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Journal of Bacteriology, June 2008, p. 3859-3868, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.00128-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Karin E. Kram, Galadriel A. Hovel-Miner, Mladen Tomich,{ddagger} and David H. Figurski*

Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received 24 January 2008/ Accepted 17 March 2008

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.

* Corresponding author. Mailing address: Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032. Phone: (212) 305-3425. Fax: (212) 305-1468. E-mail: dhf2{at}columbia.edu

{triangledown} Published ahead of print on 28 March 2008.

{ddagger} Present address: Merck & Co., Inc., 126 E. Lincoln Ave., RY80Y-210, Rahway, NJ 07065.

Journal of Bacteriology, June 2008, p. 3859-3868, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.00128-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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