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 '.

The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G؉C content significantly lower than that expected for P. syringae, indicates that we have located a plasmidborne pathogenicity island equivalent to those found in mammalian pathogens.Varietal resistance to halo-blight disease of bean (Phaseolus vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola (Pph) is determined by gene-for-gene interactions involving five resistance (R) genes in the host and five matching avirulence (avr) genes in the pathogen. Depending on the presence or absence of functional avr genes, nine races of Pph have been distinguished (1, 2). The avr genes matching R1, R2, and R3 have been cloned and sequenced. Their full designations are avrPphF.R1, avrPphE.R2, and avrPphB.R3; the terminal R gene designation will not be used here (3-5). Both avrPphE and avrPphB are chromosomal, whereas avrPphF is located on a large plasmid in those races that cause the hypersensitive reaction (HR) in cultivars of bean with the matching R1 gene.

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (Pph) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo-blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in Pph. Strain RW60 of Pph, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classi®ed as encoded by avirulence genes.

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We showed that a bacterial avirulence (avr) gene function, avrPpiA1, from the pea pathogen Pseudomonas syringae pv pisi, is recognized by some, but not all, genotypes of Arabidopsis. Thus, an avr gene functionally defined on a crop species is also an avr gene on Arabidopsis. The activity of avrPpiA1 on a series of Arabidopsis genotypes is identical to that of the avrRpm1 gene from P.s. pv maculicola previously defined using Arabidopsis. The two avr genes are homologous and encode nearly identical predicted products. Moreover, this conserved avr function is also recognized by some bean and pea cultivars in what has been shown to be a gene-for-gene manner. We further demonstrated that the Arabidopsis disease resistance locus, RPM1, conditioning resistance to avrRpm1, also conditions resistance to bacterial strains carrying avrPpiA1. Therefore, bean, pea, and conceivably other crop species contain functional and potentially molecular homologs of RPM1.

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 HR11302A, 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 pTiKl2 of Thiobacillus intermedius and other ColE2-related plasmids. However, both peptides were 100 residues longer than any of the known ColE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of a t least three incompatibility determinants, designated IncA, lncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the rulAB 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.

S U M M A R YWild-type Streptomyces coelicolor A 3(2), and many mutant and recombinant derivatives of it, are of the IF (Initial Fertility) type. At an early step in the production of recombinant strains from some of the first derivatives of A3 (2), a variant fertility type arose (NF: Normal Fertility), and subsequently IF and NF segregated within the pedigree of stock cultures. IF x IF crosses are about Ioo-fold less fertile than N F X N F or I F X N F crosses, but the clearest distinction between IF and NF is achieved by crossing with a strain of the previously described UF (Ultra-Fertile) type, when the difference in fertility approaches I ooo-fold.The IF strains give rise to UF strains with a high spontaneous frequency, and the frequency is increased by ultraviolet or X-irradiation but not appreciably by N-methyl-N'-nitro-N-nitrosoguanidine. NF strains do not give rise to UF variants with a high frequency.The difference between IF and NF is determined by a chromosomal locus near the 9 o'clock position on the linkage map. There is no evidence for the infectious conversion of one type of strain to the other in a mixed culture.In crosses with an NF strain, both IF and UF strains contribute the whole chromosome to the effective merozygotes, and the NF strain contributes the fragment, which obligatorily includes the g o'clock region. However, whereas in UF x NF crosses there is obligate inheritance of the g o'clock region of the NF genome by all sexually produced progeny, this is not true of I F X N F crosses .