single experiment [3]. The microarray technology has been successfully used to explore transcriptional responses in several bacteria. However, convoluted regulatory cascades, multi-TF regulation of certain genes, and various indirect effects on the transcription and abundance of mRNA make the observed regulatory responses too complex for a direct top-down analysis. The chromatic immunoprecipitation approach is now increasingly used for the investigation of genome-wide DNA-binding of global TFs in bacteria [3]. At the same time, a growing number of complete prokaryotic genomes allows us to extensively use comparative genomics approaches to infer conserved cis-acting regulatory elements (e.g. TFBSs and riboswitches) in regulatory networks of numerous groups of bacteria ([4-15], also reviewed in [1]). These and other previous studies enabled us to define and prototype a general workflow of the knowledge-driven approach for the comparative-genomic reconstruction of regulons. Two major components of this analysis are (i) propagation of previously known regulons from model organisms to others and (ii) ab initio prediction of novel regulons (see Methods for more details). This approach is different, and in many ways complementary to the two most common alternative approaches to the TRN reconstruction: (i) the data-driven approach, top-down regulatory network reconstruction from microarray data [16]; and (ii) the computation-driven approach, ab initio automated identification and clustering of conserved DNA motifs [17] . Shewanella spp. are Gram-negative facultative anaerobic g-proteobacteria characterized by a remarkable versatility in using a variety of terminal electron acceptors for anaerobic respiration (reviewed in [18]). Isolated from various aquatic and sedimentary environments worldwide, the Shewanella demonstrate diverse metabolic capabilities and adaptation for survival in extreme conditions (Fig. 1) [19]. Although the model species Shewanella oneidensis MR-1 is a subject of extensive genetics and physiological studies, as well as genome-scale transcriptomics and proteomics approaches [18,20-22], our experimental knowledge of transcriptional regulation in S. oneidensis is limited to the Fur, ArcA, TorR, Crp, and EtrA (Fnr) TFs controlling iron metabolism and anaerobic respiration [23-29]. In addition, the novel NrtR regulon for NAD cofactor metabolism was inferred by comparative genomics and experimentally validated in S. oneidensis[11]. Availability of multiple closely-related genomes from the Shewanella genus (Fig. 1) provided a basis for the reconstruction of the metabolic and regulatory networks using comparative genomics. Recently, we have applied the comparative genomic approach to predict novel pathways and regulons for the N-acetylglucosamine and lactate utilization [30,31], and to reconstruct two novel regulons f (...truncated)