Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality. Although the intrinsic virulence of P. aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease. In this study, the virulence of 35 P. aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia. The virulence of individual isolates differed significantly from one to another in each of these assays. Increased virulence was associated with the secretion of ExoU, a toxin transported by the P. aeruginosa type III secretion system. Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence. Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P. aeruginosa.

Most known virulence determinants of Pseudomonas aeruginosa are remarkably conserved in this bacterium's core genome, yet individual strains differ significantly in virulence. One explanation for this discrepancy is that pathogenicity islands, regions of DNA found in some strains but not in others, contribute to the overall virulence of P. aeruginosa. Here we employed a strategy in which the virulence of a panel of P. aeruginosa isolates was tested in mouse and plant models of disease, and a highly virulent isolate, PSE9, was chosen for comparison by subtractive hybridization to a less virulent strain, PAO1. The resulting subtractive hybridization sequences were used as tags to identify genomic islands found in PSE9 but absent in PAO1. One 99-kb island, designated P. aeruginosa genomic island 5 (PAGI-5), was a hybrid of the known P. aeruginosa island PAPI-1 and novel sequences. Whereas the PAPI-1-like sequences were found in most tested isolates, the novel sequences were found only in the most virulent isolates. Deletional analysis confirmed that some of these novel sequences contributed to the highly virulent phenotype of PSE9. These results indicate that targeting highly virulent strains of P. aeruginosa may be a useful strategy for identifying pathogenicity islands and novel virulence determinants.

Key to Pseudomonas aeruginosa’s ability to thrive in a diversity of niches is the presence of numerous genomic islands that confer adaptive traits upon individual strains. We reasoned that P. aeruginosa strains capable of surviving in the harsh environments of multiple hosts would therefore represent rich sources of genomic islands. To this end, we identified a strain, PSE9, that was highly virulent in both animals and plants. Subtractive hybridization was used to compare the genome of PSE9 to the less virulent strain PAO1. Nine genomic islands were identified in PSE9 that were absent in PAO1; seven of these had not been previously described. One these seven islands, designated PAGI-5, has already been shown to carry numerous interesting ORFs, including several required for virulence in mammals. Here we describe the remaining six genomic islands, PAGI-6, -7, -8, -9, -10, and -11, which include a prophage element and two Rhs elements.

Abstract:The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfurcontaining molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. K m s for SSO 3 2-and CN -were 2.7 mM and 32 mM, respectively, and k cat was 64 s-1 . The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.