THE GENES INVOLVED IN OSMOPROTECTION AND DIMETHYLGLYCINE SYNTHESIS SEEMS TO BE NOT GENETICALLY LINKED IN PSEUDOMONAS AERUGINOSA

ANGELA T. LISA; JAVIER FERNANDO MARISCOTTI; ANA LUZ SERRA; CARLOS E. DOMENECH; GLORIA I. LUCCHESI

Bruselas

Congreso; Pseudomonas 2001; 2001

Federation of European Microbiological Societies

In Pseudomonas aeruginosa choline or betaine employed as the sole carbon and nitrogen source induced a phospholipase C, acid phosphatase and cholinesterase. Through their coordinate action, the bacteria may break down various compounds or cells membranes; hence it is possible to include choline among the factors promoting the pathogenesis of this microorganism. As a step to elucidate the metabolism of choline and its relationship with the above enzymes, we selected a Tn5-¬751 mutant, strain ALS-96, which failed to metabolize choline, betaine or carnitine but grew on dimethylglycine as sole carbon and nitrogen sources. This mutant was capable of incorporate choline, convert it to betaine and use it as an osmoprotectant under osmotic stress conditions. ALS-96 was found to be deficient in betaine homocysteine methyl transferase (BHMT) activity. Concomitantly, it was capable to grow on dimethylglycine as sole carbon and nitrogen sources, and to produce cholinesterase, acid phosphatase and phospholipase C activities at levels similar as those detected in the wild type cells. The physical presence and a single insertion of Tn5-751 in the chromosome of ALS-96 were demonstrated by Southern hybridization experiments. By exploiting SalI restriction sites within Tn5-¬751 that separate the two resistance gene markers, the IS50L arm of the transposon (with neighboring chromosomal DNA) was cloned into pBlueScript II SK and used to transformed E. coli DH5α cells. Restriction analysis showed that a kanamycin resistance clone contained a plasmid, pJM1, with an insert harboring a Tn5-751 insertion near one of its ends. The nucleotide sequence of the 814 pb DNA insert showed an homology of 98% with the section 293 of 529 of the complete genome sequence of P. aeruginosa PAO1 which encodes a hypothetical and conserved protein named PA3081. This section is far away from the genes of operon bet (Pseudomonas Genome Project [http//www.pseudomonas.com]). Therefore, it is suggested that genes involved in osmoprotection and dimethylglycine synthesis seems to be not genetically linked in Pseudomonas aeruginosa.