No abstract

Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

Pseudomonas putida GR12-2R3 is a rifampicin-resistant derivative of a cold-tolerant, nitrogen-fixing bacterium isolated from the roots of grasses growing in the Canadian high arctic. It colonizes canola (Brassica campestris) roots and promotes canola root and shoot growth under gnotobiotic conditions and in the field. TnphoA insertion mutagenesis was used to isolate derivatives of strain GR12-2R3 that had reduced abilities to promote canola root elongation (PRE− mutants). Within a pool of 10 290 TnphoA insertion mutants, 1.4% expressed active PhoA fusion proteins (PhoA+). Among 20 PhoA+ mutants, 6 were PRE− and 4 of those strains secreted PhoA activity into the culture medium. PhoA+ strain PG269 showed PhoA activity, 25% cell associated, that was induced by canola seed exudate. The ability of this strain to promote canola root elongation was similar to that of the parent strain. Like other pseudomonads, strain GR12-2R3 utilizes a wide range of sugars, amino acids, and other compounds as carbon and nitrogen sources. Twenty-one TnphoA insertion mutants, all PhoA−, were unable to utilize a specific nutrient. That group included strains that could not utilize arabinose (Ara−, three mutants) or glycerol and other compounds (Csu−, three mutants) as carbon source and strains that could not utilize glycine (Gut−, eight mutants), histidine (Hut−, three mutants), or proline (Put−, four mutants) as nitrogen source. One Ara− mutant, three Gut− mutants, and one Csu− mutant were PRE−. Five of the mutant strains examined in detail (two PhoA− PRE−, one PhoA− PRE+, one PhoA+ PRE−, and one PhoA+ PRE+) grew less well than strain GR12-2R3 in LB and (or) seed exudate medium. Further characterization of the TnphoA target genes in selected PRE− strains is expected to yield additional insight regarding the molecular basis for the interaction between P. putida GR12-2R3 and canola.Key words: Pseudomonas putida, plant growth promoting rhizobacterium, TnphoA.