The Stress Responses of Probiotic Lactobacilli and a Bifidobacterium With Special Emphasis on Clp Family Proteins
Year: 2008
Type: dissertation
Abstract: The use of food products containing probiotic microorganisms is of increasing economic importance. The health promoting effects of selected probiotic strains has been substantiated in controlled clinical studies. The probiotic nature of the health promoting bacteria is not well studied compared to the virulence of pathogenic bacteria. Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, such as temperature changes and low pH. Virulence and stress responses are closely related in several Gram-positive bacteria while extremely little is known about the possible overlap of stress responses and the probiotic nature of the bacteria. The available data concerning stress responses of lactobacilli and bifidobacteria mainly cover physiological changes in these bacteria when subject to stress, such as high temperature and low pH, and their ability to survive in different challenges. ClpATPases are a family of stress proteins that are known as virulence factors in a number of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, and regulators of several vital biological processes in Gram-positive bacteria with a low G+C content. In the first part of this thesis, clpL ATPase encoding genes and their protein products were characterized in two potentially probiotic lactobacilli, Lactobacillus rhamnosus E-97800 and L. gasseri ATCC 33323. Southern blot analysis revealed that among four L. rhamnosus strains only L. rhamnosus E-97800 carried two clpL genes, assigned as clpL1 and clpL2. Expression of both genes were induced after heat stress >20- and 3-fold, respectively. The clpL2 region was found to be mobilized after prolonged cultivation of E-97800 at a high temperature. The sequence analyses revealed that clpL2 shared 98 % identity to L. plantarum clpL gene, clpL2 is flanked by inverted repeat highly identical to the repeats of a functional insertion element in a L. plantarum strain. The data indicate that L. rhamnosus E-97800 has acquired clpL2 region via horizontal gene transfer, propably the donor being a lactobacillar strain. Moreover, the low G+C content (40 %) of clpL2 compared to clpL1 (49 %) and to the average L. rhamnosus entries at GenBank (48 %) together with high identity of clpL2 to the L. plantarum clpL gene indicate that the clpL2 containing element has been transposed relatively recently. Homology searches using clpL genes as query sequences revealed that the number of paralogous clpL genes varies among lactic acid bacteria (LAB). However, the putative selective advantage of this extra clpL paralog to host bacteria remains to be studied, since the stress tolerance of the clpL2-deficient strain was not altered compared to its parental strain. We demonstrated that a CIRCE element, which is known to mediate HrcA-dependent regulation, is located upstream of the clpL1 in L. rhamnosus and clpL in L. gasseri which together with the strong induction fold of clpL1 during heat stress suggest HrcA-mediated regulation of clpL genes. Moreover, we showed that purified HrcA protein is able to specifically bind to the promoter region of clpL in L. gasseri. Thus, the expression of clpL is most likely regulated by the HrcA/CIRCE system in these lactobacilli representing a novel regulon. In the second part of this work, two-dimensional electrophoresis-based (2-DE) tools were applied to investigate stress responses in selected probiotic bacteria. Since probiotic bacteria are adapted to grow in an environment rich in nutrients, the optimization of growth conditions for efficient metabolic labelling to examine protein synthesis kinetics in defined media is needed. A semi-defined medium for metabolic labelling with [35S]methionine for Bifidobacterium longum was developed. This medium was shown to support efficient protein radiolabelling. In addition, chemically defined media and experimental conditions supporting efficient protein radiolabelling with [35S]methionine were developed, and proved to be applicable for a number of lactobacillar strains to investigate their stress responses. Fluorescence 2-D difference gel electrophoresis (DIGE) was applied to study the heat shock response of L. gasseri ATCC 33323. In addition to classical chaperons DnaK and GroEL, four Clp AAA+ (ATPases associated with a variety of cellular activities) ATPases were detected and found to be increased in abundance after a heat shock. One of these, clpL, was deleted by using a thermosensitive vector. It was shown that the functional clpL gene is essential for the development of constitutive and induced thermotolerance in L. gasseri. The expression of several HSPs (heat shock proteins) was at the same level in both clpL deficient and its parental strain indicating that ClpL is not involved in modulation of the heat shock response in L. gasseri. Instead, ClpL probably prevents aggregation of non-native proteins generated by stress and thus the clpL gene might have industrial potential.
Source: Työväentutkimus Vuosikirja
Author: Aki Suokko
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W612652532