Communication is a process of sensing cell density and activating different adaptive mechanisms, which involves the exchange of specific signal molecules. It is known that cell density plays a key role in communication, while the influence of other factors (i.e. salinity, temperature) is less understood. We discussed quantitative methods for signal detection and microbial responses to environmental stresses in four model systems that include two Gram negative bacteria Vibrio sp. and C. jejuni, a Gram positive bacterium B. subtilis and a yeast S. cerevisiae.
B.04 Guest lecture
COBISS.SI-ID: 3812984The effect of temperature on signal molecules production in Vibrio sp. was studied in diploma thesis. Signal molecules isolated from the spent media of the wild type strain Vibrio sp. were extracted with acidified ethyl acetate. AHL molecules present in spent media were determined with TLC and biosensor strain E.coli JM109 (pSB401). For detection of AI-2 molecules a biosensor strain Vibrio harveyi BB170 was used. The results indicate that Vibrio sp. produces one type of AHL molecules, 3-oxohexanoyl-L-homoserine lactone (OHHL). It also produces AI-2 signal molecule. Production of AHL and AI-2 molecules is temperature sensitive.
D.11 Other
COBISS.SI-ID: 3946872The method for monitoring QS molecules during fermentation, which was developed in the project, was presented with an invited lecture at the international meeting of experts on yeast bioflavour production (2nd European Yeast Flavour Workshop, Cost Action FA0907 BIOFLAVOUR). The main contribution of the presented methodology is miniaturisation, efficacy and rapidness of the detection of yeast aromatic alcohols. Moreover, despite the miniaturisation, the method has still high sensitivity and specificity. The fermentation is processed in 2 ml bioreactors and detection is performed with HPLC and phenyl column of new generation. There is no need for special isolation of analites (e.g. extraction) or purification, therefore the optimized process exceedingly shorten, simplify and last but not the least reduce the cost of analysis (for approx. 100 – 1000 fold when compared with other methods in the field).
B.04 Guest lecture
COBISS.SI-ID: 3920248The mechanism of quorum sensing (QS) in Bacillus subtilis is well understood, however the influence of the physico-chemical factors on QS remains to be elucidated. We studied the influence of temperature (24, 37 and 51 °C) on concentration of the major signaling peptide of B. subtilis, ComX, and transcription of its gene comX and the comQ gene, which encodes isoprenyl transferase, ComQ, responsible for the processing and isoprenylation of mature ComX at the triptophane residue and srfA expression. HPLC results suggested that the concentration of ComX in the growth medium increases with temperature of cultivation. In contrast, based on quantitative PCR analyses of mRNA concentrations, transcription of comX, comQ and srfA was negatively regulated by temperature at 51 °C. The ratio of comX mRNA copies at 24 and 51 °C was 4 ± 2, which is in contrast to concentrations of ComX in growth media. Therefore, expression of all three studied genes did not coincide with the growth rate and the final cell densities, which were higher at 51 than at 24 °C. Results suggest that ComX is not sufficient to overcome the high temperature induced inhibition of srfA expression.
D.11 Other
COBISS.SI-ID: 4114296Mutual interactions between yeasts and their influence on production of aromatic compounds important for wine aroma are not well understood and the research on yeast communication is still in its infancy. Saccharomyces cerevisiae secretes the communication molecules phenylethanol and tryptophol, which have been suggested to play in quorum sensing. They are present in the media in micromolar concentrations and therefore their detection and quantification requires analytical methods with high detection sensitivity. In our experimental work a simple, quick and broadly accessible method for the detection of phenylethanol, tryptophol and tyrosol was developed. The method is based on high pressure liquid chromatography in combination with a highly specific phenyl column and fluorescence detector. We assumed that communication molecules are secreted only by viable cells and by counting them by Image J, a computer based program we were able to obtain the data that were based only on live cells, which lead to implementation of a new parameter named production rate of communication molecules. Using this parameter we observed that production of aromatic alcohols is synchronized with transcription of ARO genes responsible for the synthesis of these communication molecules. Furthermore, the production of communication molecules was found to be dependent on cell density and their concentration was the highest at the end of the exponential phase. Next, the effect of ethanol, nitrogen, sulphur dioxide and oxygen on the production rate of communication molecules was investigated. Results indicated that the production rate of phenylethanol, tryptophol and tyrosol decreases with increasing ethanol concentrations. The addition of 1000 μM concentration of phenylethanol and tryptophol at the start of mixed and single culture fermentation with S. cerevisiae and C. zemplinina had no effect on cell viability or fermentation dynamics but significantly changed the fermentation aromatic profile. This is the first report addressing the effect of phenylethanol and tryptophol on the fermentation process and providing the first evidence on the role of communication molecules in formation of the wine aroma.
D.11 Other
COBISS.SI-ID: 4201080