Cyanobacteria toxins in the Salton Sea

Background

Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.

Results

Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m² of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.

Conclusion

Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.     

Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.     

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H₂O₂, and the rpoB mutant frequencies were calculated. The frequency of the H₂O₂-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.     

The action of 4-hydroxyaminobiphenyl in Escherichia coli: cytotoxic and mutagenic effects in DNA repair deficient strains

The cytotoxic and mutagenic effects of 4-hydroxyaminobiphenyl (N-OH-ABP) were studied using Escherichia coli strains with different repair capacities. N-OH-ABP was equally cytotoxic for uvrA and recA mutants as well as in wild-type cells while polA mutants strains proved particularly sensitive to its toxicity. In contrast, the mutation frequency in the uvrA strains tested was elevated to 30–400-fold the wild-type values. We suggest that aminobiphenyl-DNA adducts responsible for mutation are repaired by UVR endonuclease but different pathways exist for removal of DNA lesions responsible for bacterial killing. From the ³²P-postlabelling analysis, it was concluded that ABP-DNA adducts can be relatively rapidly repaired in wild-type strains, while persisting in the uvrA strains.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

The dependence of postreplication repair on uvrB in a recF mutant of Escherichia coli K-12.

Summary Mutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated vir and wild type rates of excision gap closure in repairing UV damage to their own DNA. The same mutants showed reduced rates of postreplication repair strand joining. When uvrA ^- recF^- or uvrB ^- recF^- strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m². We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF may be similar. An intermediate in postreplication repair may accumulate in the uvr ^- recF^- strain.     

New Rapid and Simple Methods for Detection of Bacteria and Determination of Their Antibiotic Susceptibility by Using Phage Mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria (“infectious centers”). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Properties of strains of Escherichia coli K12 carrying mutant lex-1 and uvrA6 alleles

Summary Strains with both uvrA6 and the lex-1 mutations are more sensitive to ultraviolet light (UV) than isogenic strains with only one of the mutations. The lex ^- uvrA^-double mutant has the same sensitivity to methyl-methane-sulfonate as the lex ^- uvrA⁺single mutant. UV-irradiated cultures of lex ^- uvrA⁺and lex ^- uvrA^-strains do not produce more streptomycinresistant mutants per survivor than unirradiated cultures. UV-irradiated cultures of a lex ⁺ uvrA^-strain produce large yields of mutants at both low (4 ergs/mm²) and high (25 ergs/mm²) doses of UV compared with the lex ⁺ uvrA⁺ strain which produce an intermediate yield of mutants at 25 ergs/mm², and a small yield at 4 ergs/mm², not significantly greater than unirradiated cultures. A dose of UV which does not induce mutations in strains with the lex-1 mutation produces only a small decrease in DNA synthesis in the lex ^- uvrA⁺strain. The results are interpreted to mean that the lex-1 mutation probably does not affect the same pathway of DNA repair as the uvrA ⁺product (i.e. excision of thymine dimers), and that the absence of UV-induced mutations in irradiated cultures of lex ^-strains is probably not due to a cessation of DNA replication.     

Survival of Shewanella oneidensis MR-1 after UV Radiation Exposure

We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D₃₇ and D₁₀ values of 5.6 and 16.5% of Escherichia coli K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m⁻², 568 J of UVB m⁻², 25 kJ of UVA m⁻², and 558 J of solar UVB m⁻², respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10⁻⁵ to 10⁻⁶. Unlike in E. coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents.     

Identification of the uvrB gene product

We have constructed plasmids carrying various restriction fragments of the biouvrB region of the Escherichia coli chromosome. By analyzing the proteins synthesized in maxicells from the uvrB⁺ plasmid pDR1494, and its derivatives containing γδ sequences inserted in the uvrB gene, we have determined that the uvrB gene is about two kilobase-pairs in length, that it is transcribed clockwise on the standard E. coli genetic map, and that it codes for a single polypeptide of M_r = 84,000. The number of uvrB polypeptides in a normal cell is estimated to be about 140.We have also found that the uvrB gene is cut by EcoRI near its promoter.     

Isolation and Mapping of t Gene Mutants of Bacteriophage T4D

A procedure for selective isolation of T4 t mutants is described. At 120 min after infection of Escherichia coli cells with a low multiplicity of T4 bacteriophage, the mixture was sedimented through a linear sucrose gradient, and infected cells that remained intact were collected as the fastest sedimenting fraction. Ten to 50% of the phage released by chloroform treatment of this fraction were t mutants. Collection of a high proportion of t mutants depended on efficient elimination of cells that would survive because of superinfection lysis inhibition. This was accomplished by early addition of anti-T4 serum and heat-killed cells to inactivate progeny wild-type phage released at the normal burst time. Of 85 t mutants that were isolated and mapped, 23 new mutations were found, 14 of which are suppressible by an rII mutation and 9 of which are suppressible by rII or amber suppressors. Two hot-spot sites for spontaneous mutations were found; 14 mutants at one site, represented by a frameshift mutation, and 12 mutants at a second site were obtained from 39 spontaneous mutants independently isolated from different parental plaques. On our map of the t gene, the distance between the farthest t mutations is 6% recombination. A nonreverting triple t mutant, constructed to contain a frameshift mutation between two amber mutations, exhibited the same t mutant phenotype observed with revertible t mutants.     

Two mini-F-encoded proteins are essential for equipartition

The par region of mini-F is both necessary and sufficient to promote equipartition of plasmid copies to daughter cells. It is approximately 2.5 kb long and contains the coding sequences for two proteins, F1 (41 kDa) and F2 (37 kDa). We isolated 13 mutants of a phage λ-mini-F hybrid that form unstable plasmids. Two of these putative Par^? mutants are fully suppressible nonsense (amber) mutants. One of the amber mutants, par-41, eliminates the synthesis of F1, generating a large nonsense fragment of the protein. The other mutant, par-36, eliminates the synthesis of F2. Thus both proteins appear to be essential for plasmid equipartition.