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 59-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 DqseB or DqseBC strains, but was restored to WT levels in the DqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the DqseBC 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 59 end of the ygiW ORF. The expression of ygiW-qseBC was increased by twofold in DihfA and DihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW-qseBC operon.

QseBC, a two‐component bacterial adrenergic receptor and global regulator of virulence in Enterobacteriaceae and Pasteurellaceae

“…() showed that the PmrB sensor phosphorylates the non‐cognate QseB response regulator, which autoregulates the qseBC operon (see below). The pmrAB genes are not present in the A. actinomycetemcomitans genome but the QseB binding site in the ygiW‐qseBC promoter is identical to the consensus PmrA binding sequence in the pmrAB operon of E. coli (Juarez‐Rodriguez et al ., ). Hence, it is possible that in the absence of PmrAB, the A. actinomycetemcomitans QseC sensor may have evolved to integrate the iron and catecholamine sensory functions of the PmrAB and QseBC TCSs of E. coli .…”

“…Similar to most other TCS, the activation of QseC leads to phosphorylation of the QseB response regulator and several studies have confirmed this either by directly demonstrating phosphate transfer (Clarke et al ., ) or by showing that site‐specific mutation of the conserved Asp 51 inhibits QseB function (Kostakioti et al ., ; Juarez‐Rodriguez et al ., ). In addition, using a mobility shift assay, Clarke & Sperandio (,b) demonstrated that phosphorylation of QseB is required for it to bind to QseB‐regulated promoters in vitro .…”

“…). In A. actinomycetemcomitans , the promoter that drives ygiW‐qseBC expression resides completely within a fragment of 138 bp upstream from the ygiW start codon (Juarez‐Rodriguez et al ., ) and two transcriptional start sites at nucleotides ‐15 and ‐53 have been mapped in this region (Juarez‐Rodriguez et al ., ). The distal initiation site at nucleotide ‐53 is the main transcriptional start site and although both sites are associated with putative ‐10 and ‐35 elements, only the upstream promoter is regulated by QseBC.…”

Functional characterization of Aggregatibacter actinomycetemcomitans QSEBC : a bacterial adrengeric receptor and global regulator of virulence.

“…CDM), and there is a basal level of activity under iron limiting conditions. In addition, our previous results and studies of QseBC in other species suggest that QseB may be activated by non-cognate sensor kinases [60,101]. This suggests that a minimal level of QseB activity is essential for A. actinomycetemcomitans and the absence of functional QseB triggers a stress response.…”

“…The histidine kinase sensor will then transfer the phosphate to the cognate response regulator causing it to change conformation and become activated. The activated response regulator can then bind to DNA to either promote or repress expression of certain genes, sometimes including its own operon [60,101]. There has also been evidence that some sensors can activate response regulators other than its own cognate response regulator [60].…”