Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus

Abstract

Two-component regulatory systems are one primary mechanism for environmental sensing and signal transduction. Annotation of the complete genome sequence of the probiotic bacterium Lactobacillus acidophilus NCFM revealed nine two-component regulatory systems. In this study, the histidine protein kinase of a two-component regulatory system (LBA1524HPK-LBA1525RR), similar to the acid-related system lisRK from Listeria monocytogenes (P. D. Cotter et al., J. Bacteriol. 181:6840-6843, 1999), was insertionally inactivated. A whole-genome microarray containing 97.4% of the annotated genes of L. acidophilus was used to compare genome-wide patterns of transcription at various pHs between the control and the histidine protein kinase mutant. The expression pattern of approximately 80 genes was affected by the LBA1524HPK mutation. Putative LBA1525RR target loci included two oligopeptide-transport systems present in the L. acidophilus genome, other components of the proteolytic system, and a LuxS homolog, suspected of participating in synthesis of the AI-2 signaling compound. The mutant exhibited lower tolerance to acid and ethanol in logarithmic-phase cells and poor acidification rates in milk. Supplementation of milk with Casamino Acids essentially restored the acid-producing ability of the mutant, providing additional evidence for a role of this two component system in regulating proteolytic activity in L. acidophilus.

FIG. 1.

Organization of LBA1524HPK-LBA1525RR 2CRS in L. acidophilus NCFM. The disrupted HPK gene is represented by a gray arrow. Putative terminator regions and their calculated free energy are indicated by hairpin structures. The start, the putative ribosome-binding site, the potential promoter, and the transcription start are indicated.

FIG. 2.

Morphology of L. acidophilus NCFM (A) and NCFM::LBA1524HPK (B) as seen under a phase-contrast microscope. Magnification, ×1,000.

FIG. 3.

(A) Survival of L. acidophilus NCK1398 (NCFM::lacL, squares) and the HPK mutant NCK1686 (circles) in MRS adjusted to pH 3.5 with lactic acid. (B) Cells were exposed to pH 5.5 (open symbols) or pH 6.8 (filled symbols) for 1 h prior to challenge at pH 3.5 (adjusted with lactic acid).

FIG. 4.

Organization of the oligopeptide transport (opp) operons in L. acidophilus NCFM. Predicted rho-independent terminators with a free energy over −10 kcal/mol (continuous line) and under −10 kcal/mol (dotted line) are indicated.

FIG. 5.

Growth of L. acidophilus NCFM (squares) and NCK1686 (NCFM::LBA1524HPK [circles]) in milk (solid symbols) and milk supplemented with yeast extract (open symbols) (A) and in milk supplemented with 0.25% Casamino Acids (B). r = 0.99.

FIG. 6.

(A) Northern blot analysis of seven genes was performed with RNA isolated in three independent experiments from L. acidophilus NCK1398 (NCFM::lacL) and NCK1686 (NCFM::LBA1524HPK) exposed to pH 6.8, 5.5, or 4.5 for 30 min. RNA ratios were calculated by densitometry analysis from data obtained from the Northern blot. (B) Comparison of expression measurements by microarray and Northern blot analysis. The correlation coefficient for each condition is given in the figure.