Brändle, M.

Obst, U.

Schwartz, T.

English

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Miriam Brändle, Thomas Schwartz, Ursula Obst Forschungszentrum Karlsruhe, Institute of Functional Interfaces, Department of Microbiology of Natural and Technical SurfacesSPECIFIC AND GLOBAL STRESS RESPONSES IN BACTERAL POPULATIONSBacteria are exposed to various stresses in the natural environment. The aim of our work was to study specific and global stress responses to starvation, osmotic changes and increasing densitiesof hygienic relevant bacteria.Generally, we investigated global parameters like total RNA amount, viability via counts and DNA contents from living bacteria.More specifically gene expression was quantificated during stress by Real-Time-PCR and Nothern Blot analysis in reference bacteria. The sigma factor rpoS of P. aeruginosa, rcsA, a member of the Rcs phosphorelay of E. coli and the penicillin binding protein (pbp5) of E. faecium and E. faecalisparticipating in peptidoglycan synthesis are the selected gene targets. The expression pattern of these genes was compared with the expression of usp (universal stress protein) as a common putative marker of stress.As fingerprint technique for detection of non gene directed stress patterns we used RAPD-PCR (randomly amplified polymorphic DNA-PCR). BACKGROUND AND METHODIC APPROACHImpacts on defined bacterial populations and their responses to starvation and osmotic shock were detected with the demonstrated methods. RAPD-PCR seems to be a promising technique for detection of stress patterns without investigation of specific genes. Further investigations of natural bacterial populations will lead to assessment concept of physiological states and to potential measures for manipulating biofilms in technical processes.CONCLUSION AND OUTLOOKB) Species specific and universal stress genesA) Species specifc genesInduction of the master regulator of stress, rpoS, and rcsA which is involved in biofilm formation during all stress exposures.Repression of pbp5, which might be a marker for VBNC-state during starvation and osmotic stress, but an induction during stationary growth phase. B) Universal stress protein (usp)usp seems to be a suitable stress marker with one exception (see rcsA and starvation).During biofilm formation there was an induction of rpoS and rcsA participating in EPS synthesis (extracellular polymeric substances) in biofilms.D) Global stress response (RAPD-PCR)For differentiation of physiological states we used cDNA and DNA as template. Transcriptom analysisGram- negative bacteria showed distinctions after treatment with sodium chloride (data not shown).E. faecalis had a stress response on osmotic shock and starvation while E. faecium showed different band patterns only at starvation, indicating a stronger stress adaption of E. faecium (Fig. right). Genome analysisThese findings were corroborated by using DNA as a template indicating that E. faecalis is more sensible to osmotic stress than E. faecium (Fig. left).RESULTSA) PhysiologyEnterococci showed an increase in total RNA (2-4 fold) but a decrease in colony counts (cfu) (1-3 fold) during starvation and osmotic shock. Decreasing numbers of cultivable E. faecalis and nearlyconstant DNA contents from living bacteria of the culture indicate a VBNC-state (viable but nonculturable). single species, biofilmstress0,5 M sodiumchloridedrinking waterexponential vs. stationary phasedifferent incubation timesreverse transcription and quantitative PCRNorthern Blot-analysisstress pattern: cDNA/ DNA amplification und electrophoretic separationcolony forming units(cfu)RNA/ DNA- isolationPMA-assays(live/dead)E. faecium  and starvation1,00E+041,00E+051,00E+061,00E+071,00E+081,00E+09control 3 days 4 days 5 daystimecfu/ mL, RNA [ng/L]1013,316,619,923,226,529,8Ct- valuescfu RNA DNAE. faecalis  and starvation1,00E+041,00E+051,00E+061,00E+071,00E+081,00E+09control 3 days 4 days 5 daystimecfu/ mL, RNA [ng/L]1013,316,619,923,226,529,8Ct- valuesKBE RNA DNA√ (pbp5)repression√ (pbp5)repression√ (pbp5)inductionE. faecalis√ (rcsA)induction√ (rcsA)repression√ (rcsA)inductionE. coli√ (rpoS)induction√ (rpoS)induction√ (rpoS)inductionP. aeruginosaspecificuspspecificuspspecificusposmotic shockstarvationstationary phaseE. faecalis                     E. faeciumNaCl: sodium chloride, DW: drinking water, M: marker, control, 2: after 3 days, : after 4 days, 4: after 5 daysM   1    2     3    4    1     2    3     4        M      1     2     3     4E. faecalis      E. faeciumM    1    2    3    4      M    1    2     3    4NaCl: sodium chloride, DW: drinking water, M: marker, control, 2: after 3 days, : after 4 days, 4: after 5 days

Specific and global stress responses in bacterial populations

https://publikationen.bibliothek.kit.edu/220079097/3815685

Specific and global stress responses in bacterial populations

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