Distinct roles of ComK1 and ComK2 in gene regulation in Bacillus cereus

Abstract

The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously.

Figure 1. Numbers of genes belonging to the clusters containing commonly up- (above) or downregulated (bellow) genes upon comK overexpression classified using COG functional categories.

Figure 2. Flow cytometric analysis of PcomK1-gfp (A,C) and PcomK2-gfp (B,D) in liquid minimal medium.

Promoter fusions in wild type (black) and in comK1(A,B) or comK2(C,D) mutant (gray). Analyses were performed as described in Experimental procedures. The numbers of cells are indicated on the y axis, and their relative fluorescence levels are indicated on the x axis on a logarithmic scale. For each experiment at least 20,000 cells were analysed. The graphs are the representative of at least three independent experiments.

Figure 3. Flow cytometric analyses of PcomGA-gfp after overexpression of comK_Bsu in the wild type containing pNWcomK_Bsu (black) and in the co-expressed comK_Bsu and comK1 (pATK31(A,C)) or comK2 (pATK32(B,C)) (gray).

The samples were analyzed in three hours after IPTG induction; panels A,B- strains without induction, panels C,D- strains after induction with IPTG. The numbers of cells are indicated on the y axis, and their relative fluorescence levels are indicated on the x axis on a logarithmic scale. For each experiment at least 20,000 cells were analysed. The graphs are the representative of at least three independent experiments.

Figure 4. Single cell analyses of PcomGA-gfp and in liquid minimal medium.

The effect of ComK_Bsu overexpression in the wild type (black) and in the comK1 (A,C) or comK2 (B,D) mutant (gray). The samples were analyzed in three hours after IPTG induction; panels A,B- strains without induction, panels C,D- strains after induction with IPTG. The numbers of cells are indicated on the y axis, and their relative fluorescence levels are indicated on the x axis on a logarithmic scale. For each experiment at least 20,000 cells were analysed. The graphs are the representative of at least three independent experiments.