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ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECFσ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism.

Pseudomonas plecoglossicida is an important pathogen for aquaculture and causes high mortality in various marine fishes. Expression of sigX was found significantly up-regulated at 18°C than at 28°C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of sigX mRNA of P. plecoglossicida, whether in in vitro or in the spleen at all sampling time points. Compared with the wild-type strain, the infection of sigX-RNAi strain resulted in the onset time delay, and 20% reduction in mortality of Epinephelus coioides, as well as alleviates in the symptoms of E. coioides spleen. Compared with wild-type strain, the gene silence of sigX in P. plecoglossicida resulted in a significant change in transcriptome of infected E. coioides. The result of gene ontology and KEGG analysis on E. coioides showed that genes of serine-type endopeptidase and chemokine signaling pathway, coagulation and complement system, and intestinal immune network for IgA production pathway were mostly affected by sigX of P. plecoglossicida. Meanwhile, the immune genes were associated with different number of miRNA and lncRNA, and some miRNAs were associated with more than one gene at the same time. The results indicated that sigX was a virulent gene of P. plecoglossicida. The up-regulation of the immune pathways made E. coioides more likely to kill sigX-RNAi strain than the wild-type strain of P. plecoglossicida, while the immune genes were regulated by miRNA and lncRNA by a complex mode.

Pseudomonas fluorescens 2P24 produces 2,4-diacetylphloroglucinol (2,4-DAPG) as the major antibiotic compound that protects plants from soil-borne diseases. Expression of the 2,4-DAPG biosynthesis enzymes, which are encoded by the phlACBD locus, is under the control of a delicate regulatory network. In this study, we identified a novel role for the outer membrane protein gene oprF , in negatively regulating the 2,4-DAPG production by using random mini-Tn5 mutagentsis. A sigma factor gene sigX was located immediately upstream of the oprF gene and shown to be a positive regulator for oprF transcription and 2,4-DAPG production. Genetic analysis of an oprF and sigX double-mutant indicated that the 2,4-DAPG regulation by oprF was dependent on SigX. The sigX gene did not affect PhlA and PhlD expression, but positively regulated the level of malonyl-CoA, the substrate of 2,4-DAPG synthesis, by influencing the expression of acetyl-CoA carboxylases. Further investigations revealed that sigX transcription was induced under conditions of salt starvation or glycine addition. All these findings indicate that SigX is a novel regulator of substrate supplements for 2,4-DAPG production. [ABSTRACT FROM AUTHOR]

OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.

Pseudomonas aeruginosa biofilm formation was increased by addition of sucrose to Luria-Bertani medium, whereas addition of NaCl to a final similar osmolarity and use of maltose instead of sucrose, were ineffective. In a previous study, we showed that the extracytoplasmic sigma factor SigX is activated in the presence of sucrose. The sucrose-mediated pellicle increase was abolished in a sigX mutant strain. Sucrose addition led to an increase in pel expression and cyclic-diguanylate (c-di-GMP) pool level production. Interestingly, these two phenotypes were strongly decreased in a sigX mutant. Since pel is not known as a SigX-target, we suspect SigX to be involved in the c-di-GMP production. We found that expression of the diguanylate cyclase PA4843 gene was increased in the presence of sucrose at least partly through SigX activity. Our study shows that sucrose itself rather than osmolarity favours the biofilm mode of P. aeruginosa through the activation of SigX. [ABSTRACT FROM AUTHOR]

The Bacillus subtilis genome encodes eleven Rap proteins, which are conserved tetratricopeptide-containing regulatory proteins. Of those characterized to date, all except RapI negatively regulate response regulators, including Spo0F, ComA and DegU, via protein–protein interactions. RapD has not yet been fully characterized. It was examined whether RapD inhibits the expression of spoIIE, srfA and aprE, which are Spo0F-, ComA- and DegU-regulated genes, respectively. It was observed that multicopy rapD inhibited srfA expression, which suggests that RapD inhibits ComA. This was reinforced by the fact that multicopy rapD also blocked the expression of rapC and rapF, which belong to the ComA regulon. The expression of rapD was reported to depend on the extracytoplasmic function sigma factor SigX. DNA microarray analysis and gel retardation assays revealed that rapD expression is directly repressed by RghR. Thus, the ComA regulon is regulated by rapD in a SigX- and RghR-dependent manner. [ABSTRACT FROM AUTHOR]

Analysis of the Bacillus subtilis genome sequence revealed two open reading frames, designated sigX and ypuN (now termed rsiX ), that are homologous to fecI and fecR, respectively, of Escherichia coli. fecI encodes a σ70-type factor that is necessary for transcription of the ferric citrate transport genes fecABCDE. fecR encodes a cytoplasmic transmembrane protein that is required for the induction of fec transport gene transcription by ferric citrate binding to the FecA outer membrane receptor protein. Investigation of the SigX and RsiX activities disclosed that they are not involved in ferric citrate utilization – since ferric citrate did not serve as an iron source for B. subtilis SG64 – or in the regulation of any other ferric siderophore transport system tested. Strains deleted for sigX or rsiX displayed no phenotype under aerobic or anoxic conditions. However, cloned sigX complemented an E. coli fecI mutant, and the Fur box upstream of sigX responded to the E. coli iron regulatory protein Fur. The purified SigX protein was required for in vitro transcription of a sigX-containing DNA fragment by the E. coli RNA polymerase core enzyme. Autoregulation of sigX was also found in vivo using a sigX'-lacZ gene fusion. RsiX inhibited SigX activity in vivo and in vitro and stabilized the SigX protein. RsiX was localized in the membrane fraction. When RsiX is present, SigX is found in the membrane fraction; in the absence of RsiX, some SigX is detectable in the cytoplasm. We conclude that SigX is a σ factor that belongs to the ECF (extracytoplasmic function) σ70-factor family. It is not known which promoters are recognized by SigX in B. subtilis. SigX may be involved in the regulation of iron metabolism, as evidenced by its activity in E. coli. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa PAO1 has an integrated Pf4 prophage in its genome, encoding a relatively well-characterized filamentous phage, which contributes to the bacterial biofilm organization and maturation. Pf4 variants are considered as superinfectives when they can re-infect and kill the prophage-carrying host. Herein, the response of P. aeruginosa H103 to Pf4 variant infection was investigated. This phage variant caused partial lysis of the bacterial population and modulated H103 physiology. We show by confocal laser scanning microscopy that a Pf4 variant-infection altered P. aeruginosa H103 biofilm architecture either in static or dynamic conditions. Interestingly, in the latter condition, numerous cells displayed a filamentous morphology, suggesting a link between this phenotype and flow-related forces. In addition, Pf4 variant-infection resulted in cell envelope stress response, mostly mediated by the AlgU and SigX extracytoplasmic function sigma factors (ECFσ). AlgU and SigX involvement may account, at least partly, for the enhanced expression level of genes involved in the biosynthesis pathways of two matrix exopolysaccharides (Pel and alginates) and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) metabolism. [ABSTRACT FROM AUTHOR]

sigX , encoding the extracytoplasmic function sigma factor SigX, was found to be highly expressed in Pseudomonas plecoglossicida at its virulent temperature. Previous RNA-seq analysis also revealed that sigX might be a key virulence gene, which could affect the Epinephelus coioides immune response against P. plecoglossicida. Despite the necessity of sigX for virulence in P. plecoglossicida , how sigX senses and response to temperature changes, and how sigX regulates P. plecoglossicida virulence remain unclear. To illuminate these, we initially analyzed the promoter regions of the sigX and found the presence of a putative CspA box. EMSA and LacZ reporter gene analysis revealed that CspA1 is required for the expression of sigX. Consistent with a role in temperature sensing, CspA1-mediated regulation of sigX is influenced by the culture temperature. Further investigations revealed that sigX is necessary for motility and biofilm formation under virulent temperature, while cspA1 expression has been reported to be induced at virulent temperature and participate in P. plecoglossicida motility and biofilm formation regulation. Meanwhile, the intracellular survival rates and immune escape rates decreased significantly after the knockout of cspA1 and sigX. Moreover, the overlap between SigX and CspA1 regulons is very limited and only one gene is predicted to be regulated by both factors, which is identified as gabD and verified via the EMSA analysis. Together, these data suggest that CspA1 contributes to virulence in a temperature-specific manner via regulating sigX expression. • CspA1 contributes to activation of sigX in P. plecoglossicida. • sigX is important for motility, biofilm production, intracellular survival and immune escape. • CspA1 regulates virulence through activating sigX. [ABSTRACT FROM AUTHOR]