Transcriptional analysis and mutagenesis of the htp fimbrial gene cluster from Pseudomonas aeruginosa PAO1

Abstract

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, is one of the most and best studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. P. aeruginosa forms biofilms through a series of interactions between the cells and adherence to surfaces, which is mediated by surface appendages such as flagella and type IV pili. A gene cluster, designated htpABCDEFGI, which appears to encode protein products with homology to those encoded by recently described novel pilus biogenesis and assembly systems, has been identified in P. aeruginosa PAO1. Since the pili produced by these systems, designated Flp, are associated with the ability of the bacteria to bind non-specifically to inert surfaces, the aims of this study were to characterize the transcriptional organization of the putative P. aeruginosa PAO1 htp gene cluster and to determine the functional importance of the htp gene cluster in the ability of P. Aeruginosa PAO1 to adhere to surfaces. In silico evidence has suggested that the pilin subunit gene flp is not part of the P. Aeruginosa htp gene cluster thought to encode proteins involved in the synthesis, assembly and export of these pili. To determine the transcriptional organization of this gene cluster, total RNA from P. aeruginosa PAO1 was analyzed by reverse transcriptase-polymerase chain reaction (RTPCR). Primers designed to amplify regions spanning gene junctions yielded amplicons at each individual gene junction from htpA to htpI, as well as an amplicon for flp. Moreover, corresponding sigma 70 (σ70) consensus sequences were identified in the intergenic region between the htpA and flp genes and promoter function of the flp and htpA upstream region was subsequently confirmed using lacZ reporter gene constructs transformed into P.aeruginosa PAO1. The results therefore indicated that the htp gene cluster is an operon transcribed as a polycistronic message, whilst the flp gene is transcribed independently as a monocistronic message. To determine the functional importance of thehtp gene cluster in P. aeruginosa PAO1, the htpD gene, encoding a putative NTPase, was inactivated by in vivo homologous recombination with an appropriately constructed allelic exchange vector to generate the isogenic mutant strain PAOHtpD. Comparative analysis of the wild-type P. aeruginosa PAO1 and mutant PAOHtpD strain revealed that the mutant strain was impaired in its ability to attach to a glass wool substratum and also in its ability to grow as a biofilm. Since the mutant PAOHtpD strain was not growth-impaired, these results indicate that the htp gene cluster plays a role in P. aeruginosa PAO1 biofilm development under the culturing conditions used in this study. Thus, it can be proposed that the flp and htp gene cluster of P. aeruginosa PAO1 may play a role in its ability to successfully colonize abiotic surfaces.