Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator.

Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharacterized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.

Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp.

A total of 137 soilborne and plant-associated bacterial strains belonging to different Pseudomonas species were tested for their ability to synthesize N-acyl-homoserine lactones (NAHL). Fifty-four strains synthesized NAHL. Interestingly, NAHL production appears to be more common among plant-associated than among soilborne Pseudomonas spp. Indeed, 40% of the analyzed Pseudomonas syringae strains produced NAHL which were identified most often as the short-chain NAHL, N-hexanoyl-L-homoserine lactone, N-(3-oxo-hexanoyl)-homoserine lactone, and N-(3-oxo-octanoyl)-L-homoserine lactone (no absolute correlation between genomospecies of P. syringae and their ability to produce NAHL could be found). Six strains of fluorescent pseudomonads, belonging to the species P. chlororaphis, P. fluorescens, and P. putida, isolated from the plant rhizosphere produced different types of NAHL. In contrast, none of the strains isolated from soil samples were shown to produce NAHL. The gene encoding the NAHL synthase in P. syringae pv. maculicola was isolated by complementation of an NAHL-deficient Chromobacterium mutant. Sequence analysis revealed the existence of a luxI homologue that we named psmI. This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to psyI and ahlI, two genes involved in NAHL production in P. syringae pv. tabaci and P. syringae pv. syringae, respectively. We identified another open reading frame that we termed psmR, transcribed convergently in relation to psmI and partly overlapping psmI; this gene encodes a putative LuxR regulatory protein. This gene organization, with luxI and luxR homologues facing each other and overlapping, has been found so far only in the enteric bacteria Erwinia and Pantoea and in the related species P. syringae pv. tabaci.

Transformation of Candida oleophila and survival of a transformant on orange fruit under field conditions.

Histidine auxotrophs of wild-type strain I-182 of Candida oleophila, produced using ethyl methanesulfonate, were transformed with plasmids containing the HIS3, HIS4 and HIS5 genes of Saccharomyces cerevisiae. Histidine auxotrophy was complemented by the HIS5 gene of S. cerevisiae. Stability of the transformants under non-selective conditions and DNA gel-blot analysis suggested that the transforming DNA had integrated into the C. oleophila genome. There were no detectable physiological differences between the wild-type and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked transformant (with a beta-glucuronidase gene) colonized wounds on oranges, and its population increased under field conditions. The identity of the genetically marked transformant was established by PCR-amplification of a portion of the beta-glucuronidase gene.

Auxin production is a common feature of most pathovars of Pseudomonas syringae.

We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species. Most of those analyzed produced IAA, especially in the presence of tryptophan. Eight strains produced high IAA concentrations in the absence of Trp. The iaaM and iaaH genes of P. savastanoi pv. savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains. Thus, IAA synthesis in most assayed strains of P. syringae and related species does not involve genes highly similar to iaaM and iaaH. In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

The use of digoxigenin-labelled probes to detect DNA sequences specific for plant pathogenic bacteria.

Southern blot hybridization is a valuable method in the assessment of the pathogenicity of bacterial strains or isolates. It is also a powerful tool for the demonstration of the presence of foreign DNA sequences in the genome of genetically-engineered plant cells. In this respect, cold, digoxigenin-labelled DNA probes can be used in place of classical radioactive probes, whether hybridizations are performed on bacterial genomic or plasmidic DNA, or on plant genomic DNA. The versatility of this cold labelling makes it suitable for the detection of unique bacterial genomic or plasmid sequences, even though these are located on large plasmids. The sensitivity of this cold probe technique also permits the detection of subpicogram quantities of DNA in plant genomic preparations. Their long term storage stability allows them to be frequently re-used over long periods of time, making this technique quite cost efficient.

A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria.

We examined the sensitivity and the specificity of three versions of the Salkowski colorimetric technique. Two of these allowed the detection of indoleacetic acid (IAA) over a low range of concentrations (0.5 to 20 (mu)g/ml), while the third permitted the detection of IAA over a range of higher concentrations (5 to 200 (mu)g/ml). Overall, the three formulations reacted not only with auxin (IAA) but also with indolepyruvic acid and indoleacetamide. Therefore, these techniques appear to be specific for IAA, indolepyruvic acid, and indoleacetamide rather than for IAA alone.

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.