Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance

Abstract

Amino acid decarboxylation-antiporter reactions are one of the most important systems for maintaining intracellular pH between physiological limits under acid stress. We analyzed the Lactobacillus acidophilus NCFM complete genome sequence and selected four open reading frames with similarities to genes involved with decarboxylation reactions involved in acid tolerance in several microorganisms. Putative genes encoding an ornithine decarboxylase, an amino acid permease, a glutamate gamma-aminobutyrate antiporter, and a transcriptional regulator were disrupted by insertional inactivation. The ability of L. acidophilus to survive low-pH conditions, such as those encountered in the stomach or fermented dairy foods, was investigated and compared to the abilities of early- and late-stationary-phase cells of the mutants by challenging them with a variety of acidic conditions. All of the integrants were more sensitive to low pH than the parental strain. Interestingly, each integrant also exhibited an adaptive acid response during logarithmic growth, indicating that multiple mechanisms are present and orchestrated in L. acidophilus in response to acid challenge.

FIG. 1.

Survival of L. acidophilus NCK1398 exposed to MRS broth adjusted to pHs 3.0 (with HCl) and 3.5 (with lactic acid) and incubated at 37°C. Symbols: •, adapted (1 h, pH 5.5) cells exposed to pH 3.5; □, nonadapted cells exposed to pH 3.5; ▵, nonadapted cells exposed to pH 3.0.

FIG. 2.

Acid stress-related genes in L. acidophilus. (A) ORF La57 and surrounding genes. (B) Gene organization of the region containing ORFs La995 and La996. (C) Gene organization of the region containing ORF La867. Disrupted genes are represented by gray arrows. Putative rho-independent terminators and their calculated free energy are indicated. Potential functions based on homologies are indicated.

FIG. 3.

Insertional inactivation of ORF La57 in L. acidophilus NCFM. (A) Diagram of the La57 locus of NCFM and NCK1678 chromosomes. La57 is represented by an arrow, and the internal fragment is denoted by a shaded box. The restriction sites PstI, BamHI, and BglII are indicated. The repeating unit represents the plasmid DNA present in various copies (n). (B) Southern hybridization analysis of NCFM and NCK1678. Chromosomal DNA was digested with BglII (lane 1, plasmid pTRK803; lane 2, NCFM; lanes 3 and 4, NCK1678). The internal fragment was used as the probe. (C and D) PCR amplification of the left (C) and right (D) junction fragments in NCK1678. Lane 1, 1-kb ladder; lane 2, NCFM; lanes 3 and 4, NCK1678. Em, erythromycin.

FIG. 4.

Survival of log-phase L. acidophilus cultures in MRS adjusted to pH 3.5 with lactic acid. Viable cell counts were performed at 40-min intervals. ▪, NCK1398 (control); •, NCK1678 (La57, amino acid antiporter); ▴, NCK1680 (La867, transcriptional regulator); ⧫, NCK1682 (La995, amino acid permease); ▾, NCK1684 (La996, ornithine decarboxylase).

FIG. 5.

Specific death rate (K) of late-stationary-phase cells of L. acidophilus after exposure to pHs 3.0, 3.5, and 4.0, adjusted with lactic acid. Viable cell counts were performed at 30-min intervals. ▪, L. acidophilus NCK1398 (control); □, NCK1678 (amino acid antiporter); ▴, NCK1680 (transcriptional regulator); , NCK1684 (ornithine decarboxylase); ⊞, NCK1682 (amino acid permease).

FIG. 6.

Specific death rates (K) of nonadapted (shaded bars) and adapted (solid bars) log-phase cells of L. acidophilus derivatives after challenge at pH 3.5 (adjusted with lactic acid). Cells were adapted at pH 5.5 for 1 h prior to exposure to pH 3.5. Correlation values (R) were >0.9. Viable cell counts were performed at 30-min intervals for 2.5 h.