Flexible comparative genomics of prokaryotic transcriptional regulatory networks

Abstract

Background Comparative genomics methods enable the reconstruction of bacterial regulatory networks using available experimental data. In spite of their potential for accelerating research into the composition and evolution of bacterial regulons, few comparative genomics suites have been developed for the automated analysis of these regulatory systems. Available solutions typically rely on precomputed databases for operon and ortholog predictions, limiting the scope of analyses to processed complete genomes, and several key issues such as the transfer of experimental information or the integration of regulatory information in a probabilistic setting remain largely unaddressed. Results Here we introduce CGB, a flexible platform for comparative genomics of prokaryotic regulons. CGB has few external dependencies and enables fully customized analyses of newly available genome data. The platform automates the merging of experimental information and uses a gene-centered, Bayesian framework to generate and integrate easily interpretable results. We demonstrate its flexibility and power by analyzing the evolution of type III secretion system regulation in pathogenic Proteobacteria and by characterizing the SOS regulon of a new bacterial phylum, the Balneolaeota. Conclusions Our results demonstrate the applicability of the CGB pipeline in multiple settings. CGB’s ability to automatically integrate experimental information from multiple sources and use complete and draft genomic data, coupled with its non-reliance on precomputed databases and its easily interpretable display of gene-centered posterior probabilities of regulation provide users with an unprecedented level of flexibility in launching comparative genomics analyses of prokaryotic transcriptional regulatory networks. The analyses of type III secretion and SOS response regulatory networks illustrate instances of convergent and divergent evolution of these regulatory systems, showcasing the power of formal ancestral state reconstruction at inferring the evolutionary history of regulatory networks.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes

“…The conserved elements of this putative network also encompass the SplB radical SAM protein involved in DNA repair (COG1533), which has been shown to be SOS regulated in several species ( 57 , 7 , 10 ), as well as a two-gene operon encoding a putative DNA base excision repair system (COG1573-COG4277) involving a uracil-DNA glycosylase (UDG) ( 7 ). Other putative members of the inferred regulatory network include a umuDC operon (COG0389-COG1974) ( 40 ) and a SOS-response associated peptidase (COG2135) regulated by LexA in the Balneolaeota ( 10 , 58 ). Evidence of regulation for other genes encoding canonical SOS proteins ( 3 ), such as RecA (COG0468) and the mismatch repair enzymes UvrC (excinuclease; COG0322) and UvrD (helicase; COG0210) is also found in several species ( Supplementary Figure S3 ).…”

“…Comparative analyses have established that error-prone polymerases constitute the conserved core SOS response across bacteria, presumably due to the need to mitigate the high mutagenic load of unregulated error-prone polymerases ( 1 , 10 , 15 ). Leveraging this insight, we performed a systematic comparative analysis of error-prone polymerase regulation in the Bacteroidetes.…”

“…Refined motif discovery was performed by activating the -pal option in MEME to restrict the search to palindromic motifs and otherwise identical parameters. Comparative genomics analyses of the regulatory networks defined by the identified motifs were performed using the CGB comparative genomics platform, using COG and PFAM for functional annotation of orthologous groups ( 10 ). CGB configuration files for regulon reconstruction are provided as supplementary material in JSON format ( Supplementary Data S1 , Supplementary Data S2 ).…”

“…For RecA, records for one representative member of each Bacteroidetes family, as well as for all the species used in motif discovery, were selected for phylogenetic inference. In both cases, a protein sequence multiple sequence alignment was generated using T-COFFEE ( 36 ), combining three CLUSTALW amino acid sequence alignments with different ( 5 , 10 , 25 ) gap opening penalties and a single T-COFFEE Lalign method amino acid sequence alignment ( 37 ). For RecA, the resulting amino acid sequence alignment was processed with Gblocks using the half-gap setting ( 38 ).…”

“…The sequencing of the first representative Bacteroidetes complete genomes ( Bacteroides thetaiotaomicron and Bacteroides fragilis ) exposed the lack of LexA homologs in these bacterial species, suggesting that this phylum lacked a conventional SOS response ( 1 ). The recent description of a conventional LexA regulon in the Bacteroidetes sister phylum Balneolaeota prompted us to investigate the SOS response in the Bacteroidetes ( 10 ).…”