Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk

Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus cells propagated in 11% skim milk (SM) during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 ORFs were differentially expressed at least in one time point. Genes differentially expressed in SM included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4h, while gene expression of Opp2 increased over time reaching its highest level at 12h, suggesting that the two systems have different specificities. Expression of a two-component regulatory system (2CRS), previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (AI-2) and adherence to mucin and intestinal epithelial cells, in vitro. A loop experimental design (Vinciotti et al., 2005) was employed in a time course experiment to monitor the transcriptional response of L. acidophilus NCFM during growth in 11% reconstituted skim milk (SM). The culture was initiated in MRS broth and transferred 3-5 times in SM before a 10% inoculation in prewarmed SM. Cells and RNA samples were obtained from two biological replicates at times of 5 min (pH= 6.4; ≈ 2.1 X 107 cfu/ml), 4h (pH= 5.8; ≈ 3.8 X 107 cfu/ml), 8h (pH= 5.4; ≈ 7.1 X 107 cfu/ml), and 12h (pH= 4.9; ≈ 1.5 X 108 cfu/ml) and compared in an unbalanced loop-design. Samples were compared as follows (Cy3 vs. Cy5): T1 (5 min) vs. T2 (4h), T2 vs. T3 (8h), T3 vs. T4 (12h), T4 vs. T2, T4 vs. T1 and T3 vs. T1.