Transcriptional regulation of the Aggregatibacter actinomycetemcomitans ygiW–qseBC operon by QseB and integration host factor proteins

Abstract

The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans. We previously showed that QseBC autoregulates the ygiW–qseBC operon. In this study, we characterized the promoter that drives ygiW–qseBC expression. Using lacZ transcriptional fusion constructs and 5′-rapid amplification of cDNA ends, we showed that ygiW–qseBC expression is driven by a promoter that initiates transcription 53 bases upstream of ygiW and identified putative cis-acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two trans-acting proteins were shown to interact with the ygiW–qseBC promoter. The QseB response regulator bound to probes containing the direct repeat sequence CTTAA-N6-CTTAA, where the CTTAA repeats flank the −35 element of the promoter. The ygiW–qseBC expression could not be detected in A. actinomycetemcomitans ΔqseB or ΔqseBC strains, but was restored to WT levels in the ΔqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the ΔqseBC strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using qseB pho−, encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of ygiW–qseBC expression. In addition, integration host factor (IHF) bound to two sites in the promoter region and an additional site near the 5′ end of the ygiW ORF. The expression of ygiW–qseBC was increased by twofold in ΔihfA and ΔihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW–qseBC operon.