Project description:The global transcriptome of the wild type Lactobacillus acidophilus NCFM strain (NCK56) was measured during exponential growth on 11 prebiotic carbohydrates and glucose to identify the specific gene cluster differentially upregulated in response to each carbohydrate.
Project description:This study reports an over 20-fold increase in the adhesive ability of Lactobacillus acidophilus NCFM to Caco-2 cells following a 1 hour incubation of cells that were concentrated ten-fold, immediately prior to adhesion. Microarray analysis of the global transcriptional response with and without exposure to the adhesion adaptive conditions revealed several genes potentially involved with adhesion to the intestinal epithelial cells and a classic stress response. Interestingly, putative genes linked to the synthesis of an interspecies signaling molecule, autoinducer-2 (AI-2), were overexpressed. Examination of the L. acidophilus NCFM genome revealed the complete pathway for AI-2 synthesis. AI-2 activity was detected in L. acidophilus NCFM during stationary growth phase using the Vibrio harveyi BB170 assay system. Using site-specific integration, an isogenic mutation was created in luxS and the resulting derivative of L. acidophilus NCFM did not produce AI-2. A 58 % decrease in adherence to Caco-2 cells was also observed by the LuxS- mutant when the cells were used for adhesion directly from logarithmic phase cultures. However, the LuxS- mutant strain still responded to adhesion adaptive conditions with significantly increased adherence indicating that additional factors contribute to the amplified adhesion response. Keywords: Culture response to specific environmental conditions
Project description:Lactobacillus acidophilus NCFM is an industrially important strain used extensively as a probiotic culture. Tolerance of the presence of bile is an attribute important to microbial survival in the intestinal tract. A whole-genome microarray was employed to examine the effects of bile on the global transcriptional profile of this strain, with the intention of elucidating genes contributing to bile tolerance. Genes involved in carbohydrate metabolism were generally induced, while genes involved in other aspects of cellular growth were mostly repressed. A 7-kb eight-gene operon encoding a two-component regulatory system (2CRS), a transporter, an oxidoreductase, and four hypothetical proteins was significantly upregulated in the presence of bile. Deletion mutations were constructed in six genes of the operon. Transcriptional analysis of the 2CRS mutants showed that mutation of the histidine protein kinase (HPK) had no effect on the induction of the operon, whereas the mutated response regulator (RR) showed enhanced induction when the cells were exposed to bile. These results indicate that the 2CRS plays a role in bile tolerance and that the operon it resides in is negatively controlled by the RR. Mutations in the transporter, the HPK, the RR, and a hypothetical protein each resulted in loss of tolerance of bile. Mutations in genes encoding another hypothetical protein and a putative oxidoreductase resulted in significant increases in bile tolerance. This functional analysis showed that the operon encoded proteins involved in both bile tolerance and bile sensitivity. Keywords: stress response We used a Reference Sample design, where each sample was compared using a dye swap to a common reference sample (early log-phase L. acidophilus cultures resuspended in fresh MRS [pH ~6.8]).
Project description:Helicobacter pylori causes chronic gastritis and avoids elimination by the immune system of the infected host. The commensal bacterium Lactobacillus acidophilus has been reported to exert beneficial effects as a supplement during H. pylori eradication therapy. In the present study, we applied whole genome microarray analysis to compare the immune response induced in murine bone marrow derived macrophages (BMDM) stimulated with L. acidophilus, H. pylori, or with both bacteria in combination Microarray expression profiling was performed to analyze stimulation of bone marrow derived macrophages with Helicobacter pylori 251, Lactobacillus acidophilus NCFM or Lactobacillus acidophilus NCFM co-stimulated with Helicobacter pylori 251 were analyzed 5 hours after infection.
Project description:The global transcriptome of the wild type Lactobacillus acidophilus NCFM strain (NCK56) was measured during exponential growth on 11 prebiotic carbohydrates and glucose to identify the specific gene cluster differentially upregulated in response to each carbohydrate. Oligoarray hybridization experiments were performed to compare the differential transcriptional profiles of the NCK56 at the early-log (OD600nm 0.3-0.5) phase when cultured in semi-synthetic medium (SSM, Barrangou et al., 2003 PNAS. 100:8957-8962, supplemented with a range of 1% (w/v) carbohydrates, at 37 M-BM-0C and harvested by centrifugation and flash freezing. A total of 12 NCK56 cultures were prepared by growing in SSM supplemented with each one of the 12 tested carbohydrates. Each culture was grown in fresh carbohydrate supplemented SSM with a 1% inoculum from an overnight culture for a total of five subcultured passages. Aliquots of cells were collected at OD600nm of 0.3-0.5 (early log-phase) for total RNA extraction and cDNA synthesis. Labeled cDNA samples from NCK56 were coupled with mono-reactive Cy3 and Cy5 dyes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), for two technical replicates on each carbohydrate (e.g. cy3-GOS and cy5-GOS). Comparative hybridizations were performed on samples representing two different carbohydrate cultures at same growth phases labeled with either cy3 or cy5 using a loop design for 12 hybridizations.