OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR

Morphological analysis of the sheathed flagellum of Brucella melitensis

<p>Abstract</p> <p>Background</p> <p>It was recently shown that <it>B. melitensis </it>is flagellated. However, the flagellar structure remains poorly described.</p> <p>Findings</p> <p>We analyzed the structure of the polar sheathed flagellum of <it>B. melitensis </it>by TEM analysis and demonstrated that the Ryu staining is a good method to quickly visualize the flagellum by optical microscopy. The TEM analysis demonstrated that an extension of the outer membrane surrounds a filament ending by a club-like structure. The Δ<it>ftcR</it>, Δ<it>fliF</it>, Δ<it>flgE </it>and Δ<it>fliC </it>flagellar mutants still produce an empty sheath.</p> <p>Conclusions</p> <p>Our results demonstrate that the flagellum of <it>B. melitensis </it>has the characteristics of the sheathed flagella. Our results also suggest that the flagellar sheath production is not directly linked to the flagellar structure assembly and is not regulated by the FtcR master regulator.</p

A novel role for RecA under non-stress: promotion of swarming motility in Escherichia coli K-12

BACKGROUND: Bacterial motility is a crucial factor in the colonization of natural environments. Escherichia coli has two flagella-driven motility types: swimming and swarming. Swimming motility consists of individual cell movement in liquid medium or soft semisolid agar, whereas swarming is a coordinated cellular behaviour leading to a collective movement on semisolid surfaces. It is known that swimming motility can be influenced by several types of environmental stress. In nature, environmentally induced DNA damage (e.g. UV irradiation) is one of the most common types of stress. One of the key proteins involved in the response to DNA damage is RecA, a multifunctional protein required for maintaining genome integrity and the generation of genetic variation. RESULTS: The ability of E. coli cells to develop swarming migration on semisolid surfaces was suppressed in the absence of RecA. However, swimming motility was not affected. The swarming defect of a ΔrecA strain was fully complemented by a plasmid-borne recA gene. Although the ΔrecA cells grown on semisolidsurfaces exhibited flagellar production, they also presented impaired individual movement as well as a fully inactive collective swarming migration. Both the comparative analysis of gene expression profiles in wild-type and ΔrecA cells grown on a semisolid surface and the motility of lexA1 [Ind-] mutant cells demonstrated that the RecA effect on swarming does not require induction of the SOS response. By using a RecA-GFP fusion protein we were able to segregate the effect of RecA on swarming from its other functions. This protein fusion failed to regulate the induction of the SOS response, the recombinational DNA repair of UV-treated cells and the genetic recombination, however, it was efficient in rescuing the swarming motility defect of the ΔrecA mutant. The RecA-GFP protein retains a residual ssDNA-dependent ATPase activity but does not perform DNA strand exchange. CONCLUSION: The experimental evidence presented in this work supports a novel role for RecA: the promotion of swarming motility. The defective swarming migration of ΔrecA cells does not appear to be associated with defective flagellar production; rather, it seems to be associated with an abnormal flagellar propulsion function. Our results strongly suggest that the RecA effect on swarming motility does not require an extensive canonical RecA nucleofilament formation. RecA is the first reported cellular factor specifically affecting swarming but not swimming motility in E. coli. The integration of two apparently disconnected biologically important processes, such as the maintenance of genome integrity and motility in a unique protein, may have important evolutive consequences

The Flagellum of Pseudomonas aeruginosa Is Required for Resistance to Clearance by Surfactant Protein A

Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A(+/+) mice, but survived in the SP-A(-/-) mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization

Discovery and profiling of small RNAs responsive to stress conditions in the plant pathogen <i>Pectobacterium atrosepticum</i>

BACKGROUND: Small RNAs (sRNAs) have emerged as important regulatory molecules and have been studied in several bacteria. However, to date, there have been no whole-transcriptome studies on sRNAs in any of the Soft Rot Enterobacteriaceae (SRE) group of pathogens. Although the main ecological niches for these pathogens are plants, a significant part of their life cycle is undertaken outside their host within adverse soil environment. However, the mechanisms of SRE adaptation to this harsh nutrient-deficient environment are poorly understood. RESULTS: In the study reported herein, by using strand-specific RNA-seq analysis and in silico sRNA predictions, we describe the sRNA pool of Pectobacterium atrosepticum and reveal numerous sRNA candidates, including those that are induced during starvation-activated stress responses. Consequently, strand-specific RNA-seq enabled detection of 137 sRNAs and sRNA candidates under starvation conditions; 25 of these sRNAs were predicted for this bacterium in silico. Functional annotations were computationally assigned to 68 sRNAs. The expression of sRNAs in P. atrosepticum was compared under growth-promoting and starvation conditions: 68 sRNAs were differentially expressed with 47 sRNAs up-regulated under nutrient-deficient conditions. Conservation analysis using BLAST showed that most of the identified sRNAs are conserved within the SRE. Subsequently, we identified 9 novel sRNAs within the P. atrosepticum genome. CONCLUSIONS: Since many of the identified sRNAs are starvation-induced, the results of our study suggests that sRNAs play key roles in bacterial adaptive response. Finally, this work provides a basis for future experimental characterization and validation of sRNAs in plant pathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2376-0) contains supplementary material, which is available to authorized users

Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

<p>Abstract</p> <p>Background</p> <p>Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. <it>Herminiimonas arsenicoxydans </it>has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism.</p> <p>Results</p> <p>In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of <it>H. arsenicoxydans </it>to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn<it>5 </it>transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted <it>aoxR </it>and <it>aoxS </it>genes, showing that the <it>aox </it>operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in <it>rpoN </it>coding for the alternative N sigma factor (σ⁵⁴) of RNA polymerase and in <it>dnaJ </it>coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the <it>rpoN </it>and <it>dnaJ </it>gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the <it>aoxAB </it>operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ⁵⁴-dependent promoter motif was identified upstream of <it>aoxAB </it>coding sequences.</p> <p>Conclusion</p> <p>These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in <it>H. arsenicoxydans</it>. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.</p

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation.

<div><p><i>Clostridium difficile</i> is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 <i>C. difficile</i> proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.</p></div

A Novel H-NS-like protein from an antarctic psychrophilic bacterium reveals a crucial role for the N-terminal domain in thermal stability

We describe here new members of the H-NS protein family identified in a psychrotrophic Acinetobacter spp. bacterium collected in Siberia and in a psychrophilic Psychrobacter spp. bacterium collected in Antarctica. Both are phylogenetically closely related to the HvrA and SPB Rhodobacter transcriptional regulators. Their amino acid sequence shares 40% identity, and their predicted secondary structure displays a structural and functional organization in two modules similar to that of H-NS in Escherichia coli. Remarkably, the Acinetobacter protein fully restores to the wild-type H-NS-dependent phenotypes, whereas the Psychrobacter protein is no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 degrees C. Moreover, in vitro experiments demonstrate that the ability of the Psychrobacter H-NS protein to bind curved DNA and to form dimers is altered at 37 degrees C. The construction of hybrid proteins containing the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein demonstrates the role of the N-terminal domain in this process. Finally, circular dichroism analysis of purified H-NS proteins suggests that, as compared with the E. coli and Acinetobacter proteins, the alpha-helical domain displays weaker intermolecular interactions in the Psychrobacter protein, which may account for the low thermal stability observed at 37 degrees C