Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light I. Effect of rec− Mutations on Liquid Holding Recovery

We have examined various derivatives of Escherichia coli K-12 for liquid holding recovery, a type of recovery originally observed in E. coli B irradiated with ultraviolet light. Although most of the K-12 derivatives tested showed relatively little or no recovery under our conditions, four of the six independent rec(-) mutants examined, those carrying recA1, rec-12, recA13, and rec-56, respectively, displayed marked recovery. These mutants are distinguished from rec(+) strains by their increased sensitivity to ultraviolet radiation and decreased ability to undergo genetic recombination. Two of them have also been reported to release large amounts of their deoxyribonucleic acid as acid-soluble material, especially after irradiation. None of the three uvr(-) mutants examined, containing uvrA6, uvrB5, or uvrC34, showed comparable liquid holding recovery. The one rec(-) uvr(-) derivative tested, carrying recA13 and uvrA6, did not appear to undergo liquid holding recovery, although recA13 uvr(+) strains did. Genetic analysis of one strain, a recA13 mutant, indicated that all the rec(+) derivatives obtained from it by conjugation, transduction and reversion, had lost the property of showing liquid holding recovery. From these results, we conclude that in E. coli K-12 the expression of liquid holding recovery depends upon certain rec(-) mutations.     

Cotransduction and Cotransformation of Genetic Markers in Bacillus subtilis and Bacillus licheniformis

Bacteriophage SP-15, a large generalized transducing phage of Bacillus, was compared with phages PBS-1 and SP-10 for the ability to cotransduce pairs of genetic markers exhibiting different degrees of linkage. When auxotrophs of B. subtilis W-23 were used as recipients, SP-15 and PBS-1 effected a much higher frequency of cotransduction than did SP-10 with markers that were not closely linked. With more closely linked loci, the differences were not as great. SP-15 cotransduced linked markers at a higher mean frequency than PBS-1, suggesting that SP-15 is able to transfer a larger fragment of the Bacillus genome than any phage heretofore described. The frequency of the joint transfer of genetic markers in B. licheniformis was lower via transforming deoxyribonucleic acid than by transduction with phage SP-10. The availability of three procedures for genetic exchange-transduction by SP-15 and SP-10 as well as transformation-each of which reveals a different degree of linkage, makes B. licheniformis 9945A especially amenable to genetic analysis.     

Suppression of Ultraviolet Sensitivity in Escherichia coli uvr502 by the su58 Missense Suppressor

Introduction of the su58 missense suppressor into the chromosome of the uvr502 mutant, either by mutation or by transduction, results in a marked increase of ultraviolet resistance of the uvr502 mutant. In the uvr(+) genetic background, the su58 suppressor causes some decrease of ultraviolet resistance and marked increase in the spontaneous mutation frequency. The presence of the su58 suppressor did not decrease the high frequency of spontaneous mutants in the population of the uvr502 strain. However, the significant increase in spontaneous mutant frequency in the uvr(+)su58 strain makes the difference between the uvr502 su58 and the uvr(+)su58 strains 18 times lower than that between the uvr502 and the uvr(+) suppressor-free strains. Since the missense suppressors act at the level of translation, the results suggest that the product of the uvr(502) gene is a protein.     

Transfer of erythromycin resistance in Staphylococcus aureus.

In the course of an outbreak of enteritis and conjunctivitis, Staphylococcus aureus was isolated from newborn infants. Strains cultured at a later phase of the outbreak differed from those found at the beginning in being resistant to several antibiotics, showing resistance to typing phages and releasing phages of the same lysis spectrum (10(9) p.f.u./ml after heating at 56 degrees C for 2 min). Transduction experiments with a strain and its cell-free lysate showed that inducible erythromycin resistance was transferable to strains isolated at the beginning of the outbreak and to laboratory strains. Plasmid origin of resistance was confirmed by (i) high transduction frequency; (ii) transduction to RN981 rec- mutants; (iii) kinetics of transduction; (iv) elimination of resistance. Mixed culture experiments yielded transductants at high frequency with resistance to erythromycin, streptomycin and tetracycline.     

The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants.

The effect of the CAM-OCT plasmid on responses to UV irradiation of Pseudomonas aeruginosa recA mutants was characterized. Mutant alleles examined included rec-1, rec-2, and recA7::Tn501. The plasmid substantially enhanced both survival and mutagenesis of RecA- cells after treatment with UV light. Survival of the RecA-(CAM-OCT) cells after UV irradiation was intermediate between that seen in the wild-type P. aeruginosa PAO1 and the increased survival seen in PAO1(CAM-OCT) cells. Mutability was quantitated by the reversion to carbenicillin resistance of strains carrying a bla(Am) mutation on a derivative of plasmid RP1. UV-induced mutagenesis of CAM-OCT carrying recA mutants occurred at levels comparable to that seen in PAO1(CAM-OCT). The ability of CAM-OCT plasmid to suppress the recombination deficiency in recA mutants was tested by assaying for bacteriophage F116L-generalized transduction of a Tn7 insertion in the alkane utilization genes of CAM-OCT. Transduction of the Tn7 insertion was not detected in RecA-(CAM-OCT) strains but was easily seen in PAO1(CAM-OCT), indicating that the plasmid does not encode a recA analog. The results indicate that the CAM-OCT UV response genes are expressed in RecA- cells, which differs from results seen with other UV response-enhancing plasmids. The results suggest that CAM-OCT either encodes several UV responses genes itself or induces chromosomal UV response genes by an alternate mechanism.     

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.     

Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium

We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors.     

Dark-Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light III. Effect of rec Mutations on Recovery of Excision-Deficient Mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to excise pyrimidine dimers from their deoxyribonucleic acid (DNA) because of a uvr mutation show a higher survival when plated on a minimal salts medium after exposure to ultraviolet radiation than when plated on a complex medium such as nutrient agar containing yeast extract. This response has been called minimal medium recovery (MMR). Recovery of uvr mutants can take place in liquid as well as on solid medium, but not in buffer or under conditions of amino acid starvation that do not permit cell growth and normal DNA replication. MMR can thus be distinguished from the recovery of recombination-deficient (rec(-)uvr(+)) derivatives of K-12 which can occur under conditions where growth is not possible. Because MMR is characteristic of excision-defective mutants, it evidently reflects a type of repair independent of excision. We have obtained genetic evidence that MMR is determined by the rec genes, which also control recombination in K-12. Cells carrying a uvr mutation together with recA13, recA56, recB21, or recC22 failed to show MMR and were more sensitive to ultraviolet radiation than either their rec(+)uvr(-) or rec(-)uvr(+) parents. The rec(+)uvr(-) derivatives obtained from recA uvr(-) strains by transduction or by reversion regained the capacity for MMR. Our results indicate that inactivation of any one of the three genes, recA, recB, or recC, prevents cells from showing MMR.     

UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E. coli B/r having the same tyr(Oc) allele and different excision-repair capabilities: uvr+ (excision repair initiated by wild-type UvrABC activity), uvrA (excision repair defective), uvrA/pdenV-7 (excision repair initiated by endonuclease V of bacteriophage T4, DenV activity), and uvr+/pdenV-7 (excision repair initiated by UvrABC and DenV activities). The production of Tyr+ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events. Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutagenic photoproducts. When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations. This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer (pyrimidine (6-4) pyrimidone) photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers.     

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.     

rec-2-dependent phage recombination in Haemophilus influenzae.

The genetic transformation mutant Rd(DB117)rec- has a pleiotropic phenotype that includes reduced levels of phage recombination. Physical mapping experiments showed that this strain has a 78.5-kbp insertion in the rec-2 gene. The rec-2 dependence of phage recombination was reexamined to determine whether the defective phenotype in Rd(DB117)rec- was due to the simple disruption of the rec-2 gene or whether trans-acting factors from the inserted DNA were responsible. Analysis of strains with transposon insertions in the rec-2 gene showed that they were also defective for phage recombination. Therefore, the phage recombination defect was due solely to the disruption of the rec-2 gene. Strain KB6 is proficient for phage recombination but has a defect in genetic transformation resembling that of Rd(DB117)rec-. The transformation defect of KB6 could be complemented by the wild-type rec-2 gene, showing that the rec-2 contributions to genetic transformation and phage recombination were uncoupled in this strain. The rec-2-dependent phenotype of KB6 suggests that the rec-2 gene participates in genetic transformation and phage recombination in different ways.     

Metabolic characterization of the viable, residually dividing and nondividing cell classes of recombination-deficient strains of Escherichia coli.

The abilities of rec+, recB- recC-, recA-, and recA- recB- rec C- strains to support growth of bacteriophage T4, to take up oxygen, and to maintain cell integrity have been measured. (i) With respect to bacteriophage T4 growth, T4 phage is produced with identical lysis time in single -step growth curves with all strains tested. rec- strains show reduced phage production (fewer infected centers), but the average burst size per infected center is the same for all strains tested. Some rec- cells are unable to produce any phage, whereas the remainder of the rec-cells produce phage as rapidly and as efficiently as rec+ cells. (ii) With respect to oxygen consumption, rec- strains are deficient relative to the rec+ control to the same extent as the deficiency in phage production by theculture. The reduction in oxygen consumption is coordinate with reduction in rate of mass increase of the rec- culture. (iii) With respect to cell integrity, rec- cultures show increased lysis as measured by release of beta-galactosidase into the medium. The kinetics of release suggest that rec- nondividing cells all eventually lyse. These results are most consistent with the idea that rec- residually dividing cells and viable cells are metabolically normal according to the parameters we have measured, whereas nondividing cells are metabolically inactive.     

Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.     

Genetic mapping of a mutation conferring sensitivity to bacteriophage Mu in Salmonella typhimurium LT2.

Two strains of Salmonella typhimurium LT2, SA1475 and MA411, were fortuitously found to be sensitive to bacteriophage Mu. The Mu-sensitivity allele of SA1475 was called musA1 and shown to be linked to the histidine operon both in conjugation and transduction experiments. The Mus allele of MA411 was unlinked to the his region and was tentatively designated musB2. Strains carrying large deletions of the his operon were also tested for Mu sensitivity; those of which the his-rib region is deleted were also sensitive to Mu. Transduction data led to the order zee-2 hisOGDCBAHFIE gnd musA. An Hfr injecting the his operon early (HfrK9) an carrying hisG9424::Tn10 delta 4 delta 11 and musA1 was isolated; this Hfr made it possible to introduce the Mus character into most derivatives of S. typhimurium LT2. Since strain SA1475 is resistant to bacteriophage P1, it could be used to select a new P1-Mu hybrid which has the host range of Mu and the transduction properties of P1.     

Generation of transducing particles in Staphylococcus aureus.

Transduction of plasmid pC194 and bacteriophage phi 11de varied inversely with the multiplicity of infection. As the multiplicity of infection decreased from 10(-1) to 10(-5) PFU/CFU, the transduction frequency of pC194 increased 10(4)-fold; the transduction frequency of phi 11de increased 300-fold with a 100-fold decrease in multiplicity of infection. Physical and genetic analysis of the transduced DNA showed that pC194 resided in the phage particle as a random, circularly permuted linear concatemer. In DNA prepared from phage that cotransduced pC194 and phi 11de, pC194 resided in the transducing phage primarily as a linear multimer of 15.8 kilobases, or about 5.4 pC194 monomers. The pC194 multimer was randomly inserted into the phi 11 genome.     

R plasmids which alter ultraviolet light-sensitivity and enhance ultraviolet light-induced mutability in Pseudomonas aeruginosa.

R plasmids pMG1, R2, R931 and pMG15 increased the survival of Pseudomonas aeruginosa exposed to ultraviolet radiation (u.v.) in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA mutant. The R plasmid RPL11 reduced u.v.-survival in the wild type, and uvr and polA mutants but did not alter the u.v.-response of a recA host. All the plasmids enhanced the level of spontaneous and u.v.-induced back mutation (Trp+) in a trpB1 strain. The effect of sublethal concentration of sodium arsenite following u.v.-irradiation was examined. It was concluded that in strains trpB1(pMG1) and trpB1(R931), u.v.-protection is determined by a recA+-dependent, arsenite-sensitive repair pathway, whereas in strains trpB1(R2) and trpB1(pMG15), u.v.-protection is determined by a recA+-dependent, arsenite-insensitive step in DNA repair.     

Genetic characterization of the inducible SOS-like system of Bacillus subtilis.

The SOS-like system of Bacillus subtilis consists of several coordinately induced phenomena (e.g., cellular filamentation, prophage induction, and Weigle reactivation of UV-damaged bacteriophage) which are expressed after cellular insult such as DNA damage or inhibition of DNA replication. Mutagenesis of the bacterial chromosome and the development or maintenance of competence also appear to be involved in the SOS-like response in this bacterium. The genetic characterization of the SOS-like system has involved an analysis of (i) the effects of various DNA repair mutations on the expression of inducible phenomena and (ii) the tsi-23 mutation, which renders host strains thermally inducible for each of the SOS-like functions. Bacterial filamentation was unaffected by any of the DNA repair mutations studied. In contrast, the induction of prophage after thermal or UV pretreatment was abolished in strains carrying the recE4, recA1, recB2, or recG13 mutation. The Weigle reactivation of UV-damaged bacteriophage was also inhibited by the recE4, recA1, recB2, or recG13 mutation, whereas levels of Weigle reactivation were lower in strains which carried the uvrA42, polA5, or rec-961 mutation than in the DNA repair-proficient strain. Strains which carried the recE4 mutation were incapable of chromosomal DNA-mediated transformation, and the frequency of this event was decreased in strains carrying the recA1, recB2, or tsi-23 mutation. Plasmid DNA transformation efficiency was decreased only in strains carrying the tsi-23 mutation in addition to the recE4, recA1, or recB2 mutation. The results indicate that the SOS-like system of B. subtilis is regulated at different levels by two or more gene products. In this report, the current data regarding the genetic regulation of inducible phenomena are summarized, and a model is proposed to explain the mechanism of SOS-like induction in B. subtilis.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.