Despite a considerable interest in prodigiosin, the mechanism of its antibacterial activity is still poorly understood. In this work, Escherichia coli cells were treated with prodigiosin to determine ...its antimicrobial effect on bacterial physiology. The effect of prodigiosin was concentration dependent. In prodigiosin treated cells above MIC value no significant DNA damage or cytoplasmic membrane disintegration was observed. The outer membrane, however, becomes leaky. Cells had severely decreased respiration activity. In prodigiosin treated cells protein and RNA synthesis were inhibited, cells were elongated but could not divide. Pre-treatment with prodigiosin improved E. coli survival rate in media containing ampicillin, kanamycin and erythromycin but not phleomycin. The results suggest that prodigiosin acts as a bacteriostatic agent in E. coli cells. If prodigiosin was diluted, cells resumed growth. The results indicate that prodigiosin has distinct mode of antibacterial action in different bacteria.
Gram-positive bacteria use peptides as auto-inducing (AI) signals to regulate the production of extracellular enzymes (e.g., proteases). ComX is an AI peptide, mostly known for its role in the ...regulation of bacterial competence and surfactant production in
. These two traits are regulated accordingly to the bacterial population size, thus classifying ComX as a quorum sensing signal. ComX also indirectly regulates exoprotease production through the intermediate transcriptional regulator DegQ. We here use this peptide-based AI system (the ComQXPA system) as a model to address exoprotease regulation by ComX in biofilms. We also investigate the potential of ComX regulated proteases to degrade the ComX AI peptide. Results indicate that ComX indeed induces the expression of
, the gene for the major serine protease subtilisin, and stimulates overall exoprotease production in biofilms of
PS-216 and several other
soil isolates. We also provide evidence that these exoproteases can degrade ComX. The ComX biological activity decay is reduced in the spent media of floating biofilms with low proteolytic activity found in the
and
mutants. ComX biological activity decay can be restored by the addition of subtilisin to such media. In contrast, inhibition of metalloproteases by EDTA reduces ComX biological activity decay. This suggests that both serine and metalloproteases, which are induced by ComX, are ultimately capable of degrading this signaling peptide. This work brings novel information on regulation of exoproteases in
floating biofilms and reveals that these proteolytic enzymes degrade the AI signaling peptide ComX, which is also a major determinant of their expression in biofilms.
Genetic competence for the uptake and integration of extracellular DNA is a key process in horizontal gene transfer (HGT), one of the most powerful forces driving the evolution of bacteria. In ...several species, development of genetic competence is coupled with cell lysis. Using
as a model bacterium, we studied the role of surfactin, a powerful biosurfactant and antimicrobial lipopeptide, in genetic transformation. We showed that surfactin itself promotes cell lysis and DNA release, thereby promoting HGT. These results, therefore, provide evidence for a fundamental mechanism involved in HGT and significantly increase our understanding of the spreading of antibiotic resistance genes and diversification of microbial communities in the environment.
Peatlands play an important role in emissions of the greenhouse gases CO2, CH4 and N2O, which are produced during mineralization of the peat organic matter. To examine the influence of soil type ...(fen, bog soil) and environmental factors (temperature, groundwater level), emission of CO2, CH4 and N2O and soil temperature and groundwater level were measured weekly or biweekly in loco over a one-year period at four sites located in Ljubljana Marsh, Slovenia using the static chamber technique. The study involved two fen and two bog soils differing in organic carbon and nitrogen content, pH, bulk density, water holding capacity and groundwater level. The lowest CO2 fluxes occurred during the winter, fluxes of N2O were highest during summer and early spring (February, March) and fluxes of CH4 were highest during autumn. The temporal variation in CO2 fluxes could be explained by seasonal temperature variations, whereas CH4 and N2O fluxes could be correlated to groundwater level and soil carbon content. The experimental sites were net sources of measured greenhouse gases except for the drained bog site, which was a net sink of CH4. The mean fluxes of CO2 ranged between 139 mg m−2 h−1 in the undrained bog and 206 mg m−2 h−1 in the drained fen; mean fluxes of CH4 were between −0.04 mg m−2 h−1 in the drained bog and 0.05 mg m−2 h−1 in the drained fen; and mean fluxes of N2O were between 0.43 mg m−2 h−1 in the drained fen and 1.03 mg m−2 h−1 in the drained bog. These results indicate that the examined peatlands emit similar amounts of CO2 and CH4 to peatlands in Central and Northern Europe and significantly higher amounts of N2O.
Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of ...prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.
Pigments such as melanin, scytonemin and carotenoids protect microbial cells against the harmful effects of ultraviolet (UV) radiation. The role in UV protection has never been assigned to the ...prodigiosin pigment. In this work, we demonstrate that prodigiosin provides a significant level of protection against UV stress in
Vibrio
sp. DSM 14379. In the absence of pigment production,
Vibrio
sp. was significantly more susceptible to UV stress, and there was no difference in UV survival between the wild-type strain and non-pigmented mutant. The pigment’s protective role was more important at higher doses of UV irradiation and correlated with pigment concentration in the cell. Pigmented cells survived high UV exposure (324 J/m
2
) around 1,000-fold more successfully compared to the non-pigmented mutant cells. Resistance to UV stress was conferred to the non-pigmented mutant by addition of exogenous pigment extract to the growth medium. A level of UV protection equivalent to that exhibited by the wild-type strain was attained by the non-pigmented mutant once the prodigiosin concentration had reached comparable levels to those found in the wild-type strain. In co-culture experiments, prodigiosin acted as a UV screen, protecting both the wild-type and non-pigmented mutants. Our results suggest a new ecophysiological role for prodigiosin.
Bacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their ...expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100-1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide.
ABSTRACT
Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated ...traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study,
Bacillus subtilis
was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (
comQ::kan
) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P
srfAA
hypersensitivity of the
comQ::kan
mutant is linked to a 168 strain-specific mutation in the P
degQ
region. Hence, the markerless Δ
comQ
mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P
srfAA
and not detected in another ComX-regulated promoter, the P
aprE
, which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P
srfAA
and P
aprE
acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production.
IMPORTANCE
DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.
DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.
Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we ...explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain
Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of
Typhimurium through the production of the
cluster-dependent polyketide bacillaene. The presence of
Typhimurium enhanced the activity of the P
promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall P
activity, and P
induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the
operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and
Typhimurium.
Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.
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