Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

Abstract

The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.

Transcriptional Activation of the tad Type IVb Pilus Operon by PypB in Yersinia enterocolitica

“…The genes of the tad locus are organized in one large transcriptional unit, and our Northern blot experiments furthermore reveal a shorter flp-carrying transcript, indicating a transcriptional terminator 3Ј of the flp gene. This finding is similar to what was described previously for A. actinomycetemcomitans and, to a certain extent, H. ducreyi (20,27), with the exception that in Y. enterocolitica, the pypB gene is part of the operon but is missing in other species outside the genus Yersinia.…”

“…3B, we were able to detect a transcript with a size of approximately 400 nt. The size of this fragment corresponds well with a monocistronic flp transcript starting from P flp , indicating a transcriptional terminator downstream of the flp gene as described previously for A. actinomycetemcomitans (20). Indeed, we identified a putative stem-loop structure in the intergenic flp-tadV region that resembles classical terminators and has a calculated ⌬G of Ϫ17.9 kcal (Fig.…”

“…In A. actinomycetemcomitans a monocistronic transcript that corresponds to the flp gene has been identified (20). To analyze if a transcriptional terminator might exist downstream of flp, we performed a Northern blot analysis using an flp-specific probe.…”

Targeted Mutagenesis in the Study of the Tight Adherence (tad) Locus of Aggregatibacter actinomycetemcomitans

“…However, the function of the IR is currently unknown. Because the tad promoter is very strong (Kram et al, 2008), if the IR is a binding site for a protein that regulates transcription, it seems likely that the protein that binds IR would be a repressor that reduces tad transcription.…”

“…Our studies have indicated that tad locus transcription is regulated by a termination cascade to maintain the correct stoichiometry of the tad gene products (Kram et al, 2008). We isolated three transcriptional terminators (T).…”