title: ygiW and qseBC are co-expressed in Aggregatibacter actinomycetemcomitans and regulate biofilm growth.
abstract: The quorum-sensing Escherichia coli regulators B and C (QseBC) two-component system were previously shown to regulate biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans and to be essential for virulence. In this study, we use RT-PCR to show that an open reading frame, ygiW, residing upstream of qseBC and encoding a hypothetical protein is co-expressed with qseBC. In addition, using a series of lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA Ends (RACE), the promoter that drives expression of the ygiW-qseBC operon and the transcriptional start site was mapped to the 372 bp intergenic region upstream from ygiW. No internal promoters drive qseBC expression independently from ygiW. However, qseBC expression is attenuated by approximately ninefold by a putative attenuator stem-loop (ΔG = -77.0 KJ/mol) that resides in the 137 bp intergenic region between ygiW and qseB. The QseB response regulator activates expression of the ygiW-qseBC operon and transcription from the ygiW promoter is drastically reduced in ΔqseB and ΔqseBC mutants of A. actinomycetemcomitans. In addition, transcriptional activity of the ygiW promoter is significantly reduced in a mutant expressing an in-frame deletion of qseC that lacks the sensor domain of QseC, suggesting that a periplasmic signal is required for QseB activation. Finally, a non-polar in-frame deletion in ygiW had little effect on biofilm depth but caused a significant increase in surface coverage relative to wild-type. Complementation of the mutant with a plasmid-borne copy of ygiW reduced surface coverage back to wild-type levels. Interestingly, deletion of the sensor domain of QseC or of the entire qseC open reading frame resulted in significant reductions in biofilm depth, biomass and surface coverage, indicating that the sensor domain is essential for optimal biofilm formation by A. actinomycetemcomitans. Thus, although ygiW and qseBC are co-expressed, they regulate biofilm growth by distinct mechanisms.
authors.affiliation: Research Group in Oral Health and Systemic Disease, University of Louisville School of Dentistry, 501 S. Preston Street, Louisville, KY 40202, USA.
authors.forename: María Dolores
authors.initials: MD
authors.lastname: Juárez-Rodríguez
authors.affiliation: Research Group in Oral Health and Systemic Disease, University of Louisville School of Dentistry, 501 S. Preston Street, Louisville, KY 40202, USA.
authors.forename: Ascención
authors.initials: A
authors.lastname: Torres-Escobar
authors.affiliation: Research Group in Oral Health and Systemic Disease, University of Louisville School of Dentistry, 501 S. Preston Street, Louisville, KY 40202, USA.
authors.forename: Donald R
authors.initials: DR
authors.lastname: Demuth
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D009876:Operon; D015661:Pasteurellaceae / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D024363:Transcription Initiation Site
chemical_list: D001426:Bacterial Proteins