Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides.

We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.     

Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).     

DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria.

The copper resistance (Cur) genes encoded on pXV10A, a 190-kb plasmid in Xanthomonas campestris pv. vesicatoria XV10, were isolated on a 44-kb cosmid clone designated pCuR1. Tn5 mutagenesis of pCuR1 indicated that a 4.0-kb region was required for copper resistance. Three restriction fragments located within the 4.0-kb region demonstrated high specificity for the Cur genes present in X. campestris pv. vesicatoria and will be useful in monitoring the presence of these genes in the environment.     

DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria.

The copper resistance (Cur) genes encoded on pXV10A, a 190-kb plasmid in Xanthomonas campestris pv. vesicatoria XV10, were isolated on a 44-kb cosmid clone designated pCuR1. Tn5 mutagenesis of pCuR1 indicated that a 4.0-kb region was required for copper resistance. Three restriction fragments located within the 4.0-kb region demonstrated high specificity for the Cur genes present in X. campestris pv. vesicatoria and will be useful in monitoring the presence of these genes in the environment.     

Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.     

Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to ultraviolet B (290-320 nm) radiation and distribution of rulAB among P. syringae pathovars

The effect of the plasmid-encoded rulAB (resistance to ultraviolet radiation) determinant on responses of Pseudomonas syringae to ultraviolet-B (UV-B) radiation and the distribution of rulAB among pathovars of P. syringae were determined. The cloned rulAB determinant and the native rulAB + plasmid pPSR1 both conferred approximately a 10-fold increase in survival on P. syringae pv. syringae FF5 following increasing doses of UV-B radiation. rulAB + P. syringae strains also maintained significantly larger epiphytic populations on leaf surfaces irradiated with UV-B. rulAB -insertional mutants, constructed in two native rulAB + strains, were from 10- to 100-fold more sensitive to UV-B radiation. The UV tolerance phenotype and the rulAB genes were widely distributed among P. syringae pathovars isolated from varied plant hosts throughout the world and within a broad range of genotypic backgrounds of P. syringae pv. syringae. With one exception, the rulAB determinant was harboured on pPT23A-like plasmids; these replicons are indigenous residents of the species P. syringae and also tend to encode determinants of importance in host–pathogen interactions.     

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae.

Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.     

Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase.

Copper-resistant strains of Xanthomonas campestris pv. juglandis occur in walnut orchards throughout northern California. The copper resistance genes from a copper-resistant strain C5 of X. campestris pv. juglandis were cloned and located on a 4.9-kb ClaI fragment, which hybridized only to DNA of copper-resistant strains of X. campestris pv. juglandis, and was part of an approximately 20-kb region which was conserved among such strains of X. campestris pv. juglandis. Hybridization analysis indicated that the copper resistance genes were located on the chromosome. Plasmids conferring copper resistance were not detected in copper-resistant strains, nor did mating with copper-sensitive strains result in copper-resistant transconjugants. Copper resistance genes from X. campestris pv. juglandis shared nucleotide sequence similarity with copper resistance genes from Pseudomonas syringae pv. tomato, P. syringae, and X. campestris pv. vesicatoria. DNA sequence analysis of the 4.9-kb fragment from strain C5 revealed that the sequence had an overall G+C content of 58.7%, and four open reading frames (ORF1 to ORF4), oriented in the same direction. All four ORFs were required for full expression of copper resistance, on the basis of Tn3-spice insertional inactivation and deletion analysis. The predicted amino acid sequences of ORF1 to ORF4 showed 65, 45, 47, and 40% identity with CopA, CopB, CopC, and CopD, respectively, from P. syringae pv. tomato. The most conserved regions are ORF1 and CopA and the C-terminal region (166 amino acids from the C terminus) of ORF2 and CopB. The hydrophobicity profiles of each pair of predicted polypeptides are similar except for the N terminus of ORF2 and CopB. Four histidine-rich polypeptide regions in ORF1 and CopA strongly resembled the copper-binding motifs of small blue copper proteins and multicopper oxidases, such as fungal laccases, plant ascorbate oxidase, and human ceruloplasmin. Putative copper ligands of the ORF1 polypeptide product are proposed, indicating that the polypeptide of ORF1 might bind four copper ions: one type 1, one type 2, and two type 3.     

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.     

62-kb Plasmids Harboring <Emphasis Type="Italic">rulAB</Emphasis> Homologues Confer UV-tolerance and Epiphytic Fitness to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.     

Association of the strA-strB genes with plasmids and transposons in the present-day bacteria and in bacterial strains from permafrost

Transposons closely related to the streptomycin resistance transposon of modem bacteria, Tn5393, were detected in the bacterial isolates from permafrost resistant to streptomycin. Many transposons studied were located on the medium-size plasmids with a narrow host range. None of the streptomycin-resistant strains isolated from permafrost contained small plasmids carrying the strA-strB genes and related to the broad host range plasmid RSF1010.     

A multi-resistance plasmid isolated from commensal Neisseria species is closely related to the enterobacterial plasmid RSF1010

pFM739, an R plasmid from Neisseria sicca that encodes penicillin, streptomycin and sulphonamide resistance, and the enterobacterial IncQ(P-4) plasmid RSF1010, which encodes streptomycin and sulphonamide resistance, were incompatible, and were mobilized by the same conjugative plasmids. Restriction mapping confirmed a high degree of similarity between both R plasmids; pFM739 carried DNA fragments corresponding to the known replication and resistance regions of RSF1010. pFM739 also carried an extra segment with the same restriction map as that described for the beta-lactamase-coding region of transposon Tn3. It is suggested that the R plasmids isolated from commensal Neisseria sp. could have resulted from transposition of a Tn3-like genetic element to an RSF1010-like plasmid, and that they contain deletion derivatives of transposon Tn3.     

Presence of STRA-STRB linked streptomycin-resistance genes in clinical isolate of Escherichia coil 2418

The streptomycin resistance of Escherichia coli 2418 strain has been shown to be associated with a 1.2-kb DNA fragment found in the naturally occurring plasmid R2418S. Here, nucleotide sequence analysis of the 1.2-kb DNA fragment revealed the presence of the strB gene which is located immediately downstream of the strA gene. Both sequences are identical to those of strA and strB genes in plasmid RSF1010. Thus, the observed resistance in the clinical isolate is due to the presence of strA-strB genes encoding streptomycin-modifying enzymes. The sequence downstream of strB gene showed a perfect homology with that of RSF1010. In addition, it contained the right inverted repeat of the transposon Tn5393 that has been suggested to be a relic of this transposon found in DNA plasmids isolated from human- and animal-associated bacteria.     

Comparative genomic analysis of the pPT23A plasmid family of Pseudomonas syringae

Members of the pPT23A plasmid family of Pseudomonas syringae play an important role in the interaction of this bacterial pathogen with host plants. Complete sequence analysis of several pPT23A family plasmids (PFPs) has provided a glimpse of the gene content and virulence function of these plasmids. We constructed a macroarray containing 161 genes to estimate and compare the gene contents of 23 newly analyzed and eight known PFPs from 12 pathovars of P. syringae, which belong to four genomospecies. Hybridization results revealed that PFPs could be distinguished by the type IV secretion system (T4SS) encoded and separated into four groups. Twelve PFPs along with pPSR1 from P. syringae pv. syringae, pPh1448B from P. syringae pv. phaseolicola, and pPMA4326A from P. syringae pv. maculicola encoded a type IVA T4SS (VirB-VirD4 conjugative system), whereas 10 PFPs along with pDC3000A and pDC3000B from P. syringae pv. tomato encoded a type IVB T4SS (tra system). Two plasmids encoded both T4SSs, whereas six other plasmids carried none or only a few genes of either the type IVA or type IVB secretion system. Most PFPs hybridized to more than one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors. The overall gene contents of individual PFPs were more similar among plasmids within each of the four groups based on T4SS genes; however, a number of genes, encoding plasmid-specific functions or hypothetical proteins, were shared among plasmids from different T4SS groups. The only gene shared by all PFPs in this study was the repA gene, which encoded sequences with 87 to 99% amino acid identityamong 25 sequences examined. We proposed a model to illustrate the evolution and gene acquisition of the pPT23A plasmid family. To our knowledge, this is the first such attempt to conduct a global genetic analysis of this important plasmid family.     

Association of the strA-strB genes with plasmids and transposons in the present-day bacteria and in bacterial strains from permafrost

Transposons closely related to the streptomycin resistance transposon of modern bacteria, Tn5393, were detected in the bacterial isolates from permafrost resistant to streptomycin. Many transposons studied were located on the medium-size plasmids with a narrow host range. None of the streptomycin-resistant strains isolated from permafrost contained small plasmids carrying the strA-strB genes and related to the broad host range plasmid RSF1010.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

Mobilization using incompatibility group P1 plasmids in strains of Pseudomonas pseudomallei and Pseudomonas mallei as potential vectors for DNA cloning]

The cells of Pseudomonas pseudomallei and Pseudomonas mallei have been shown to serve as recipients for the plasmid RSF1010 and its recombinant derivatives pVA1 and pVA4. The conjugative plasmids RP1 and pTH10 of the incompatibility group P1 are able to mobilize the nontransmissive vector plasmids for conjugation transfer into Pseudomonas pseudomallei and Pseudomonas mallei strains. The SmR determinant of the plasmid RSF1010 is expressed in the latter strains. These data makes the mentioned vector plasmids the candidates for DNA cloning in these strains.     

Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions

Tn5 insertion mutants of Xanthomonas campestris pv. vesicatoria were inoculated into tomato and screened for reduced virulence. One mutant exhibited reduced aggressiveness and attenuated growth in planta. Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X. campestris pv. vesicatoria. The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X. campestris pv. vesicatoria 91-118. Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants. The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv. syringae and OpgH of Erwinia chrysanthemi. Based on homology, the new locus was designated opgHXcv. Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.