Restoration induced by catalase in irradiated microorganisms

1. E. coli, strain K-12, and B. megatherium 899, irradiated in strict but still undefined physiological conditions with certain heavy doses of ultraviolet light, are efficiently restored by catalase, which acts on or fixes itself upon the bacteria in a few minutes. This restoration (C. R.), different from photorestoration, is aided by a little visible light. 2. At 37° the restorability lasts for about 2 hours after UV irradiation; the restored cells begin to divide at the same time as the normal survivors. 3. C. R. is not produced after x-irradiation. 4. B. megatherium Mox and E. coli, strain B/r show little C. R.; E. coli strain B shows none. None of these three strains is lysogenic, whereas the two preceding catalase-restorable strains are. 5. Phage production in the system "K-12 infected with T2 phage" is restored by catalase after UV irradiation, whereas phage production in the system "infected B" is not. 6. With K-12, catalase does not prevent the growth of phage and the lysis induced by UV irradiation (Lwoff's phenomenon). 7. Hypotheses are discussed concerning: (a) the chemical nature of this action of catalase; (b) a possible relation between C. R. and lysogenicity of the sensitive bacteria; (c) the consequences of such chemical restorations on the general problem of cell radiosensitivity.     

Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli.

The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic. UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain. The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host. The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta pathway) phenotype

Summary Plasmid pPGH1 originating from Pseudomonas putida strain H carries all the genes required for the degradation of phenol (or cresols) via the meta cleavage pathway. Besides mobilization of pPGH1 by a plasmid of the incompatibility group P-1, hybrid plasmids conferring the Phl⁺ phenotype could be selected, when R68.45 was the conjugative plasmid. The hybrids contain the complete R68.45 and part of pPGH1. Integration of Phl-DNA of pPGH1 into R68.45 occurred exclusively via the IS21 region of R68.45.Dedicated to Udo Taubeneck on the occasion of his 60th birthday     

Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera.

Summary R. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.     

Transfer of kanamycin resistance mediated by plasmid R68.45 in Paracoccus denitrificans.

Plasmid R68.45 mediates the transfer of kanamycin resistance from Pseudomonas aeruginosa to Paracoccus denitrificans. Kanamycin resistance could be transferred from one strain of P. denitrificans to another, thus opening up the possibility of using R68.45 as a sex factor in P. denitrificans.     

Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property

Summary Plasmid R46 (an R factor conferring resistance to ampicillin, sulfonamides, streptomycin and tetracycline) reduces the bactericidal effect of UV irradiation but increases its mutagenic effect (reversion of hisG46), and raises the frequency of spontaneous reversion (mutator effect). Putative deletion mutants of R46 were obtained by transduction of the plasmid, then two successive conjugal transfers. Plasmids of five of six deletion classes, each with a different combination of drug resistance traits, retained conjugative ability and the UV-protecting, mutagenesis-enhancing and mutator effects of R46. (pKM101, used in the Ames system to enhance responsiveness to chemical mutagens, is one such mutant of R46.) Plasmids of a sixth class, represented by pKM115, conferred resistance only to streptomycin and were non-conjugative. All of several such plasmids (of independent origin) had a much stronger mutator effect than did R46, but lacked UV-protecting ability and did not enhance the mutagenic effect of UV irradiation. We infer that R46 possesses: (i) a gene, uvp, which increases capacity for error-prone repair of UV-damaged DNA, and thus causes both UV protection and enhancement of UV mutagenesis; (ii) gene(s) whose action in the absence of gene uvp greatly increases the frequency of spontaneous reversion of hisG46. A plasmid of another incompatibility group, pLS51, has UV-protecting and mutagenesis-enhancing effect but lacks the mutator property; introduction of pLS51 into a clone of hisG46 carrying a pKM115-type plasmid greatly reduced its spontaneous reversion rate, as expected if pLS51 also has a uvp gene able to modulate the mutator effect of R46-derived gene(s) in the pKM115-type plasmid.     

Transfer of chromosomal genes mediated by plasmid r68.45 in Rhodopseudomonas sphaeroides.

Plasmid R68.45 was transferred from Pseudomonas aeruginosa PAO25 to the photosynthetic species Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides by selection for resistance to antibiotics. R. sphaeroides strains carrying the plasmid could transfer the plasmid and also chromosomal genes to other strains of R. sphaeroides.     

Isolation and characterization of R-plasmid variants with enhanced chromosomal mobilization ability in Escherichia coli K-12

Enhanced Chromosome Mobilizing (ECM) plasmids derived from the IncP-1 plasmid R68 were isolated in Escherichia coli K-12 by the same methods which have given similar plasmids such as R68.45 in Pseudomonas aeruginosa. The chromosome mobilizing properties of such plasmids in E. coli were similar to those of R68.45 but while retaining the ability to transfer to P. aeruginosa they did not mobilize the chromosome of that organism. Restriction enzyme analysis of two such plasmids, pMO163 and pMO168, showed that they both possessed an additional segment of DNA. With pMO163, an addition of 0.8 kb is located near the TnA region and is characterized by the cleavage site pattern SmaI-HpaI-PstI-BamHI. For pMO168, the additional DNA segment is located at a different site, about 4.0 kb anti-clockwise from the EcoRI site. It was also characterized by the sites SmaI-(HpaI-PstI)-BamHI. No sequence homology has been found between the additional segments of either pMO163 or pMO168 and IS21 of R68.45. However homology of these additional segments was found with the E. coli K-12 chromosome suggesting that pMO163 and pMO168 arise by the acquisition of a transposable element from the E. coli K-12 chromosome.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Relationship between Pigment Producibility and Drug Resistance in Serratia marcescens

Among the clinical isolates of Serratia marcescens, non-pigmented cells appeared more frequently from pigmented, drug-resistant strains than from pigmented, drug-sensitive strains. Transfer of R plasmid from Escherichia coli to pigmented strains caused spontaneous loss of pigment producibility, whereas such spontaneous loss never occurred in fresh cultures of drug-sensitive strains. The non-pigmented strain was a better recipient of R plasmid from E. coli than was the pigmented strain. R plasmid was transferred from the non-pigmented strain to the pigmented strain at a higher frequency than from E. coli to the pigmented strain. The results of the present investigation suggest that transfer of R plasmid may be one of the reasons for the significant increase of non-pigmented, drug-resistant strains of S. marcescens in nature.     

Transfer of nodulation ability in Rhizobium using R68.45 derived plasmids

Summary Nodulation ability was transferred from Rhizobium meliloti L5.30 to the non-nodulating mutant Rhizobium trifolii 24K using plasmid R68.45. Transconjugants selected for the carbenicillin resistance (cb ^r) marker became simultaneously capable of nodulating clover and showed changes in phage sensitivity. Besides the indigenous plasmid of 90 MD (pUCS201), the nodulating transconjugants harbored the newly introduced plasmid pUCS202 (ca. 40 MD). After treatment of the transconjugants with curing agents the simultaneous loss of antibiotic resistance and ability to form nodules were associated with the disappearance of pUCS202. nod and cb ^r genes were cotransferred into R. trifolii strains by conjugation and transformation. There is genetic evidence that the nod gene(s) was integrated into R68.45.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using Bradyrhizobium japonicum USDA 123 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 × 10^-5 in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using B. japonicum USDA 438 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 × 10^-6). Mercury additions to soil, ranging from 10 to 50 μg/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.     

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli

Summary An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

H-NS Regulates DNA Repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30°C and nonfunctional at 37 to 40°C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40°C postirradiation, shows up to 30-fold higher survival than when incubated at 30°C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40°C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40°C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40°C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Properties of Mitomycin C-sensitive Mutants of Escherichia coli K-12

Strains hypersensitive to mitomycin C (MC) were isolated from Escherichia coli K-12 after treatment with nitrosoguanidine. Of 43 MC-sensitive strains tested for their ultraviolet light (UV) sensitivity and for their ability to reactivate UV-inactivated λ phage, 38 were found to be insensitive to UV irradiation and to be able to reactivate UV-irradiated bacteriophage λ. Some properties of the MC-sensitive, uvr⁺ mutants were analyzed. Synthesis of deoxyribonucleic acid (DNA) in MC-sensitive, uvr⁺ mutants was inhibited at a lower concentration of MC than in the wild-type strain. Mutant cells, labeled with ³H-thymidine and then exposed to MC, released radioactivity as low molecular weight compounds. The amount of radioactivity released was the same as that from the wild-type strain. MC-sensitive, uvr⁺ mutants, as well as the corresponding wild-type strain, were equally susceptible to induction of prophage 80 by UV irradiation. However, MC induction of prophage was achieved in MC-sensitive, uvr⁺ mutants at a lower concentration of the antibiotic than in the wild-type strain. Genetic experiments indicated that a gene controlling MC sensitivity is located close to that determining lactose fermentation of E. coli. It is situated on episome F′13, and the wild type is dominant to the MC-sensitive allele.     

Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine.

A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine. It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance. Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45.