Identification of the Alternative Sigma Factor SigX Regulon and Its Implications for Pseudomonas aeruginosa Pathogenicity

Abstract

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...

Sucrose favorsPseudomonas aeruginosapellicle production through the extracytoplasmic function sigma factor SigX

“…In these conditions, the pel gene cluster was not identified as a putative SigX target (Gicquel et al ., ). In a study integrating chip‐on‐chip experiments and transcriptomic assays based on P. aeruginosa PA14 and its isogenic sigX mutant, pel was not identified as a direct SigX target (Blanka et al ., ). Furthermore, we were unable to find a putative SigX consensus binding motif (Blanka et al ., ) in the promoter region of pelA (data not shown).…”

“…SigX is an ECF sigma factor that is involved in fatty acid synthesis (Boechat et al ., ; Blanka et al ., ) and in biofilm formation (Gicquel et al ., ; Blanka et al ., ). As we have previously shown that SigX activity was increased in high sucrose‐containing medium (Bouffartigues et al ., ), we suspected SigX to be involved in the molecular mechanism linking sucrose and biofilm formation.…”

Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition

“…6). A study showed that osmotic stress changes the fatty acid profile of P. aeruginosa through an alternative sigma factor (SigX)-dependent influence on accAB transcription (36). However, high salt concentrations might also stimulate the Wsp cascade through an alteration of the mechanical properties of the cell membrane because of the introduction of unsaturated fatty acids (Fig.…”

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition

“…Cell cultures were grown and maintained at 37°C in the presence of 5% CO 2 . Host cell viability was monitored during live bacterial infections and flagellin incubation assays and were performed directly on collected A549-Gluc cell supernatants as previously described (34,35). Percent viability was calculated as the relative light unit (RLU) ratio between infected and uninfected controls.…”