1887

Abstract

The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen . We previously showed that QseBC autoregulates the operon. In this study, we characterized the promoter that drives expression. Using transcriptional fusion constructs and 5′-rapid amplification of cDNA ends, we showed that expression is driven by a promoter that initiates transcription 53 bases upstream of and identified putative -acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two -acting proteins were shown to interact with the 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 expression could not be detected in Δ or Δ strains, but was restored to WT levels in the Δ mutant when complemented by single copy chromosomal insertion of . Interestingly, partially complemented the Δ strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using , encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of 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 ORF. The expression of was increased by twofold in Δ and Δ strains of , suggesting that IHF is a negative regulator of the operon.

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2014-12-01
2019-11-17
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