Expression of transcription activator ComK of Bacillus subtilis in the heterologous host Lactococcus lactis leads to a genome-wide repression pattern: a case study of horizontal gene transfer

Abstract

Horizontal gene transfer (HGT) is generally considered a possible mechanism by which bacteria acquire new genetic properties. Especially when pathogenicity genes are involved, HGT might have important consequences for humans. In this report we describe a case study of HGT in which a transcriptional activator, ComK of Bacillus subtilis, was introduced into a heterologous host species, Lactococcus lactis. ComK is the central regulator of competence development, activating transcription by binding to a ComK-binding site, a so-called K-box. Interestingly, L. lactis does not contain a comK gene, but it does contain almost 400 putatively functional K-boxes, as well as homologues of a number of competence genes. In this study, the effect of HGT of B. subtilis comK into L. lactis was investigated by determining the effects on the transcription profile using DNA microarray analyses. Production of wild-type ComK was shown to stimulate the transcription of 89 genes and decrease the expression of 114 genes. Notably, potential direct effects (i.e., genes preceded by a K-box) were found mainly among repressed genes, suggesting that ComK functions as a repressor in L. lactis. This is a remarkable difference between L. lactis and B. subtilis, in which ComK almost exclusively activates transcription. Additional DNA microarray analyses with a transcription activation-deficient but DNA-binding ComK variant, ComKDeltaC25, demonstrated that there were similar effects on gene regulation with this variant and with wild-type ComK, confirming that the direct effects of ComK result from interference with normal transcription through binding to available K-boxes. This study demonstrates that horizontal gene transfer can have dramatic effects that are very different than those that are expected on basis of the original functionality of a gene.

FIG. 1.

Overview of a K-box. ComK-regulated genes are characterized by the presence of a K-box upstream of their promoter (arrow). Each K-box consists of two AT-boxes, which are separated by a flexible spacing. This results in positioning of both AT-boxes on the same side of the DNA helix with an interval of two, three, or four helical turns, depending on the size of the spacing, between the start of the two AT-boxes.

FIG. 2.

Growth and ComK production of the strains tested. (A) Independent cultures of L. lactis NZ9000(pNZ8048), L. lactis NZ9000(pNZ-His₆-ComK), and L. lactis NZ9000(pNZ-His₆-ComKΔC25) were grown as biological replicates. The moment of induction with supernatant of nisin-producing strain L. lactis NZ9700 at a 1:1,000 dilution is indicated by an arrow. Two growth rates were distinguished after induction of ComK production. For the organisms in the upper box the growth rates of the cultures were normal; for the organisms in the lower box the growth rates of the cultures were decreased. Samples for RNA isolation were harvested after 2 h of induction. OD₆₀₀, optical density at 600 nm. (B) Western blotting was performed to detect the His-tagged ComK proteins using an anti-His antibody. Samples of three cultures per strain were loaded on a gel. Lanes NZ, L. lactis NZ9000(pNZ8048); lanes wt, L. lactis NZ9000(pNZ-His₆-ComK); lanes 25, L. lactis NZ9000(pNZ-His₆-ComKΔC25).

FIG. 3.

ComK negatively affects transcription of ywcC and tig. β-Galactosidase (Betagal) assays were performed to verify the negative effects of ComK production on transcription of ywcC and tig, using a P_ywcC-lacZ fusion (A) and a P_tig-lacZ fusion (B) as reporters, respectively. As a control for significant regulation, the mild effect of ComK on P_ctrA-lacZ, which just missed the arbitrary cutoff level, was determined (C). At the end of the exponential growth phase, expression of wtComK or ComKΔC25 was induced. β-Galactosidase activity was determined at 30-min intervals until 2 h after induction. For each strain, four independent cultures were grown, and the average activity (in Miller units) is shown along with the standard deviation for the average. Symbols: ⧫, no ComK (empty plasmid pNZ8048); ▴, wtComK; ▪, ComKΔC25.