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Candida albicans has been previously shown to stimulate the production of Pseudomonas aeruginosa phenazine toxins in dual-species colony biofilms. Here, we report that P. aeruginosa lasR mutants, which lack the master quorum sensing system regulator, regain the ability to produce quorum-sensing-regulated phenazines when cultured with C. albicans. Farnesol, a signalling molecule produced by C. albicans, was sufficient to stimulate phenazine production in [LasR.sup.-] laboratory strains and clinical isolates. P. aeruginosa [DELTA]lasR mutants are defective in production of the Pseudomonas quinolone signal (PQS) due to their inability to properly induce pqsH, which encodes the enzyme necessary for the last step in PQS biosynthesis. We show that expression of pqsH in a [DELTA]lasR strain was sufficient to restore PQS production, and that farnesol restored pqsH expression in [DELTA]lasR mutants. The farnesol-mediated increase in pqsH required RhlR, a transcriptional regulator downstream of LasR, and farnesol led to higher levels of N-butyryl-homoserine lactone, the small molecule activator of RhlR. Farnesol promotes the production of reactive oxygen species (ROS) in a variety of species. Because the antioxidant Nacetylcysteine suppressed farnesol-induced RhlR activity in [LasR.sup.-] strains, and hydrogen peroxide was sufficient to restore PQS production in las mutants, we propose that ROS are responsible for the activation of downstream portions of this quorum sensing pathway. LasR mutants frequently arise in the lungs of patients chronically infected with P. aeruginosa. The finding that C. albicans, farnesol or ROS stimulate virulence factor production in lasR strains provides new insight into the virulence potential of these strains. DOI 10.1099/mic.0.037911-0

Carnitine is a quaternary amine compound prevalent in animal tissues, and a potential carbon, nitrogen and energy source for pathogens during infection. Characterization of activities in Pseudomonas aeruginosa cell lysates has previously shown that carnitine is converted to 3-dehydrocarnitine (3-dhc) which is in turn metabolized to glycine betaine (GB), an intermediate metabolite in the catabolism of carnitine to glycine. However, the identities of the enzymes required for carnitine catabolism were not known. We used a genetic screen of the P. aeruginosa PAl4 transposon mutant library to identify genes required for growth on carnitine. We identified two genomic regions and their adjacent transcriptional regulators that are required for carnitine catabolism. The PA5388-PA5384 region contains the predicted P. aeruginosa carnitine dehydrogenase homologue along with other genes required for growth on carnitine. The second region identified, PA 1999-PA2000, encodes the [alpha] and [beta] subunits of a predicted 3-ketoacid CoA-transferase, an enzymic activity hypothesized to be involved in the first step of deacetylation of 3-dhc. Furthermore, we confirmed that an intact GB catabolic pathway is required for growth on carnitine. The PA5389 and PA1998 transcription factors are required for growth on carnitine. PA5389 is required for induction of the PA5388-PA5384 transcripts in response to carnitine, and the PA 1999-PA2000 transcripts are induced in a PA1998-dependent manner and induction appears to depend on a carnitine catabolite, possibly 3-dhc. These results provide important insight into elements required for carnitine catabolism in P. aeruginosa and probably in other bacteria.

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PAl4 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-[sup.13]C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and [sup.13]C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAOI and PAl4 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.

Research has been conducted on strain K1 which is the member of the delta proteobacteria. The isolation results of this bacterium from anaerobic soil enrichments are discussed.

The study of purified recombinant yeast protein from Saccharomyces cerevisiae that is a Fe(II)-dependent sulfonate /alpha-ketoglutarate dioxygenase is presented. Results show that disruption of the encoding enzyme gene affects negatively the use of sulfur sources by S. cerevisiae.