Role of nucleotide excision repair and photoreactivation in the solar UVB radiation survival of Pseudomonas syringae pv. syringae B728a.

AIMS: To assess the role of DNA repair and photoreactivation in the solar radiation survival of the plant pathogen and leaf surface epiphyte Pseudomonas syringae pv. syringae (Pss). METHODS AND RESULTS: Mutants of Pss B728a, with insertional mutations within the nucleotide excision repair gene uvrA, photolyase gene phr, or uvrA phr double mutants, were constructed to examine the importance of individual repair mechanisms in solar UV radiation (UVR) survival. The survival of either the uvrA mutant or the phr mutant was reduced by approx. 10(2)-fold following exposure to a dose of 4.5 kJ m(-2) solar UVB (290-320 nm wavelengths) while the uvrA phr double mutant was reduced >10(6)-fold by the same dose. We constructed a transcriptional fusion between the Pss recA promoter and gfp to examine the induction of the SOS response in wild-type and mutant strains. Initiation of the recA mediated SOS response was more rapid and peaked at higher levels in mutant strains suggesting both increased DNA damage in mutant strains and also that photoreactivation and nucleotide excision repair remove DNA damage as it is incurred which is reflected in a delay of recA expression. Visualization of expression of B728a cells containing the recA::gfp reporter on UVB-irradiated bean leaves highlighted the movement of cells to intercellular spaces over time and that SOS induction was detectable when leaves were irradiated 48 h following leaf inoculation. CONCLUSIONS: This study indicated that solar UVB is detrimental to Pss B728a, DNA repair mechanisms play an important role in strain survival and expression of the SOS regulon on leaf surfaces contributes to survival of UVR-exposed cells during plant colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: This work links previous laboratory-based UVR analyses with solar UVB dose-response analyses and highlights the role of photoreactivation in delaying induction of the SOS response following solar irradiation. Knowledge of population dynamics following direct solar irradiation will enhance our understanding of the biology of Pss in the phyllosphere.

The role of pigmentation, ultraviolet radiation tolerance, and leaf colonization strategies in the epiphytic survival of phyllosphere bacteria.

Phenotypic mechanisms that enhance bacterial UVR survival typically include pigmentation and DNA repair mechanisms which provide protection from UVA and UVB wavelengths, respectively. In this study, we examined the contribution of pigmentation to field survival in Clavibacter michiganensis and evaluated differences in population dynamics and leaf colonization strategies. Two C. michiganensis pigment-deficient mutants were significantly reduced in UVA radiation survival in vitro; one of these mutants also exhibited reduced field populations on peanut when compared to the wild-type strain over the course of replicate 25-day experiments. The UVR-tolerant C. michiganensis strains G7.1 and G11.1 maintained larger epiphytic field populations on peanut compared to the UVR-sensitive C. michiganensis T5.1. Epiphytic field populations of C. michiganensis utilized the strategy of solar UVR avoidance during leaf colonization resulting in increased strain survival on leaves after UVC irradiation. These results further demonstrate the importance of UVR tolerance in the ability of bacterial strains to maintain population size in the phyllosphere. However, an examination of several bacterial species from the peanut phyllosphere and a collection of environmental Pseudomonas spp. revealed that sensitivity to UVA and UVC radiation was correlated in some but not all of these bacteria. These results underscore a need to further understand the biological effects of different solar wavelength groups on microbial ecology.

Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv. syringae A2.

Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv. syringae A2, and is a member of the recently described pPT23A plasmid family. We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs. Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements. pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395. The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene. Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P. syringae. Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P. syringae pv. syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection. Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P. syringae pathovars. The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants.

Effect of solar UV-B radiation on a phyllosphere bacterial community.

The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (Arachis hypogeae L.) was examined in field studies using plants grown under UV-B-transmitting (UV-B+) or UV-B-excluding (UV-B-) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B+ plots (n = 368) were significantly more UV tolerant than those from the UV-B- (n = 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B+ and UV-B- treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were Bacillus coagulans, Clavibacter michiganensis, and Curtobacterium flaccumfaciens. We selected C. michiganensis as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of C. michiganensis G7.1, especially following high UV-C doses (300 and 375 J m(-2)), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for C. michiganensis T5.1.

Construction and analysis of photolyase mutants of Pseudomonas aeruginosa and Pseudomonas syringae: contribution of photoreactivation, nucleotide excision repair, and mutagenic DNA repair to cell survival and mutability following exposure to UV-B radiation.

Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2. Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m(-2) followed by a recovery period under photoreactivating conditions. A phr uvrA mutant of P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m(-2). Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 using the cloned phr gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant rulAB. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing rulAB indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which rulAB only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 x 10(-5) and 2.1 x 10(-3), respectively, following a UV-B dose of 1,550 J m(-2). The construction and characterization of phr mutants in the present study will facilitate the determination of the roles of light and dark repair systems in organisms exposed to solar radiation in their natural habitats.

Sequence diversity of rulA among natural isolates of Pseudomonas syringae and effect on function of rulAB-mediated UV radiation tolerance.

The rulAB locus confers tolerance to UV radiation and is borne on plasmids of the pPT23A family in Pseudomonas syringae. We sequenced 14 rulA alleles from P. syringae strains representing seven pathovars and found sequence differences of 1 to 12% within pathovar syringae, and up to 15% differences between pathovars. Since the sequence variation within rulA was similar to that of P. syringae chromosomal alleles, we hypothesized that rulAB has evolved over a long time period in P. syringae. A phylogenetic analysis of the deduced amino acid sequences of rulA resulted in seven clusters. Strains from the same plant host grouped together in three cases; however, strains from different pathovars grouped together in two cases. In particular, the rulA alleles from P. syringae pv. lachrymans and P. syringae pv. pisi were grouped but were clearly distinct from the other sequenced alleles, suggesting the possibility of a recent interpathovar transfer. We constructed chimeric rulAB expression clones and found that the observed sequence differences resulted in significant differences in UV (wavelength) radiation sensitivity. Our results suggest that specific amino acid changes in RulA could alter UV radiation tolerance and the competitiveness of the P. syringae host in the phyllosphere.

Regulation of the rulAB mutagenic DNA repair operon of Pseudomonas syringae by UV-B (290 to 320 nanometers) radiation and analysis of rulAB-mediated mutability in vitro and in planta.

The effects of the rulAB operon of Pseudomonas syringae on mutagenic DNA repair and the transcriptional regulation of rulAB following irradiation with UV-B wavelengths were determined. For a rulB::Km insertional mutant constructed in P. syringae pv. syringae B86-17, sensitivity to UV-B irradiation increased and UV mutability decreased by 12- to 14-fold. rulAB-induced UV mutability was also tracked in phyllosphere populations of B86-17 for up to 5 days following plant inoculation. UV mutability to rifampin resistance (Rif(r)) was detected at all sampling points at levels which were significantly greater than in nonirradiated controls. In P. aeruginosa PAO1, the cloned rulAB determinant on pJJK17 conferred a 30-fold increase in survival and a 200-fold increase in mutability following a UV-B dose of 1,900 J m(-2). In comparative studies using defined genetic constructs, we determined that rulAB restored mutability to the Escherichia coli umuDC deletion mutant RW120 at a level between those of its homologs mucAB and umuDC. Analyses using a rulAB::inaZ transcriptional fusion in Pseudomonas fluorescens Pf5 showed that rulAB was rapidly induced after UV-B irradiation, with expression levels peaking at 4 h. At the highest UV-B dose administered, transcriptional activity of the rulAB promoter was elevated as much as 261-fold compared to that of a nonirradiated control. The importance of rulAB for survival of P. syringae in its phyllosphere habitat, coupled with its wide distribution among a broad range of P. syringae genotypes, suggests that this determinant would be appropriate for continued investigations into the ecological ramifications of mutagenic DNA repair.

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.

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.

Resistance to ultraviolet light in Pseudomonas syringae: sequence and functional analysis of the plasmid-encoded rulAB genes.

The indigenous plasmids, pPSR1 and pPSR5, were each shown to confer resistance to ultraviolet light (UV) in Pseudomonas syringae (Ps) pv. syringae FF5. The UV-resistance (UVR) determinant was subcloned from a cosmid library of pPSR1, and sequence analysis revealed the presence of two ORFs, designated rulAB which are homologous to the Escherichia coli umuDC mutagenic DNA repair systems and other plasmid-encoded UVR operons. Amino acid (aa) alignments indicated that RulAB are most closely related to the RumAB proteins from plasmid R391, sharing 40.5% and 48.6% aa identity with RumA and RumB, respectively. UV sensitivity assays with the cloned rulAB genes indicated that the expression of UVR in Ps required a functional recA gene.

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Molecular analysis of closely related copper- and streptomycin-resistance plasmids in Pseudomonas syringae pv. syringae.

The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv. syringae hosts was examined. Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P. syringae pv. syringae plasmid. Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments. An analysis of the host P. syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA. Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393. All plasmids hybridized to two additional probes derived from P. syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences. Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P. syringae pv. syringae FF5. The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria.

Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants.

Gene transfer within bacterial communities has been recognized as a major contributor in the recent evolution of antibiotic resistance on a global scale. The linked strA-strB genes, which encode streptomycin-inactivating enzymes, are distributed worldwide and confer streptomycin resistance in at least 17 genera of gram-negative bacteria. Nucleotide sequence analyses suggest that strA-strB have been recently disseminated. In bacterial isolates from humans and animals, strA-strB are often linked with the suIII sulfonamide-resistance gene and are encoded on broad-host-range nonconjugative plasmids. In bacterial isolates from plants, strA-strB are encoded on the Tn3-type transposon Tn5393 which is generally borne on conjugative plasmids. The wide distribution of the strA-strB genes in the environment suggests that gene transfer events between human, animal, and plant-associated bacteria have occurred. Although the usage of streptomycin in clinical medicine and animal husbandry has diminished, the persistence of strA-strB in bacterial populations implies that factors other than direct antibiotic selection are involved in maintenance of these genes.

Distribution of the streptomycin-resistance transposon Tn5393 among phylloplane and soil bacteria from managed agricultural habitats.

The distribution of the strA-strB streptomycin-resistance (Smr) genes associated with Tn5393 was examined in bacteria isolated from the phylloplane and soil of ornamental pear and tomato. Two ornamental pear nurseries received previous foliar applications of streptomycin, whereas the tomato fields had no prior exposure to streptomycin bactericides. Although the recovery of culturable Smr bacteria was generally higher from soil, the highest occurrence of Smr was observed in phylloplane bacteria of an ornamental pear nursery that received 15 annual applications of streptomycin during the previous 2 years. Twenty-two and 12% of 143 Gram-negative phylloplane and 163 Gram-negative soil isolates, respectively, contained sequences that hybridized to probes specific for the strA-strB Smr genes and for the transposase and resolvase genes of Tn5393. These sequences were located on large plasmids (> 60 kb) in 74% of the isolates. The 77 Smr Gram-positive bacteria isolated in the present study showed no homology to the Tn5393-derived probes. Although the repeated use of a single antibiotic in clinical situations is known to favor the development of strains with resistance to other antibiotics, we found no evidence that intensive streptomycin usage in agricultural habitats favors the development of resistance to tetracycline, an antibiotic also registered for disease control on plants. The detection of Tn5393 in bacteria with no prior exposure to streptomycin suggests that this transposon is indigenous to both phylloplane and soil microbial communities.

Expression of the strA-strB streptomycin resistance genes in Pseudomonas syringae and Xanthomonas campestris and characterization of IS6100 in X. campestris.

Expression of the strA-strB streptomycin resistance (SMr) genes was examined in Pseudomonas syringae pv. syringae and Xanthomonas campestris pv. vesicatoria. The strA-strB genes in P. syringae and X. campestris were encoded on elements closely related to Tn5393 from Erwinia amylovora and designated Tn5393a and Tn5393b, respectively. The putative recombination site (res) and resolvase-repressor (tnpR) genes of Tn5393 from E. amylovora, P syringae, and X. campestris were identical; however, IS6100 mapped within tnpR in X. campestris, and IS1133 was previously located downstream of tnpR in E. amylovora (C.-S Chiou and A. L. Jones, J. Bacteriol. 175:732-740, 1993). Transcriptional fusions (strA-strB::uidA) indicated that a strong promoter sequence was located within res in Tn5393a. Expression from this promoter sequence was reduced when the tnpR gene was present in cis position relative to the promoter. In X. campestris pv. vesicatoria, analysis of promoter activity with transcriptional fusions indicated that IS6100 increased the expression of strA-strB. Analysis of codon usage patterns and percent G+C in the third codon position indicated that IS6100 could have originated in a gram-negative bacterium. The data obtained in the present study help explain differences observed in the levels of SMr expressed by three genera which share common genes for resistance. Furthermore, the widespread dissemination of Tn5393 and derivatives in phytopathogenic prokaryotes confirms the importance of these bacteria as reservoirs of antibiotic resistance in the environment.

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.