Development of flow cytometry technique for detection of thinning of peptidoglycan layer as a result of solvent production by Clostridium pasteurianum

Abstract

Clostridium pasteurianum forms acetic and butyric acids in an initial growth phase, which is a typical feature of clostridial acetone-butanol fermentation where an initial accumulation of acids is followed by production of solvents 1-butanol, acetone and ethanol. The initiation of the solvent production coupled with endospore formation leads to decrease of cell-wall thickness; thinner cell wall is more resistant against solvents and dyes. These changes can be observed by the method based on adaptation of Gram staining. The cell wall of G+ bacteria allows the entry of hexidium iodide and rhodamine 123, whereas the outer membrane of G- bacteria does not allow the uptake and therefore G+ bacteria are stained with higher fluorescence intensity than G- bacteria. The ratio of fluorescence intensity (FI) to forward scatter (FSC) was determined to correspond to G+ bacteria when clostridia were producing less solvents. The significant drop of the ratio FI to FSC to the level corresponding to G- bacteria is detected after initiation of solvent production.

Characterization of a Glucosamine/Glucosaminide N -Acetyltransferase of Clostridium acetobutylicum

“…The Gram-positive firmicute C. acetobutylicum is an important solvent producer strain and widely used for metabolic engineering. The initiation of the solvent production in this organism is coupled with endospore formation and associated with the thinning of the peptidoglycan layer, which apparently results in the increased resistance against the solvents (15). A better knowledge of cell wall modifications, turnover, and recycling is mandatory for optimization of solvent production in C. acetobutylicum by rational approaches.…”

Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry

“…In coriander oil, linalool, its major component, is found mainly in the form of its S (+) enantiomer (Casabianca et al, 1998), which is known to cause increased permeability only in negatively charged membranes (Trombetta et al, 2005). Moreover, alcohols have been related to a decrease of Gram-positive bacterial cell wall thickness, causing an increased resistance of bacteria to compounds such as solvents and dyes (Linhová et al, 2010). In conclusion, the S enantiomer of linalool could cause the disruption of the negatively charged Gramnegative bacterial outer membrane whereas its properties as an alcohol could lead to an increased resistance in Gram-positive bacteria due to bacterial cell wall thickening as described above.…”

Draft Genome Sequence of Clostridium pasteurianum NRRL B-598, a Potential Butanol or Hydrogen Producer

“…This strain has been used in only a few studies (2–7); however, it might be a useful platform for further genetic modification because it is not sensitive to oxygen, has versatile sugar-fermenting and proteolytic abilities, seems to be genetically stable in comparison with other clostridia, and tolerates minor changes in fermentation conditions.…”