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.

Fig. 1.

Transcriptional analysis of the ygiW-qseBC locus. (a) Schematic representation of ygiW-qseBC genes on the A. actinomycetemcomitans 652 chromosome. Boxes 1, 3, 5, 6, 7 and 8 and thick lines 2 and 4 represent the products obtained from RT-PCR using the primers described in Methods. (b) Ethidium bromide-stained agarose gel showing the RT-PCR products represented by boxes 1, 3 and 5 and thick lines 2 and 4 in (a) obtained using cDNA as the template in the presence (+) or absence (−) of RT. Additional control reactions were conducted using genomic DNA as the template (d) or using primers specific for the 16S rRNA gene. Product sizes are as follows: 1 (324 bp), 2 (373 bp), 3 (428 bp), 4 (423 bp), 5 (550 bp) and 16S RNA (302 bp). (c) Ethidium bromide-stained agarose gel showing the RT-PCR products represented by boxes 6, 7 and 8 in (a) obtained using cDNA as the template in the presence (+) or absence (−) of RT. Additional control reactions were conducted using genomic DNA as the template (d) or using primers specific for the 16S rRNA gene. Product sizes are as follows: 6 (430 bp), 7 (1101 bp), 8 (1845 bp) and 16S RNA (302 bp).

Fig. 2.

Transcriptional reporter plasmids. (a) Schematic representation of the fusions of different DNA fragments containing the ygiW-qseBC operon to the promoterless lacZ gene. (b) Growth of all strains was monitored by measuring OD₆₀₀ at the indicated time intervals. (c) β-Gal activity for each construct is expressed as Miller units and was measured in A. actinomycetemcomitans transformed individually with each plasmid and grown in BHI medium as described in Methods. The values are means±sd of results from three independent experiments.

Fig. 3.

β-gal activity of wild-type A. actinomycetemcomitans 652 and isogenic ifΔygiW, ΔqseB, ΔqseBC and qseCΔpr mutants harbouring pDJR29. (a) Graphic representation of the transcriptional reporter plasmids pDJR29. (b) Growth of each strain was monitored by measuring OD₆₀₀ at the indicated time intervals. (c) β-gal activity for each construct is expressed as Miller units and was measured in A. actinomycetemcomitans transformed individually with each plasmid and grown in BHI medium as described in Methods. The values are means±sd of results from three independent experiments.

Fig. 4.

Three-dimensional renditions of biofilms formed by wild-type A. actinomycetemcomitans 652, an isogenic mutant harbouring an in-frame deletion in ygiW (ifΔygiW), ifΔygiW complemented [ifΔygiW(pDJR28)], by deletion mutants lacking qseC (ΔqseC) or the sequence encoding the periplasmic sensor domain of qseC (qseCΔpr). Biofilms were cultured in open-flow cells as described in Methods and analysed using confocal laser scanning microscopy. Image stacks were assembled and analysed with Volocity image analysis software. The yellow scale bars in the x–y, x–z and y–z sections represent 50 µm.

Fig. 5.

Genomic organization of the ygiW-qseBC locus in A. actinomycetemcomitans and related loci in Pasteurellaceae species A. pleuropneumoniae, H. influenzae and P. multocida and Enterobacteriaceae species E. coli and S. Typhimurium. Gene transcription orientation is indicated by open arrows.