Regulation of twitching motility in Pseudomonas aeruginosa by the las and rhl quorum-sensing systems and the distribution of the narG gene of Escherichia coli among denitrifying bacteria

Denitrification is a bacterial process in which nitrate is converted to nitrogen gas. The respiratory nitrate reductase (NR) enzyme catalyzes the first step in the pathway of bacterial denitrification. DNA probes constructed from the narG gene of Escherichia coli, which codes for the alpha-subunit of the enteric respiratory nitrate reductase (NR), were used in this study to determine the genetic relatedness of this enzyme among known nitrate reducing bacteria and environmental isolates. Of the 15 known nitrate reducing bacteria in our survey, nine representing six genera, were shown to share homology to the narG gene of E. coli under stringent hybridization conditions. Thirty-nine environmental strains, isolated from denitrifying enrichments were also tested for homology with narG. The enrichments were prepared from four different sites, including a pristine fen, a degraded wetland, an agricultural field, and stream sediment. Of the 39 isolates, 35 gave a positive hybridization signal in colony blots under stringent conditions. Southern blots of restricted genomic DNA from each of these 35 isolates were performed and a total of nine different banding patterns, or genotypes, were observed. None of the banding patterns were unique to a specific environmental site. Nitrate uptake studies were conducted to further characterize each of the genotypes. This survey suggests that the narG gene of E. coli is widely distributed among a variety of enteric nitrate-reducing and denitrifying bacteria.; Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl.{09}Both the las and rhl quorum-sensing systems are also required for type-4 pili dependent twitching motility and infection by the pili-specific phage, D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility were partially restored by exposure of the autoinducer deficient mutants to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pili-specific phage are believed to be dependent on the extension and retraction of polar type-4 pili. Western blot analysis of whole cell lysates and ELISAs of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to the wild type. It appears that rhll plays a crucial role in the export and assembly of surface type-4 pili twitching motility and phage infection by affecting the export and assembly of surface type-4 pili. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1, via the last gene, were defective in the maintenance of cellular monolayers and migrating packs of cells.