Identification in Listeria monocytogenes of MecA, a homologue of the Bacillus subtilis competence regulatory protein

Abstract

We identified in Listeria monocytogenes a gene encoding a protein homologous to MecA, a regulatory protein acting with ClpC and ComK in the competence pathway of Bacillus subtilis. In L. monocytogenes, MecA is involved, along with ClpC and ClpP, in the downregulation of a 64-kDa secreted protein. In B. subtilis, the MecA protein of L. monocytogenes behaves as a regulatory protein, controlling the transcription of comK and comG. Complete or disrupted ComK homologues were also found in L. monocytogenes. However, we failed to detect competence in various strains of L. monocytogenes, including those with intact ComK. Our results suggest that the functions of MecA in the saprophytes L. monocytogenes and B. subtilis have presumably diverged in response to their respective ecological niches.

FIG. 1

(A) MecA regions in B. subtilis and L. monocytogenes LO28. Small arrows with asterisks indicate the positions of the primers used for PCR amplification of the mecA and yjbD regions. (B) Northern blot analysis of total RNA extracted from strain LO28 grown in BHI broth at 37 and 42°C. RNA samples were separated and hybridized with an intragenic yjbD probe (403 bp) (lanes 1 to 5) or mecA probe (584 bp) (lanes 6 to 10). These probes were obtained by PCR from the chromosomal DNA of LO28: yjbD, primers 5′-CCCGATAAGGAGTGTGAATG-3′ and 5′-GCGCTTCACGTAGTTGATACG-3′; mecA, primers 5′-CCCTTCATTGTCAATGAC-3′ and 5′-ACTAACGGCATTGTCAATG-3′. Lanes: 1 and 6, 37°C, exponential phase; 2 and 7, LO28, 37°C, stationary phase; lanes 3 and 8, LO28, 42°C, exponential phase; 4 and 9, LO28, 42°C, stationary phase; 5 and 10, mecA mutant, 37°C, exponential phase. The locations and sizes of mRNAs are indicated by arrows.

FIG. 2

Growth of L. monocytogenes LO28 mecA mutant at 37°C and complementation. Symbols: ●, LO28/pAT18; ○, LO28/pAT18-mecA; ■, LO28 mecA mutant/pAT18; □, LO28 mecA mutant/pAT18-mecA. Bacteria were grown in BHI medium, and the optical density (OD) at 600 nm was measured at various intervals.

FIG. 3

A secreted 64-kDa protein accumulates in the culture supernatant of L. monocytogenes in the absence of MecA, ClpC, or ClpP. Bacteria were cultured in BHI medium until mid-log growth phase, and supernatants were collected and filtered. Supernatant protein extracts were prepared by trichloroacetic acid precipitation as previously described (13). Extracts with equal protein concentrations were applied to a sodium dodecyl sulfate–10% polyacrylamide gel and subjected to electrophoresis. Lane 1, molecular size marker; lane 2, LO28; lane 3, LO28 oppA mutant (unpublished data); lane 4, LO28 prfA mutant (12); lane 5, LO28 mecA mutant; lane 6, LO28 clpC mutant; lane 7, LO28 clpP mutant.