Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

e Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma () factors. The largest group of alternative factors is that of the extracytoplasmic function (ECF) factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can be distinguished by its exceptional high capability to adapt and survive in various and challenging habitats and hosts, including animals, plants, and the human host. The necessary means for bacterial adaptation processes critically rely on sensing and quickly responding to the specific extracellular conditions encountered. One common way to achieve rapid activation of genes in response to fluctuating environmental conditions is the use of extracytoplasmic function (ECF) sigma () factors that are especially abundant in P. aeruginosa (1, 2). ECF factors serve as important regulators, and they are increasingly recognized as factors regulating expression of virulence genes and virulence-associated genes (3-5). The activity of most of the ECF factors are modulated by inner membrane sensor proteins that act as antisigma factors. An off-switch of the anti-sigma factor in response to specific environmental changes thereby presumably leads to the release of the cognate factor and thus allows recruitment of the RNA polymerase to initiate expression of the specific factordependent gene regulon (6). So far, cell envelope stress, iron limitation, and oxidative stress have been demonstrated to play a pivotal role during host infection and were described to activate ECF factors (7,8). In addition to th...