A molecular switch controlling competence and motility: competence regulatory factors ComS, MecA, and ComK control sigmaD-dependent gene expression in Bacillus subtilis

Abstract

Bacillus subtilis, like many bacteria, will choose among several response pathways when encountering a stressful environment. Among the processes activated under growth-restricting conditions are sporulation, establishment of motility, and competence development. Recent reports implicate ComK and MecA-ClpC as part of a system that regulates both motility and competence development. MecA, while negatively controlling competence by inhibiting ComK, stimulates sigmaD-dependent transcription of genes that function in motility and autolysin production. Both ComK-dependent and -independent pathways have been proposed for MecA's role in the regulation of motility. Mutations in mecA reduce the transcription of hag. encoding flagellin, and are partially suppressed by comK in both medium promoting motility and medium promoting competence. Reduced sigmaD levels are observed in mecA mutants grown in competence medium, but no change in sigmaD concentration is detected in a comK mutant. The comF operon, transcription of which requires ComK, is located immediately upstream of the operon that contains the flgM gene, encoding the sigmaD-specific antisigma factor. An insertion mutation that disrupts the putative comF-flgM transcription unit confers a phenotype identical to that of the comK mutant with respect to hag-lacZ expression. Expression of a flgM-lacZ operon fusion is reduced in both sigD and comK mutant cells but is abolished in the sigD comK double mutant. Reverse transcription-PCR examination of the comF-flgM transcript indicates that readthrough from comF into the flgM operon is dependent on ComK. ComK negatively controls the transcription of hag by stimulating the transcription of comF-flgM, thereby increasing the production of the FlgM antisigma factor that inhibits sigmaD activity. There likely exists another comK-independent mechanism of hag transcription that requires mecA and possibly affects the sigmaD concentration in cells undergoing competence development.

FIG. 1

ComK-dependent and -independent MecA control of hag expression. High cell density and nutritional stress stimulate expression of the comS gene. ComS interaction with MecA-ClpC results in release of ComK, which activates comF-flgM operon transcription, as well as the expression of other late competence operons. Antisigma factor FlgM negatively controls ς^D, resulting in reduced expression of hag and other genes of the ς^D-regulon. MecA affects the ς^D protein level, particularly in cells grown in medium that promotes competence development.

FIG. 2

Expression of hag-lacZ in the wild type and mecA, comK, and mecA comK, and srf (comS) mutants. Cells of each strain were grown in 2XYT (A) or CM (B and C), and samples were collected at the indicated times. hag-directed β-galactosidase activity was determined as described in Materials and Methods and in accordance with published protocols. Symbols: ▵, LAB2723 (comK hag-lacZ); ○, LAB2607 (hag-lacZ); •, LAB2724 (comK mecA hag-lacZ); ■, LAB2722 (mecA hag-lacZ); □, LAB2944 (hag-lacZ srf).

FIG. 3

Level of ς^D protein in the wild type (wt) and mecA, comK, and mecA comK mutants grown in rich medium. Cells precultured in 2XYT liquid medium were grown in 2XYT medium at 37°C. Samples were harvested at T₀ and T₂ (at the end of exponential growth and 2 h after the end of exponential growth, respectively). Cell extracts with equal protein concentrations were applied to SDS–12% polyacrylamide gels and subjected to electrophoresis. The resolved protein was electrotransferred to nitrocellulose and analyzed by the Western blotting procedures described in Materials and Methods. (A). Lane MW contained molecular size markers. The other lanes contained samples from cultures of LAB2607 (wild type), LAB2722 (mecA), LAB2723 (comK), and LAB2724 (mecA comK) cells collected at T₀ and T₂ of the growth curve. (B) Western blot band intensity determined by scanning of the image of the stained blot and quantification by the NIH-Image computer program. The values presented are percentages of the level of protein in the wild-type strain at T₂. The standard deviation was calculated from three independent experiments. (C) Levels of hag-directed β-galactosidase in cultures used to obtain extracts for Western blot analysis.

FIG. 4

Levels of ς^D protein in the wild type (WT) and mecA, comK, and mecA comK mutants grown in CM. Cells precultured in DSM agar plates were grown in CM at 37°C. Samples were harvested at the same time points as in Fig. 3. Analysis of the protein extracts was conducted as described in the legend to Fig. 3. (A) Western blot of extracts of LAB2607 (wild type [WT]), LAB2722 (mecA), LAB2723 (comK), and LAB2724 (mecA comK) cell samples collected at T₀ and T₂ of the growth curve. (B) Western blot band intensity determined and presented as described in the legend to Fig. 3.

FIG. 5

Structure of the comF-flgM operon and construction of a comF-flgM::pJL010 insertion mutant. (A) flgM is located within an operon containing the promoter P_D-1, immediately downstream of the comF operon. comF is a B. subtilis late competence operon. Transcription of comF is driven by a single ς^A-type promoter, utilization of which is dependent on ComK. The flgM operon contains orf139, flgM, orf160, and flgK. (B) The mutant was constructed by insertion of a plasmid (pJL010) carrying comFC and orf139 fragments by a single-recombination mechanism into the region containing the junction between the comF and flgM operons.

FIG. 6

Expression of hag-lacZ in wild-type and comK and comF-flgM::pJL010 mutant cells grown in CM. Cells were precultured on DSM agar plates and then grown in CM liquid at 37°C. hag-directed β-galactosidase (β-gal) specific activity of samples collected at the indicated time points was determined as described in Materials and Methods and in the legend to Fig. 3. Symbols: ▵, LAB2819 (SPβhag-lacZ); ○, LAB2921 (SPβhag-lacZ comK); ■, LAB3932 (SPβhag-lacZ comF-flgM::pJL010).

FIG. 7

flgM operon (orf139)-lacZ fusion expression in comK and sigD mutant cells. Cells of the wild-type and the comK, sigD, and comK sigD mutant strains bearing plasmid pJL011 [flgM (orf139)-lacZ] integrated at the flgM locus were grown in CM. Samples were collected at 30-min intervals for assay of β-galactosidase activity. Symbols: ○, LAB2995 [flgM (orf139)-lacZ]; ▴, LAB2997 [sigD flgM (orf139)-lacZ]; □, LAB2996 [comK flgM (orf139)-lacZ]; •, LAB2998 [sigD comK flgM (orf139)-lacZ].

FIG. 8

RT-PCR products from comF-flgM and flgM operon transcripts. At the top is a diagram of the comFC-orf139 region of the comF-flgM operon. P_D1 indicates the location of the flgM promoter utilized by the ς^D form of RNA polymerase. The arrows indicate the oligonucleotide primers (numbered 1 through 3) used to prime RT and to amplify cDNA and PCR products. The bottom panel is a photograph of an ethidium bromide-stained 1% agarose gel on which the PCR and RT-PCR products were resolved. In lanes 1 and 2, the template for PCR was JH642 chromosomal DNA. In lanes 3 to 6, the template for PCR and RT-PCR was RNA from strain LAB2917 (comK::neo). In lanes 7 to 10, the template for PCR and RT-PCR was from JH642 (wild-type) cells. MW, molecular weight markers. Lanes: 1, PCR product of primers 1 and 2 (no reverse transcriptase); 2, PCR product of primers 1 and 3; 3, PCR using primers 1 and 2; 4, PCR using primers 1 and 3; 5, RT-PCR using primers 1 and 2; 6, RT-PCR using primers 1 and 3; 7, PCR using primers 1 and 2; 8, PCR using primers 1 and 3; 9, RT-PCR using primers 1 and 2; 10, PCR using primers 1 and 3.