Differential Transcriptional Regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK Operons by Integration Host Factor Protein

We previously showed that the Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons are regulated by LsrR and cyclic AMP receptor protein (CRP) and that proper regulation of the lsr locus is required for optimal biofilm growth by A. actinomycetemcomitans. Here, we identified sequences that reside immediately upstream from both the lsrA and lsrR start codons that closely resemble the consensus recognition sequence of Escherichia coli integration host factor (IHF) protein. A. actinomycetemcomitans IHF alpha and IHF beta were expressed and purified as hexahistidine fusion proteins, and using electrophoretic mobility shift assays (EMSAs), the IHF alpha-IHF beta protein complex was shown to bind to probes containing the putative IHF recognition sequences. In addition, single-copy chromosomal insertions of lsrR promoter-lacZ and lsrA promoter-lacZ transcriptional fusions in wild-type A. actinomycetemcomitans and Delta ihfA and Delta ihfB mutant strains showed that IHF differentially regulates the lsr locus and functions as a negative regulator of lsrRK and a positive regulator of lsrACDBFG. Deletion of ihfA or ihfB also reduced biofilm formation and altered biofilm architecture relative to the wild-type strain, and these phenotypes were partially complemented by a plasmid-borne copy of ihfA or ihfB. Finally, using 5'rapid amplification of cDNA ends (RACE), two transcriptional start sites (TSSs) and two putative promoters were identified for lsrRK and three TSSs and putative promoters were identified for lsrACDBFG. The function of the two lsrRK promoters and the positive regulatory role of IHF in regulating lsrACDBFG expression were confirmed with a series of lacZ transcriptional fusion constructs. Together, our results highlight the complex transcriptional regulation of the lsrACDBFG and lsrRK operons and suggest that multiple promoters and the architecture of the lsrACDBFG-lsrRK intergenic region may control the expression of these operons.