|Wechter, William - Pat|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/23/2007
Publication Date: 4/1/2007
Citation: Smeianov, V.V., Wechter, W.P., Broadbent, J.R., Hughes, J.E., Rodriguez, B., Steele, J.L. 2007. Comparative High-Density Microarray Analysis of Gene Expression During Growth of Lactobacillus Helveticus in Milk vs. Rich Culture Medium. Applied and Environmental Microbiology, 73:2661-2672. Interpretive Summary: Lactobacillus helveticus CNRZ32 is a bacterium used by industry to modulate cheese flavor. This bacterium is used as a starter for numerous dairy-based consumer products including yogurt, Mozzarella and Swiss cheese, as well as a flavor adjuvant for a wide variety of cheese type products. Using high-density microarrays, gene expression of this bacterium during growth in milk as compared to growth in an artificial laboratory medium was determined. Twenty-two genes were shown to be significantly modulated during growth in milk. These genes, encoding enzymes important in the enzymatic utilization of sugars and other chemicals as well as transportation of these compounds into and out of the cell, will be extremely useful for investigating expanded or new uses for this bacterium.
Technical Abstract: Lactobacillus helveticus CNRZ32 is used by industry to modulate cheese flavor. Compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 169 genes potentially important for growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate expression of these genes during growth in milk and MRS medium using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays where the non-interrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA was isolated from cells were grown in double-steamed skim milk or in MRS to mid-log phase, converted to cDNA, then labeled with Cy-3 and hybridized to the microarrays. Statistical analysis of array data from annotated gene probes identified 52 genes that were over-expressed during growth of CNRZ32 in milk (P<0.05), and 22 of these genes showed up-regulation after applying Bonferroni adjustment at 0.1 overall cutoff value. Data from the tiled microarrays identified numerous additional genes that were significantly up-regulated in milk versus MRS. Collectively, array data showed growth of L. helveticus CNRZ32 in milk induced genes encoding cell-envelope proteinases, oligopeptide transporters, endopeptidases, as well as enzymes for lactose, cysteine and hypothetical phosphoserine utilization pathways, de novo synthesis and/or salvage pathways for purines and pyrimidines, and other functions. These results improve our understanding of L. helveticus CNRZ32 physiology and the contribution of this bacterium to cheese properties, and also identify interesting targets for functional genetics studies.