Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate

Abstract

Mucoid Pseudomonas aeruginosa strain FRD, a sputum isolate from a cystic fibrosis patient, was used to develop a genetic system. The mucoid appearance is due to the biosynthesis of the exopolysaccharide alginate and is a potential virulence factor of the organism. The sex factor plasmid FP2 was used for uninterrupted genetic exchange to investigate the nature of spontaneous mutations which produce frequent alginate-negative (Alg-) derivatives. Crosses between Alg+ donors and Alg- recipients demonstrated linkage between alginate genes and chromosomal markers. Crosses between an Alg- donor and Alg- recipients produced Alg+ recombinants at frequencies that varied, depending on the recipient strains used. This indicated that more than one genetic locus was associated with spontaneous mutation leading to loss of the mucoid character. Three classes of Alg- mutants were identified. Genetic exchange experiments showed that the loci of the alginate (alg) mutations of the three mutant classes are in the same region of the chromosome. The sex factor plasmid R68.45 was used for nonpolarized chromosome transfer and demonstrated close linkage between chromosomal markers (his-1, met-1) and alg markers. This was consistent with the data obtained in FP2-mediated crosses. Thus, the evidence obtained indicated that the alg genes which undergo frequent mutation are chromosomal, that several loci are involved, and that these alg loci are apparently clustered on the chromosome.