Pseudomonas syringae pv. phaseolicola can be separated into two genetic lineages distinguished by the possession of the phaseolotoxin biosynthetic cluster.

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox(+)). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox(-)). These Tox(-) isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox(+) and Tox(-) isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox(+) isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox(-) isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox(-) isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox(+), but not among Tox(-) isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox(+) and Tox(-) P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.

Location and activity of members of a family of virPphA homologues in pathovars of Pseudomonas syringae and P. savastanoi.

Summary virPphA is a major determinant of the pathogenicity of Pseudomonas savastanoi pv. phaseolicola to Phaseolus bean. A family of homologues of virPphA was detected in pathovars of P. savastanoi and P. syringae. We examined the structure and activity of alleles designated virPphA, virPphA(Pgy), and virPphA(Psv) from P. savastanoi pathovars phaseolicola, glycinea, and savastanoi, respectively, and avrPtoB from P. syringae pv. tomato. Sequencing showed that the virPphA(Pgy) homologue had a 48-bp central deletion in the open reading frame (ORF) compared with virPphA and virPphA(Psv), but otherwise all three P. savastanoi alleles had > 98% identity at the DNA level. By contrast, AvrPtoB from P. syringae pv. tomato strain DC3000 was predicted to have only 51% amino acid similarity with VirPphA. All ORFs have an upstream hrp-box promoter indicating potential regulation by HrpL. Each cloned homologue was introduced into the P. savastanoi pv. phaseolicola strain RW60, which lacks a native plasmid carrying virPphA as part of a pathogenicity island (PAI), and which is not pathogenic on bean. The homologues all restored virulence, as measured by the development of water-soaked lesions in bean pods, and increased bacterial populations in leaves compared with RW60 alone. RW60 harbouring virPphA or virPphA(Psv) elicited a strong hypersensitive reaction (HR) in soybean cv. Osumi; the presence of avrPtoB caused a weak HR, but virPphA(Pgy) did not affect the null reaction observed in soybean with RW60 alone. A second effector gene, avrPphD, was detected on the genomic clones carrying virPphA(Pgy) and virPphA(Psv). avrPphD was also present in both P. savastanoi pv. phaseolicola and P. syringae pv. tomato, but elsewhere in their genomes. Comparison of the genomic locations of virPphA and other effectors found in the P. savastanoi pv. phaseolicola PAI revealed the greatest divergence of the sequences surrounding virPphA to be in P. syringae pv. tomato.

Highly conserved sequences flank avirulence genes: isolation of novel avirulence genes from Pseudomonas syringae pv. pisi.

DNA sequences flanking two avr genes (avrPpiA1 and avrPpiB1) from Pseudomonas syringae pv. pisi show a high degree of similarity. Specific primers designed from the conserved regions were used in PCR amplifications with all P. syringae pv. pisi races. As well as amplifying the expected avrPpiA- and avrPpiB-containing fragments, two additional fragments were amplified: one contained a single open reading frame (ORF1) and was found in races of genomic group II (2, 3A, 4A and 6); the second fragment contained two open reading frames (ORF2 and ORF3), separated by 658 nt, and was detected in all races. All three ORFs had G+C ratios (46.9-48 mol%) that were significantly less than that for P. syringae and each was preceded by a potential hrp box promoter. In P. syringae pv. phaseolicola, ORF1 and ORF2 each elicited a strong non-host hypersensitive reaction on bean leaves; ORF1 was designated avrPpiG, the product of which had strong similarity to AvrRxv, AvrBsT and YopP. ORF2 was identical to a gene, designated avrPpiC, previously isolated from P. syringae pv. pisi race 5. ORF3 was always found in association with avrPpiC and both were detected in a wide range of P. syringae pathovars. In contrast, avrPpiG was only detected in strains of P. syringae pv. pisi genomic group II and P. syringae pv. coronafaciens (ICMP 3113). In P. syringae pv. pisi, avrPpiG was plasmid-borne and avrPpiC and ORF3 were chromosomal. This conservation of flanking sequences has implications for the horizontal transfer of avirulence and virulence genes, suggesting that specific regions of the bacterial genome act as sites for their integration/excision.

Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes.

Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains). Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri. IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex'. The 'P: putida complex' included P: putida and P: fulva. The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P. savastanoi and P. syringae. The 'P. fluorescens complex' was further divided into two subpopulations, the 'P. fluorescens lineage' and the 'P. chlororaphis lineage'. The 'P. fluorescens lineage' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the 'P. chlororaphis lineage' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the 'P. fluorescens complex'.

Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons.

Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.