Regulation of ykrL ( htpX ) by Rok and YkrK, a Novel Type of Regulator in Bacillus subtilis

Abstract

Expression of ykrL of Bacillus subtilis , encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL : Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a P ykrL - gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated P ykrL (membrane protein overproduction, dissipation of the membrane potential, and salt and phenol stress) point to the involvement of YkrL in membrane protein quality control.

Reexamining Transcriptional Regulation of the Bacillus subtilis htpX Gene and the ykrK Gene, Encoding a Novel Type of Transcriptional Regulator, and Redefining the YkrK Operator

“…6B of reference 17). Since one of the three proposed YkrK binding sites happened to overlap the Ϫ35 box of the ykrK A promoter, they predicted that ykrK expression might be subject to autoregulation (17). However, we found in this study that disruption of the ykrK gene had no effect on expression of ykrK-bgaB ( Fig.…”

“…A recent report by Marciniak et al showed that expression of the B. subtilis htpX gene was under dual negative control by the Rok repressor and YkrK, a novel type of transcriptional regulator (17). However, we have found that some of the propositions and predictions in the report by Marciniak et al (17) were controversial. In the present study, we have identified the authentic promoters of htpX and ykrK, redefined the YkrK operator, and recharacterized transcriptional regulation of htpX and ykrK.…”

“…HtpX was recently proposed to be one among a group of proteins that was associated with the MreB cytoskeleton of B. subtilis (15). A recent report by Marciniak et al showed that expression of the B. subtilis htpX gene was under dual negative control by the Rok repressor and YkrK, a novel type of transcriptional regulator (17). However, we have found that some of the propositions and predictions in the report by Marciniak et al (17) were controversial.…”

“…Marciniak et al claimed to identify the sequence 5=-TGAWCTTA-3= (where W represents A or T) as the YkrK binding motif (17). This motif is present within the ykrK-htpX intergenic region at three locations: downstream of the predicted A promoter of htpX, overlapping with the Ϫ35 box of the predicted A promoter of ykrK, and, with more deviation from the consensus, between these two predicted promoters (see Fig.…”

“…S1 in the supplemental material). Marciniak et al recently reported that environmental osmotic stress and overproduction of membrane proteins could strongly induce expression of the htpX-gfp fusion in B. subtilis (17). When heat induction of htpX was investigated by Marciniak et al, gfp was also used as the reporter gene and the green fluorescent protein (GFP) fluorescence of the htpX-gfp fusion was monitored by flow cytometry (17).…”