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.     

The specificity of an immune serum to the exocellular lipopolysaccharide of Pseudomonas wieringae

The serum obtained to exocellular lipopolysaccharide (ELPS) of Pseudomonas wieringae selectively agglutinated strains of pathovar of P. syringae and did not agglutinated strains of P. cichorii, P. solanacearum, P. gladioli pv. allicola, P. fluoroviolaceus, strains of nonphytopathogenic pseudomonads as well as bacteria of the genera Erwinia, Bacillus, Xanthomonas, Klebsiella. Consequently, the antigen determinant common with antigen of the species Pseudomonas syringae is present in the composition of ELPS.     

Long-term effects of inducible mutagenic DNA repair on relative fitness and phenotypic diversification in Pseudomonas cichorii 302959

Mutagenic DNA repair (MDR) employs low-fidelity DNA polymerases capable of replicating past DNA lesions resulting from exposure to high-energy ultraviolet radiation (UVR). MDR confers UVR tolerance and activation initiates a transient mutator phenotype that may provide opportunities for adaptation. To investigate the potential role of MDR in adaptation, we have propagated parallel lineages of the highly mutable epiphytic plant pathogen Pseudomonas cichorii 302959 with daily UVR activation (UVR lineages) for ~500 generations. Here we examine those lineages through the measurement of relative fitness and observation of distinct colony morphotypes that emerged. Isolates and population samples from UVR lineages displayed gains in fitness relative to the ancestor despite increased rates of inducible mutation to rifampicin resistance. Regular activation of MDR resulted in the maintenance of genetic diversity within UVR lineages, including the reproducible diversification and coexistence of “round” and “fuzzy” colony morphotypes. These results suggest that inducible mutability may present a reasonable strategy for adaptive evolution in stressful environments by contributing to gains in relative fitness and diversification.     

Growth parameter components of adaptive specificity during experimental evolution of the UVR-inducible mutator Pseudomonas cichorii 302959

Background

Mutagenic DNA repair (MDR) transiently increases mutation rate through the activation of low-fidelity repair polymerases in response to specific, DNA-damaging environmental stress conditions such as ultraviolet radiation (UVR) exposure. These repair polymerases also confer UVR tolerance, intimately linking mutability and survival in bacteria that colone habitats subject to regular UVR exposure.

Methodology/Principal Findings

Here, we investigate adaptive specificity in experimental lineages of the highly UVR-mutable epiphytic plant pathogen Pseudomonas cichorii 302959. Relative fitness measurements of isolates and population samples from replicate lineages indicated that adaptive improvements emerged early in all lineages of our evolution experiment and specific increases in relative fitness correlated with distinct improvements in doubling and lag times. Adaptive improvements gained under UVR and non-UVR conditions were acquired preferentially, and differentially contributed to relative fitness under varied growth conditions.

Conclusions

These results support our earlier observations that MDR activation may contribute to gains in relative fitness without impeding normal patterns of adaptive specificity in P. cichorii 302959.     

Protein and enzymatic criteria for the characterization of phytopathogenic Pseudomonas fluorescens

Polyacrylamide gel electrophoresis of proteins was carried out to characterize eight bacterial strains belonging to the genus Pseudomonas. The sampling included three species (P. cichorii, P. viridiflava and P. syringae), with three pathovars for this last species (pv. pisi, pv. syringae, pv. tomato). Several molecular markers were evaluated: native proteins, denatured proteins, esterases, superoxide dismutases (SOD) and polyphenoloxidases (PPO). Each species or pathovar of Pseudomonas was clearly differentiated by esterase patterns. SOD, PPO and native protein patterns allowed strains of P. cichorii, P. viridiflava and P.s. pv. tomato also to be distinguished. Strains of P.s. pv. pisi and P.s. pv. syringae were identical for these criteria. Denatured protein patterns of these two pathovars and P. viridiflava were similar.     

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.     

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 × 10⁻⁵ and 2.1 × 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.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

The lipopolysaccharides of Pseudomonas solanacearum i Pseudomonas cichorii

Structure analysis by the methods of methylation, 1H- and 13C-n. m. r. spectroscopy has shown that O-specific polysaccharides of typical strains of Pseudomonas solanacearum (biovar I) and P. cichorii are identical by their structure and constructed of branched pentasaccharide repeating links which include three residues of rhamnose (one of them is in the branching node), one residue of beta-xylose (it occupies terminal position) and one reside of N-acetyl-beta-glucosamine. The other strain of P. solanacearum of biovar I and two strains belonging to biovars III and IV also produce structure-similar O-specific polysaccharides, constructed of linear tetrasaccharide repeating links which include three residues of alpha-L-rhamnose and one residue of N-acetyl-alpha-D-glucosamine.     

Plasmid modification of radiation and chemical-mutagen sensitivity in Pseudomonas aeruginosa.

The R factor pMG2 protects Pseudomonas aeruginosa against the lethal effects of ultraviolet (u.v.) and gamma irradiation, and methyl methanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine treatment. Enhanced survival occurs in strains of uvr+ rec+ (wild-type) genotype and a variety of uvr rec+ type mutants. No protection occurs in a rec A-type mutant. The plasmid also enhances u.v.-induced mutagenesis. These effects appear to be due to host-cell controlled plasmid-determined DNA repair function(s). Studies on P. aeruginosa strains deficient in DNA polymerase I (polyA) suggest that a plasmid-determined repair resynthesis function may be responsible for increased u.v.-survival and enhanced u.v.-mutability in pMG2-containing bacteria.     

The pyoverdins of Pseudomonas syringae and Pseudomonas cichorii

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.     

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild‐type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild‐types attained similar population densities. Given the importance of the methyl‐directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild‐type phenotype.     

Comparative analysis of UV-induced mutability of ten different codon units in position 211 of the Escherichia coli trpA gene

Summary To study the influence of the local base, composition upon UV-induced mutability the reversion frequencies of ten trpA mutants with known codon sequences at position 211 were compared. Comparison of mutant strains reverting by the same base substitution type but with different dipyrimidinic, sequences reveals the mutagenic character of the pyrimidine-pyrimidine (6-4) photoproduct. The codons GAC and GAT, both reverting by AT-GC transitions and AT-CG transversions in the middle position and harboring the dipyrimidinic sequence CT in the opposite strand, differ, in their reversion frequencies sixfold. This difference can only be due to the influence of the different bases in the third codon position upon the mutability of adjacent second bases.     

Effect of repair deficiency and R plasmids on spontaneous and radiation-induced mutability in Pseudomonas aeruginosa.

The effect of R plasmids on spontaneous and radiation (ultraviolet and gamma)-induced mutability in Pseudomonas aeruginosa was studied in strains containing the radiation-sensitive markers polA3 or rec-2 and the revertable auxotrophic markers hisO27 and trpB1. In the absence of an R plasmid, the radiation-induced mutability was dependent on the recA+ genotype and independent of the polA+ genotype, whereas spontaneous mutability was similar in all genetic backgrounds. R plasmids pPL1, R2, and pMG15 increased the ultraviolet radiation survival and ultraviolet-induced mutability of wild-type and polA host cells but did not alter either effect in a recA mutant. These R plasmids also increased the gamma radiation survival and gamma-induced mutability of wild-type host cells bud pMG15 also enhanced the level of spontaneous mutagenesis in wild-type host cells but not in a polA or recA mutant. These data suggested that a common plasmid gene product(s) may participate in various recA-dependent, error-prone deoxyribonucleic acid repair pathways of P. aeruginosa. The properties of a mutant R plasmid, pPL2, originally selected because it lacked enhanced ultraviolet-induced mutability, supported this conclusion.     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.     

Deoxyribonucleic Acid Homologies Among Some Pseudomonas Species

Phylogenetic relationships among a number of strains belonging to the genus Pseudomonas were explored by the use of in vitro deoxyribonucleic acid (DNA) hybridization. The fluorescent nomenspecies (P. fluorescens, P. putida, P. aeruginosa, P. cichorii, P. syringae, and related species), as well as the nonfluorescent species P. stutzeri, P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, were shown to belong to a single DNA homology complex which is isolated from other Pseudomonas species that have been studied [P. cepacia (= P. multivorans), P. caryophylli, P. marginata (= P. alliicola), P. pseudomallei, P. acidovorans, P. testosteroni, P. solanacearum, P. diminuta, P. facilis, P. delafieldii, P. saccharophila, P. palleronii]. A limited numerical analysis of the phenotypic properties of the examined strains supported, with some exceptions, their previous allocation to nomenspecies and biotypes. The internal structure and nomenclature of the "P. fluorescens homology complex" are discussed.