CHARACTERIZATION OF THE 33A9 LOCUS OFPSEUDOMONAS AERUGINOSA: ITS ROLE IN BACTERIAL VIRULENCE
E DRENKARD1, BG RICHTER1, FM AUSUBEL1, RM TOMPKINS2 and LG RAHME2
1Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA; 2Department of Medicine, Harvard Medical School and Department of Surgery and Shriners Burns Institute, Massachusetts General Hospital, Boston 02114, USA
Background and objectives
Based on the identification of some clinical strains of P. aeruginosa that cause disease in both plants and animals, Rahme et al.  developed a novel in vivo pathogenesis system that utilizes a human clinical isolate of P. aeruginosa (strain UCBPP-PA14) that is infectious in both a mouse full skin thickness burn model and in a leaf infiltration model in a variety of plants. By screening 2500 TnphoA transposon-generated mutants of P. aeruginosa strain UCBPP-PA14 using a plant leaf infiltration assay, a group of seven previously unknown P. aeruginosa pathogenicity-related genes was identified . One of the mutants identified as less pathogenic in the plant system, the P. aeruginosa mutant 33A9, was chosen for further characterization because it also exhibited no morbidity or mortality in a mouse burn model, representing a new and important P. aeruginosa virulence factor.
Results and conclusions.
We describe the characterization of the new pathogenesis-related gene 33A9 identified by mutation in a screen for non-virulent P. aeruginosa mutants using a plant leaf assay. Mutant 33A9 elicited reduced symptoms and growth on Arabidopsis thaliana ecotype Lagostera (LI-0), and no mortality in a mouse burn model. Complete DNA sequence analysis of the region that contains the mutation showed no homology to other known virulence-related genes. Protease, elastase and phospholipase activity assays, as well as assays that measured the ability of the mutant to produce the secondary metabolite pyocyanin, did not show any differences compared with the wild-type strain UCBPP-PA14, indicating that the mutation was not affecting these previously identified pathogenicity factors. The 33A9 mutant was observed, however, to overproduce biofilm, a structure composed of bacterial colonies enclosed in a exopolysaccharide matrix that protects the bacteria from adverse environmental conditions and from biological and biochemical antibacterial agents. The data obtained from the biofilm assays also indicated that biofilm production was growth-phase regulated and that the 33A9 gene was playing a role in that regulation. Expression assays further confirmed that the expression of 33A9 was growth-phase dependent, and that 33A9 was involved in the regulation of two genes encoded in two open reading frames located upstream and downstream of 33A9. Our results suggest that 33A9 could play a role as a growth-phase regulator of multiple genes involved in bacterial pathogenesis. In addition, data obtained from the sequence of the region that surrounds 33A9 suggested that the 33A9 locus could be part of a new pathogenicity island in P. aeruginosa.
1. Rahme LG et al., 1995. Science 268, 1899-1902.
2. Rahme LG, Tan MW, Le L et al., 1997. Proceedings of the National Academy of Sciences, USA 94, 13245-13250.