Transcriptional analysis of the 5′ terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans

摘要

Fresh isolates of the oral bacterial pathogen Actinobacillus actinomycetemcomitans exhibit a fimbriated, rough colony phenotype. Evidence suggests that the fimbrial subunit gene flp is part of a cluster of 14 genes (flp to tadG) thought to encode proteins involved in the synthesis, assembly and export of these fimbriae. To determine the transcriptional organization of the 5′ terminus of this gene cluster, total RNA from rough and smooth phenotype variants of A. actinomycetemcomitans strain 283 were analysed by RT-PCR. Primers designed to amplify regions spanning gene junctions or multiple genes yielded amplicons at each individual gene junction from flp to tadD for both the rough and smooth variants. Semi-quantitative RT-PCR of the rcpA to tadZ amplicon revealed that significantly more mRNA was transcribed from the rough than the smooth variant. Longer amplicons encompassing flp to tadZ (3·9?kb) and tadA to tadD (2·1?kb) were also detected, but only from the rough variant. Rapid amplification of cDNA ends (RACE) was used to identify the 5′ end of the mRNA containing flp. Antisense primers located within rcpC, orfB and flp-2 enabled amplification of a RACE product that was subsequently isolated and subcloned into pGEM-T. DNA sequencing indicated that the 5′ end of the mRNA was located at a G or T nucleotide ?102 to ?101 nt upstream of flp. Corresponding σ70 consensus sequences were located at ?10 (TATAAT) and ?35 (TTGCAT) relative to the transcription start site. These data confirm that the flp gene cluster is an operon transcribed as a polycistronic message commencing from a G or T nucleotide located in the intergenic region upstream of flp. Promoter function of the flp upstream region was confirmed using a lacZ reporter gene construct transformed into Escherichia coli. RT-PCR analysis further suggests that although transcription does occur in both the rough and smooth variants, full-length transcripts are rapidly degraded or are significantly downregulated in the smooth variant.