Functional analysis of the ComK protein of Bacillus coagulans

Abstract

The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis- and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNA-uptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B.subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis.

Figure 1. Survey on the presence of competence genes and the alignment of ComK protein sequences from various Bacillus strains.

(A) Results of BLAST searches were visualized with Genesis 1.6 software: white is absent (with E-value of E–0), dark blue is present (E-valueB. subtilis protein sequences against translated protein database of a given genome. Protein names are indicated on the right. Bsu, B. subtilis; Bli, B. licheniformis, Bam, B. amyloliquefaciens, Bce, B. cereus Bco, B. coagulans. Question marks denote small ORFs where identification is uncertain using the available bioinformatic tools that can miss homologues. (B) Multiple alignment of ComK homologues. Black background represents conserved amino acids and grey background represents similar amino acids. Alignment was performed using ClustalW , and presented using Boxshade 3.21 program. The N- and C-terminal deletions analyzed by Susanna et al are marked (ΔN9 and ΔC25, respectively). Boxed amino acid residues indicate the residues involved in interaction with MecA . Alpha-helices and beta-sheets of B. subtilis ComK protein are indicated with rectangles and arrows under the alignment, respectively.

Figure 2. Single cell analysis of PcomGA_Bsu-gfp in the presence of comK_Bco in B. subtilis.

Samples were taken 2 hours after the transition point between the exponential and stationary growth phase. (A) Flow cytometric analyses of comGA_Bsu expression in wild type (red line), ΔcomK mutant (blue line), ΔcomK strain with the comK_Bco containing plasmid pATK4 (green line), and ΔcomK strain with the empty plasmid (black line). The relative 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 analyzed. The graph is the representative of at least three independent experiments. (B) Light-microscopic phase-contrast picture (top row) and fluorescence image (bottom row) of cells. Strains used from left to right are wild type, ΔcomK mutant, ΔcomK with pATK4, and ΔcomK with pEM53, respectively.

Figure 3. Expression from the comK_Bco and comGA_Bco promoters in B. subtilis.

Single cell analysis of B. subtilis strains containing plasmids with promoter-less gfp (A), with PcomK_Bco-gfp fusion (B), and the PcomGA_Bco-gfp reporter (C). Samples were taken at the indicated time points given in hours relative to the transition point between the exponential and stationary growth phase (T0). The single cell expression pattern in the wild type strain is indicated with light grey, the ΔcomK mutant is designated with dark grey, and the ΔcomK strain with the comK_Bco containing plasmid pATK4 is shown in white. The relative 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 analyzed. The graph is the representative of at least three independent experiments.

Figure 4. Purification of ComK_Bco protein and its DNA binding ability.

(A) SDS-PAGE analysis of overexpression and purification of ComK_Bco protein from E. coli. Non-induced and 0.1 mmol l⁻¹ IPTG induced cell extracts are loaded on the first and second lanes, respectively, while purified MBP-ComK_Bco is run in the third lane. (B) Immunoblot analysis of the purified MBP-ComK_Bco protein using α-ComK_Bsu antibodies. Marker sizes are indicated on the left of the blot. (C–G) Gel retardation assay with the purified MBP-ComK_Bco protein on the comK_Bco (C), comGA_Bco (D), comC_Bco (E), and comFA_Bco (F) promoter fragments. Lane 1 contains no protein, lanes 2–5 contain 2.2 µmol l⁻¹ to 275 nmol l⁻¹ purified MBP-ComK_Bco at 2 fold dilutions, respectively. The promoter fragment of rpsD_Bco is used as negative control with no apparent binding by MBP-ComK_Bco (G). Free probes (P), shifted bands (S), and signal specific to the wells of the gel (W) are indicated.

Figure 5. Gel retardation assay with ComK_Bsu and ComK_Bco.

The binding of MBP-ComK_Bsu (A–D) and MBP-ComK_Bco (E–F) was assayed at a doubling concentration of the proteins from 125 nmol l⁻¹ to 1 µmol l⁻¹ (lanes 2 to 5, respectively). Lane 1 of each picture lacks any added protein. DNA binding was detected on promoters of comK_Bco (A), comGA_Bco (B), comK_Bsu (C and E), and comGA_Bsu (D and F) genes. Free probes (P), shifted bands (S), and signal specific to the wells of the gel (W) are indicated.