Sequence Analysis of the Mobile Genome Island pKLC102 of Pseudomonas aeruginosa C

ABSTRACT The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNALys genes, pKLC102 is incorporated into the tRNALys gene only close to the pilA locus. Targeting of the other tRNALys copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNAGly-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

ABSTRACT Null cyclic β-1,2-glucan synthetase mutants (cgsmutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuatedB. abortus S19 strain. Although the cgsmutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected withB. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by thecgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

Molecular Cloning and Characterization of cgs, theBrucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB andAgrobacterium tumefaciens chvB Mutants

ABSTRACT The animal pathogen Brucella abortus contains a gene,cgs, that complemented a Rhizobium melilotinodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved inRhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor inBrucella infection.

Action de la désertomycine sur la synthèse des polymères pariétaux du Saccharomyces uvarum

The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of β(1,3)-glucans linked among themselves by β(1,6) bonds. Mannan formation was not inhibited; such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action.Key words: desertomycin, wall, yeast.