GDPmannose dehydrogenase and biosynthesis of alginate-like polysaccharide in a mucoid strain of Pseudomonas aeruginosa.

GDPmannose dehydrogenase (EC 1.1.1.132) in a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis was identified by demonstrating the NAD-linked formation of GDPmannuronate from GDPmannose mediated by a cell extract of the organism. Nonmucoid mutant strains did not contain GDPmannose dehydrogenase, which suggests that the enzyme is involved in the biosynthesis of alginate-like polysaccharide by P. aeruginosa.

Phenotypic differences among clinically isolated mucoid Pseudomonas aeruginosa strains.

Mucoid strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis or urinary tract infections displayed many phenotypic differences. The ratios of D-mannuronosyl to L-guluronosyl moieties of the extracellular alginate-like polysaccharides produced by the 19 strains examined varied from 99 to 0.8; the acetyl content of the polymers varied from 0.38 to 0.02 mol per mole of uronosyl residue. The strains also differed with regard to the stability of the mucoid phenotype. Of 15 isolates from patients with cystic fibrosis, 7 displayed stable mucoid phenotypes; 8 isolates were unstable and reverted to the nonmucoid phenotype at high frequency. The four strains isolated from patients with urinary tract infections were also unstable. Strains from urinary tract infections expressed the mucoid phenotype on six different media, both minimal and complex, whereas cystic fibrosis-associated strains varied widely with regard to medium-dependent expression of the mucoid phenotype. Of 15 cystic fibrosis strains, 5 were mucoid on each of six different media, 4 were mucoid on five media, 1 was mucoid on four media, 4 were mucoid on three media, and 1 yielded mucoid colonies on only one of the six media tested. There was no obvious correlation among polysaccharide structure, stability of the mucoid phenotype, and medium-dependent expression of the mucoid phenotype for any of the 19 strains investigated. These data suggest that although mucoid strains of P. aeruginosa must share some common property related to their ability to colonize their host, this property seems to be unrelated to polysaccharide composition, medium-dependent expression of the mucoid phenotype, or stability of the mucoid phenotype.

Isolation and characterization of Hfr strains of Erwinia amylovora.

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.