Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: A guide for prokaryotic and eukaryotic investigation

Zhao

et al. 2015

Appl Environ Microbiol

dAlthough fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a ␤-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome-and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. Prebiotics are defined as nondigestible food ingredients that selectively stimulate the growth and activity of beneficial microbial strains residing in the host gastrointestinal tract (GIT) (1). Among the sugars that are qualified as prebiotics, fructooligosaccharides (FOS) are fructose polymers of diverse lengths that can be either derivatives of simple fructose polymers or fructose moieties attached to a sucrose molecule (2). Because of the linkage configuration, FOS are not digested in the upper GIT and have been shown in vivo to beneficially modulate the composition of the intestinal microbiota by preferentially increasing the numbers of bifidobacteria and lactobacilli (3, 4).Despite considerable commercial and research interest in the beneficial effects of FOS, the molecular basis of FOS metabolism by specific members of the intestinal microbiota has only recently been examined. In order to understand the influence of environmental conditions on genome-wide gene expression levels, wholegenome DNA microarrays have often been used to survey the gene expression patterns of strains in the presence and absence of oligosaccharides (2, 5-8). On the basis of in silico analysis of the Lactobacillus acidophilus NCFM genome sequence, Barrangou et al. (2) identified a multiple-sugar metabolism (msm) operon that was involved in the metabolism of FOS. The msm operon encodes an ATP-dependent binding cassette-type transport system and a cytoplasmic ␤-fructosidase, which mediates FOS uptake and intracellular hydrolysis. Moreover, expression of the operon was i...

Section: Discussionsupporting

confidence: 91%

Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

Zhao

et al. 2015

Appl Environ Microbiol

Journal of Industrial Microbiology and Biotechnology

“…It has also been reported that longer chains more easily span the width of the bilayer, thereby promoting acyl chain packing and making the membrane environment more gel-like; in contrast, shorter chains are unable to span the bilayer and cannot form hydrophobic interactions with other lipids and proteins. As a result, the fluidity and instability of the bilayer increase due to the motion of the free acyl chain ends [7]. Therefore, the increased fatty acid length contributes to the decrease in cell membrane fluidity, which is in agreement with our results in terms of the pyrene excimerization rate (K a ) and rotational diffusion of DPH.…”

Section: Discussionsupporting

confidence: 90%

Lactobacillus casei combats acid stress by maintaining cell membrane functionality

Zhang

Wang

et al. 2012

161

Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress.

“…One possible, indirect indicator of the activity of bacterial cells may be the integrity of cellular membranes (Cao-Hoang et al 2008). It is assumed that bacteria are potentially active if their cellular membrane is intact, and potentially dead if their cellular membrane is damaged (Decamp and Rajendran 1998).…”

Section: Determination Of Bacteria With Intact Functioning Cytoplasmmentioning

confidence: 99%

Selected fluorescent techniques for identification of the physiological state of individual water and soil bacterial cells — review

Lew

Mieszczyński

et al. 2010

Folia Microbiol

Stimulated by demands of the natural environment conservation, the need for thorough structural and functional identification of microorganisms colonizing different ecosystems has contributed to an intensive advance in research techniques. The article shows that some of these techniques are also a convenient tool for determination of the physiological state of single cells in a community of microorganisms. The paper presents selected fluorescent techniques, which are used in research on soil, water and sediment microorganisms. It covers the usability of determination of the dehydrogenase activity of an individual bacterial cell (CTC+) and of bacteria with intact, functioning cytoplasmic membranes, bacteria with an integrated nucleiod (NuCC+) as well as fluorescent in situ hybridization (FISH).