The availability of these genes and the development of a method for gene disruption and replacement in S. nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.

Pseudomonas aeruginosa is a gram-negative rod that is ubiquitous in nature. P. aeruginosa is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts. In cystic fibrosis patients, P. aeruginosa is the leading cause of death. In this study, the evolutionary genetic relationships among 17 P. aeruginosa isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone groEL. The P. aeruginosa isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere. Phylogenetically, clinical and environmental isolates clustered together with one another on the mdh gene tree. At the groEL locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments. Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase. Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions. Among the clinical isolates examined, the 15 virulence regions were variably present. The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions. Of the four genomic islands (the flagellum island and PAGI-1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI-1, -2, and -3 were absent from all isolates tested. Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen.The remarkable ability of Pseudomonas aeruginosa to adapt and thrive in a wide variety of environments is due in part to its extensive genetic versatility, which contributes significantly to its potential as a pathogen. P. aeruginosa is an opportunistic pathogen that is a common cause of hospital-acquired infections, particularly infecting patients with predisposing factors, such as burn victims, immunocompromised hosts, or those with metabolic disorders. In cystic fibrosis (CF) patients, P. aeruginosa is believed to be a major contributory factor to chronic lung infections and is thought to form biofilms and to adhere to human mucin in the lower respiratory tract (46, 53). Once P. aeruginosa colonizes the CF patient's lung, it cannot be eradicated even by the most aggressive antibiotic therapy. It is likely that as part of colonization, invasion, and survival in the human host, P. aeruginosa utilizes a unique range of genes; however, the precise repertoire of virulence genes required is unclear.Several studies have examined the genetic diversity of P. aeruginosa isolates from CF patients (9,13,30,40,44,49). In these studies, it was deter...

The Vibrio seventh pandemic island-II is a 26?9 kb genomic island present in Vibrio cholerae El Tor and O139 serogroup isolates that shows homology to a 43?4 kb genomic island in V. vulnificus Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera. In this study the 7?5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V. cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26?9 kb genomic island (GEI) encompassing VC0490-VC0516. The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene. Interestingly, V. cholerae ORFs VC0493-VC0498, VC0504-VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510-VV0516, VV0518-VV0525 and VV0560, which also encodes an integrase, respectively. Some ORFs showed amino acid identities greater than 90 % between the two species in these regions. In V. vulnificus strain YJ016, a 43?4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509-VV0560 was identified and named V. vulnificus island-I (VVI-I). The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes. There was synteny and homology between the 59 region of V. cholerae VSP-II and the 59 region of V. vulnificus VVI-I; however, VVI-I contained an additional 31?5 kb of DNA between VV0526 and VV0560 in strain YJ016. A second V. vulnificus strain, CMCP6, did not contain the 43?4 kb VVI-I; in this strain two ORFs were found between the 59 and 39 flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I. INTRODUCTIONThe genus Vibrio, which belongs to the Gram-negative c-Proteobacteria, is ubiquitous in marine and estuarine environments. There are 71 recognized species within the genus Vibrio, of which 12 species are pathogenic to humans, in the current List of Bacterial Names with Standing in Nomenclature (Euzéby, 1997;Farmer et al., 2003). Vibrio cholerae is the most important human pathogen belonging to the genus since it is the aetiological agent of the deadly diarrhoeal disease cholera. Cholera is endemic in many areas of the world, particularly those areas where clean drinking water is absent. V. cholerae pathogenic isolates contain two main virulence factors, cholera toxin (CT) and toxin co-regulated pilus (TCP), both of which help facilitate cholera spread and pathogenesis (Sears & Kaper, 1996;Taylor et al., 1987). CT, the main cause of the explosive diarrhoeal disease, is encoded on a filamentous bacteriophage CTXw (Waldor & Mekalanos, 1996). The receptor for CTXw on the V. cholerae cell, TCP, which is an essential intestinal colonization factor, is also encoded on a mobile genetic element named the Vibrio pathogenicity island (VPI) (Waldor & Mekalanos, 1996;Kovach et al., 1996;Karaolis et al., 1998;Taylor et al., 1987). To dat...

Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces.

Amphotericin Biosynthesis in Streptomyces nodosus: Deductions from Analysis of Polyketide Synthase and Late Genes Figure 3 in this article (Chem. Biol. 8, 713-723) contains errors. In the drawings of the acyl intermediates, the methyl stereochemistry and the alcohol stereochemistry introduced by extension module 2 are incorrect. The alcohol stereochemistry introduced by extension module 12 is also incorrectly drawn. In each case, the stereochemistry is opposite to what would be inferred from the absolute structure of amphotericin B. The corrected figure is printed below.