The characteristics of autolysins associated with cell separation in Bacillus subtilis.

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

Microfluidic dielectrophoretic cell manipulation towards stable cell contact assemblies.

Cell contact formation, which is the process by which cells are brought into close proximity is an important biotechnological process in cell and molecular biology. Such manipulation is achieved by various means, among which dielectrophoresis (DEP) is widely used due to its simplicity. Here, we show the advantages in the judicious choice of the DEP microelectrode configuration in terms of limiting undesirable effects of dielectric heating on the cells, which could lead to their inactivation or death, as well as the possibility for cell clustering, which is particularly advantageous over the linear cell chain arrangement typically achieved to date with DEP. This study comprises of experimental work as well as mathematical modeling using COMSOL. In particular, we establish the parameters in a capillary-based microfluidic system giving rise to these optimum cell-cell contact configurations, together with the possibility for facilitating other cell manipulations such as spinning and rotation, thus providing useful protocols for application into microfluidic bioparticle manipulation systems for diagnostics, therapeutics or for furthering research in cellular bioelectricity and intercellular interactions.

Growth, chain formation, and toxin production by southern Brazilian Pseudo-nitzschia isolates under laboratory conditions.

Pseudo-nitzschia is a diatom genus capable of producing the neurotoxin domoic acid (DA), which has been related to mortalities of marine vertebrates, and the amnesic shellfish poisoning (ASP) in human consumers of contaminated bivalves. This study reports DA production among Pseudo-nitzschia strains isolated from shellfish farming areas in southern Brazil. Twenty-seven cultures of potentially toxigenic Pseudo-nitzschia species were established. Growth, stepped-chain formation, and DA production were evaluated in static, intermittently illuminated (12:12 photoperiod) batch cultures for 12 selected strains, and under continuous light and/or turbulence for a single Pseudo-nitzschia calliantha strain. Growth rate ranged from 0.16 to 0.39 day-1 among the 12 strains. Only P. calliantha and P. cf. multiseries yielded detectable levels of intracellular DA, reaching up to 0.054 fg cell-1 in late exponential and 0.15 pg cell-1 in early stationary phase, respectively. Continuous light impaired cell growth, and turbulence enhanced step-chain formation by threefold during exponential growth phase, but no DA production was detected under both conditions. The effect of turbulence on chain formation should be further evaluated in the field, once particle size is expected to affect the ingestion of toxic cells and thus toxin accumulation by certain DA vectors. The low cell toxicity revealed herein under laboratory conditions is in accordance with the low frequency of DA contamination episodes in south Brazilian waters. Nevertheless, monitoring of Pseudo-nitzschia abundance and DA presence in farming areas is continuously required to assure the quality of local shellfish products.

Test parameters and cell chain length of Streptococcus thermophilus affect the microbial adhesion to hydrocarbons assay: a methodical approach.

The microbial adhesion to hydrocarbons (MATH) test is one of the most common method to determine the hydrophobicity of cell surfaces. Despite its prevalence, no standard test parameters are used in literature, making a direct comparison of data almost impossible. Criticism also focuses on test parameters that may mask hydrophobic interactions and hence lead to erroneous test results. We methodically investigated the impact of different MATH test parameters on the calculation of the cell surface hydrophobicity of Streptococcus thermophilus, a widespread exopolysaccharide-producing lactic acid bacterium used in the production of fermented milk products. Besides composition and ionic strength of the buffer used for cell re-suspension, we observed a pronounced time dependency of the turbidity of the cell suspension during phase separation due to sedimentation and/or cell lysis. A new modification of the MATH assay was applied to enable the determination of cell surface hydrophobicity of long chain-forming bacteria. As the cell surface hydrophobicity was not altered during exponential growth phase, we assume that the cell surface and its capsular exopolysaccharide layer are not changed during cultivation.