Comparative high-density microarray analysis of gene expression during growth of Lactobacillus helveticus in milk versus rich culture medium

Abstract

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by 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 in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.

FIG. 1.

Correlation of expression ratios from microarray profiling and RT-PCR. Total RNA was extracted from milk-grown and MRS-grown cultures of Lactobacillus helveticus CNRZ32 and served as a template for cDNA synthesis to be used in microarrays and RT-PCR experiments. The calculated expression ratios (n-fold changes) obtained from log-transformed data of 14 genes (Table 1) are shown for microarray experiments (horizontal axis) and RT-PCR (vertical axis). The best-fit linear regression curve is shown along with the calculated equation.

FIG. 2.

Map of selected metabolic pathways in Lactobacillus helveticus CNRZ32. The different enzymatic steps are represented by the correspondent gene designations. Dotted arrows indicate several enzymatic steps or the absence of data regarding a particular gene. Arrows next to the gene symbols indicate either the upregulation (up arrow) or the downregulation (down arrow) of this gene during the growth in milk compared with growth in MRS; two opposite arrows indicate no change in the expression. glcU, gene encoding glucose/ribose uptake protein; fbaA, gene encoding fructose-1,6-biphosphate aldolase; tpiA, gene encoding triosephosphate isomerase; gapA, gene encoding glyceraldehydes 3-phosphate dehydrogenase; pgk, gene encoding phosphoglycerate kinase; pgm, gene encoding phosphoglyceromutase; eno, gene encoding enolase; glpF, gene encoding the glycerol uptake facilitator; hyp1677-2, gene encoding hypothetical membrane serine/threonine-protein kinase (membrane translocator); serA, gene encoding phosphoglycerate dehydrogenase; serB, gene encoding hypothetical phosphoserine phosphatase (ycsE); cysE, gene encoding serine O-acetyltransferase; cysK, gene encoding cysteine syntase; ATase2/NifS, gene encoding aminotransferase class V/cysteine desulfurase; cbl, gene encoding cystathionine β-lyase. Hypothetical transporter proteins are indicated by rectangles.

FIG. 3.

Schematic representation of a locus containing genes of serine metabolism in Lactobacillus helveticus CNRZ32. Abbreviations: serA, gene encoding phosphoglycerate dehydrogenase; serC, gene encoding phosphoserine aminotransferase; pgm, gene encoding phosphoglycerate mutase; tnpA, gene encoding transposase; tnpB, gene encoding transposase; hyp1677-2, gene encoding hypothetical membrane serine/threonine-protein kinase (membrane translocator); ISL2, insertion sequence.