Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.
The stressosome is a protein complex that senses environmental stresses and mediates the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The stressosome locus is found in 44% of Gram-negative Vibrio vulnificus isolates. However, V. vulnificus does not possess SigB. Nonetheless, in nutrient-limited media, the stressosome modulates gene transcription and bacterial behaviour. In this work, the expression of the stressosome genes was proven during stationary phase in nutrient-rich media and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. The construction of a stressosome mutant lacking the genes encoding the four proteins constituting the stressosome complex (VvRsbR, VvRsbS, VvRsbT, VvRsbX) allowed us to examine the role of this complex in vivo. Extensive phenotypic characterisation of the ΔRSTX mutant in nutrient-rich media showed that the stressosome does not contribute to growth of V. vulnificus. Moreover, the stressosome did not modulate the tolerance or survival response of V. vulnificus to the range of stresses tested, which included ethanol, hyperosmolarity, hypoxic, high temperature, acidity and oxidative stress. Furthermore, the stressosome was dispensable for motility and exoenzyme production of V. vulnificus in nutrient-rich media-. Therefore, in conclusion, although stressosome gene transcription occurs in nutrient-rich media, the stressosome neither has an essential role in stress responses of V. vulnificus, nor does it seem to modulate these activities in these conditions. We hypothesise that the stressosome is expressed in nutrient-rich conditions as a sensor complex, but that activation of the complex does not occur in this environment.
The stressosome is a protein complex that has been demonstrated to sense environmental stresses and mediate the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The in vivo characterisation of this complex has never been performed in Vibrio vulnificus or any other bacteria that do not possess SigB. The elucidation of the role of the stressosome in V. vulnificus would provide elements to elaborate a functional model of the complex in a Gram-negative bacterium and identify the regulatory output in the absence of SigB. The stressosome locus is only found in 44% of Vibrio vulnificus isolates raising the question as to whether the role of stressosome is essential or modulatory in this bacterial species. In this work, the expression of the stressosome genes was proven in nutrient-replete conditions and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. Moreover, the construction of a stressosome mutant lacking the four genes constituting the stressosome complex allowed us to examine the role of this complex in vivo. The initial established mutagenesis strategy relied on rifampicin-resistant V. vulnificus to select recombinant bacteria. Our data clearly showed that the influence of the RifR allele on stress and virulence characteristics overshadowed any effects of the stressosome. Therefore, we established an alternative mutagenesis strategy with a non-modified V. vulnificus parental strain and a DAP auxotrophic E. coli donor strain. Extensive phenotypic characterisation of the successfully-generated mutant in nutrient-replete conditions showed that the stressosome does not significantly contribute to the growth, of V. vulnificus. The stressosome did not modulate the response of V. vulnificus to the range of stresses tested: Ethanol, osmolarity, temperature, and salinity. Furthermore, the stressosome is dispensable for motility and exoenzyme production of V. vulnificus.
The stressosome is a protein complex that has been demonstrated to sense environmental stresses and mediate the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The in vivo characterisation of this complex has never been performed in Vibrio vulnificus or any other bacteria that do not possess SigB. The elucidation of the role of the stressosome in V. vulnificus would provide elements to elaborate a functional model of the complex in a Gram-negative bacterium and identify the regulatory output in the absence of SigB. The stressosome locus is only found in 44% of Vibrio vulnificus isolates raising the question as to whether the role of stressosome is essential or modulatory in this bacterial species. In this work, the expression of the stressosome genes was proven in nutrient-replete conditions and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. Moreover, the construction of a stressosome mutant lacking the four genes constituting the stressosome complex allowed us to examine the role of this complex in vivo. The initial established mutagenesis strategy relied on rifampicin-resistant V. vulnificus to select recombinant bacteria. Our data clearly showed that the influence of the RifR allele on stress and virulence characteristics overshadowed any effects of the stressosome. Therefore, we established an alternative mutagenesis strategy with a non-modified V. vulnificus parental strain and a DAP auxotrophic E. coli donor strain. Extensive phenotypic characterisation of the successfully-generated mutant in nutrient-replete conditions showed that the stressosome does not significantly contribute to the growth, of V. vulnificus. The stressosome did not modulate the response of V. vulnificus to the range of stresses tested: Ethanol, osmolarity, temperature, and salinity. Furthermore, the stressosome is dispensable for motility and exoenzyme production of V. vulnificus.
The stressosome is a protein complex that senses environmental stresses and mediates the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The stressosome locus is found in 44% of Gram-negative Vibrio vulnificus isolates. However, V. vulnificus does not possess SigB. Nonetheless, in nutrient-limited media, the stressosome modulates gene transcription and bacterial behaviour. In this work, the expression of the stressosome genes was proven during stationary phase in nutrient-rich media and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. The construction of a stressosome mutant lacking the genes encoding the four proteins constituting the stressosome complex (VvRsbR, VvRsbS, VvRsbT, VvRsbX) allowed us to examine the role of this complex in vivo. Extensive phenotypic characterisation of the ΔRSTX mutant in nutrient-rich media showed that the stressosome does not contribute to growth of V. vulnificus. Moreover, the stressosome did not modulate the tolerance or survival response of V. vulnificus to the range of stresses tested, which included ethanol, hyperosmolarity, hypoxic, high temperature, acidity and oxidative stress. Furthermore, the stressosome was dispensable for motility and exoenzyme production of V. vulnificus in nutrient-rich media-. Therefore, in conclusion, although stressosome gene transcription occurs in nutrient-rich media, the stressosome neither has an essential role in stress responses of V. vulnificus, nor does it seem to modulate these activities in these conditions. We hypothesise that the stressosome is expressed in nutrient-rich conditions as a sensor complex, but that activation of the complex does not occur in this environment.
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