Effect of tsl mutations in decreasing radiation sensitivity of a recA- strain of Escherichia coli K-12.

It has been shown previously that the radiation sensitivity of lexA- strains of Escherichia coli K-12 can be suppressed by thermosensitive mutations (designated tsl) that are closely linked to the lexA locus and are thought to be intragenic suppressors of lexA mutations (Mount et al., 1973). When a recA mutation is crossed into a suppressed tsl- strain, the extreme radiation sensitivity usually conferred by a recA mutation is decreased, but there is no detectable change in genetic recombination deficiency. Increased resistance to UV in the tsl-reA-strains depends upon ability to synthesize active uvrA+ product.     

Partial suppression of the LexA phenotype by mutations (rnm) which restore ultraviolet resistance but not ultraviolet mulability to Escherichia coli B/r uvrA lexA

In Escherichia coli, lexA mutations eliminate expression of UV-inducible functions, causing pleiotropic effects which include sensitivity to ultraviolet (UV) light and loss of UV mutability. Selection for UV resistance, after 5-bromouracil (BU) treatment of E. coli B/r uvr A lexA-102, has yielded derivatives more resistant than lexA but still refractory to UV mutagenesis. The mutation responsible for the UV-resistant UV-nonmutable phenotype (rnm) is cotransducible with malB to about the same extent as is lexA-102 and is tightly linked to lexA-102 in at least one strain. The rnm mutation may therefore be an intragenic partial suppressor of the LexA phenotype. In addition to increased UV resistance and lack of UV mutability, rnm strains show improved ability to perform postreplication repair and to control postirradiation DNA degration compared to the lexA parent. We ascribe the properties of rnm mutants to their having reacquired control of Exonuclease V activity without having reacquired UV-inducible error-prone postreplication repair. We relate our results to current interpretations of UV mutagenesis and to models of coordinate regulation of UV-inducible functions.     

Role of the supX gene in ultraviolet light-induced mutagenesis in Salmonella typhimurium.

Salmonella typhimurium strains with supX mutations are more sensitive than wild type to killing by ultraviolet (UV) irradiation. Studies with strains bearing the leuD21 mutation revealed that inactivation of the supX locus by a nonsense mutation or a deletion results in a complete lack of ability to produce induced Leu+ reversion mutations after UV irradiation. Suppression of the nonsense supX mutation or the presence of an Escherichia coli K-12 F'-borne supX+ allele restored the capacity for induced reversions and increased cell survival after UV irradiation. Introduction of plasmid pKM101 into supX mutant strains also restored their capacity for UV mutagenesis as well as increased survival. The possible nature of the supX gene product and mechanisms by which it may affect expression of the inducible SOS error-prone repair system are considered.     

Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12.

When thymidine-requiring lexA- strains were starved for thymidine, the kinetics of survival were similar to those of a nearly isogenic lexA+ strain. The size distribution of cells in the lexA- and lexA+ cultures were, however, quite different. Whereas most of the cells in the starved lexA+ cultures grew into long filamentous forms (longer than 4.0 mum), many of the lexA- cells were found to have a normal rod shape (4.0 mum or shorter). It was shown that lexA- cells undergo more divisions during thymidine starvation than lexA+ cells. Furthermore, using an autoradiographic method to analyze deoxyribonucleic acid (DNA) distribution in the starved cells, we demonstrated that cells without DNA are produced in both normal and starved lexA- cultures at a much higher frequency than in lexA+ cultures. Some of these cells may be produced by breakdown of DNA, but we favor the hypothesis that they result from an abnormal cell division process. Since lexA mutations are dominant, we conclude that a diffusible product decreases the synthesis or activity of an inhibitor of cell division in lexA- strains when DNA synthesis is blocked by thymidine starvation.     

Effects of DNA-repair processes on the induction of genetic duplications in bacteria by ultraviolet light

A straightforward positive selection for genetic duplication is possible in strains of Salmonella typhimurium that carry the aroC321 allele. Strains with a single copy of this allele require phenylalanine, tyrosine and tryptophan for growth. Such strains give rise to tryptophan prototrophs, which still require phenylalanine and tyrosine, through the formation of a duplication that includes about 30% of the chromosome. We have constructed strains that permit the simultaneous study of duplications and mutations and have used these strains to explore the effects of DNA repair processes on the induction of duplications by ultraviolet light (UV). UV causes dose-dependent increases in the frequency of duplications in bacteria. The exposure required to induce duplications is much less in a delta uvrB strain than in repair-proficient strains, suggesting that duplications result from DNA lesions that are subject to excision repair. The photoreversibility of UV-induced preduplication lesions implicates pyrimidine dimers in the induction of duplications. Unlike its effect on the induction of mutations, the error-prone repair process associated with plasmid pKM101 does not enhance the induction of duplications. The prevention of duplication-formation by a recA mutation suggests that the formation of duplications involves recombinational events. Taken together, the data indicate that the same DNA lesions can be mutagenic and recombinagenic in bacteria, but that the two effects involve different pathways of processing DNA damage.     

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hyper-mutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group (rad6-1) or a mutation in excision repair (rad1-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.     

Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12.

We describe the isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. These mutations, designated spr(Am), were isolated and characterized in a lexA tif sfi genetic background. They abolished the sensitivity of the strain to UV light and resulted in high rates of synthesis of recA protein. Phage lambda+ failed to lysogenize the strains as observed with similar strains carrying non-amber spr mutations described previously, thereby indicating a constitutive expression of the phage induction pathway. Introduction of an amber suppressor mutation into a strain bearing the spr(Am) mutation restored expression of the LexA mutant phenotype. We conclude that spr mutations either inactivate or prevent synthesis of the lexA gene product and that loss of this product results in constitutive expression of the E. coli induction system in the tif sfi genetic background.     

Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli.

Two closely linked genes, mrdA and mrdB, located at ca. 14.2 min on the Escherichia coli chromosomal linkage map, seen to be responsible for the normal rod shape and mecillinam sensitivity of E. coli. The product of mrdA was concluded to be penicillin-binding protein 2, because mrdA mutations caused formation of thermosensitive penicillin-binding protein 2. The product of the mrdB gene is unknown. At 42 degrees, C, mutation in either of these genes caused formation of spherical cells and mecillinam resistance. Both mutations was recessive, and complementation, as detected in +-/-+ meroheterodiploids having the wild-type phenotype, provided strong evidence that the two mutations are in different complementation groups. P1 transduction suggested that the most plausible gene order is leuS-mrdA-mrdB-lip. The rodA mutation reported previously seems to be similar to the mrdB murations, but the identities of the two have not yet been proven.     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI, active in mutation-specific reversion, and uvsC, a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli.     

The mechanism of untargeted mutagenesis in UV-irradiated yeast

Summary The SOS error-prone repair hypothesis proposes that untargeted and targeted mutations in E. coli both result from the inhibition of polymerase functions that normally maintain fidelity, and that this is a necessary precondition for translesion synthesis. Using mating experiments with excision deficient strains of Baker's yeast, Saccharomyces cerevisiae, we find that up to 40% of cyc1–91 revertants induced by UV are untargeted, showing that a reduction in fidelity is also found in irradiated cells of this organism. We are, however, unable to detect the induction or activation of any diffusible factor capable of inhibiting fidelity, and therefore suggest that untargeted and targeted mutations are the consequence of largely different processes. We propose that these observations are best explained in terms of a limited fidelity model. Untargeted mutations are thought to result from the limited capacity of processes which normally maintain fidelity, which are active during replication on both irradiated and unirradiated templates. Even moderate UV fluences saturate this capacity, leading to competition for the limited resource. Targeted mutations are believed to result from the limited, though far from negligible, capacity of lesions like pyrimidine dimers to form Watson-Crick base pairs.     

Evidence that UV-inducible error-prone repair is absent in Haemophilus influenzae Rd, with a discussion of the relation to error-prone repair of alkylating-agent damage.

Haemophilus influenzae Rd and its derivatives are mutated either not at all or to only a very small extent by ultraviolet (UV) radiation, X-rays, methyl methanesulfonate, and nitrogen mustard, though they are readily mutated by such agents as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and nitrosocarbaryl. In these respects H. influenzae Rd resembles the lexA mutants of Escherichia coli that lack the SOS or reclex UV-inducible error-prone repair system. This similarity is further brought out by the observation that chloramphenicol has little or no effect on post-replication repair after UV irradiation. In E. coli, chloramphenicol has been reported to considerably inhibit post-replication repair in the wild type but not in the lexA mutant. Earlier work has suggested that most or all the mutations induced in H. influenzae by NC result from error-prone repair. Combined treatment with NC and either X-rays or UV shows that the NC error-prone repair system does not produce mutations from the lesions induced by these radiations even while it is producing them from its own lesions. It is concluded that the NC error-prone repair system or systems and the reclex error-prone system are different.     

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.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI , active in mutation-specific reversion, and uvsC , a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli. Received: 23 September 1996 / Accepted: 2 December 1996     

Effect of transforming DNA on growth and frequency of mutation of Streptococcus pneumoniae.

We studied the effect of the presence of homologous transforming DNA on the growth of several transformable strains of Streptococcus pneumoniae and on the frequency of mutation of these strains to various antibiotic resistances. We observed no effect on growth until the strains became competent, when growth was depressed. At the end of the competence period, some strains showed recovery to varying degrees, whereas others showed evidence of cell death. Growth was also depressed by the presence of DNA from Escherichia coli, indicating that recombination was not likely to be the cause of the observed effect. Furthermore, cell death was not caused by the induction of a prophage. Several of the strains showed increased mutation frequencies during the competence period, although treatment with E. coli DNA gave no such effect, indicating that the mutagenesis was due to recombination. We observed no mutagenesis due to UV irradiation of the strains. The possibility that integration of the transforming DNA may produce lesions which induce error-prone repair is discussed. Furthermore, a strain that showed no mutability by transforming DNA, indicating the presence of a more efficient repair system, gave evidence of producing higher amounts of the hex system when competent, and the possible relationship between these properties is discussed.     

Bacteriophage lambda mutants (lambdatp) that overproduce repressor.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.     

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.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Inducible UV repair potential of Pseudomonas aeruginosa PAO

Pseudomonas aeruginosa PAO lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. aeruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a recA-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aeruginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa PAO allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recA102 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.     

The genetic characterization of lexB32, lexB33 and lexB35 mutations of Escherichia coli: location and complementation pattern for UV resistance.

Summary Mutants of LexB have been isolated by their resistance to lysogenic induction by thymine starvation, their resistance to thymine starvation and on the basis of their UV sensitivity. Here, three mutations identified originally in strains lacking mutagenic response to UV-irradiation, unmB (Kato and Shinoura, 1977), have been further characterized, mapped by P1-mediated transduction with srl into the recA-tif-zab-lexB cluster at the lexB position and analysed for complementation with various lexB and recA mutations. From the results it was concluded that unmB mutations are identical to lexB mutations; consequently these mutations have been termed lexB32, lexB33 and lexB35.The mutations lexB33 and lexB35 do not complement any of the other lexB mutations and define therefore a new complementation type. The lexB32 mutation, which like the lexB34 mutation, results in moderate UV sensitivity has a complementation pattern similar to that of lexB34. However, unlike lexB34 the lexB32 behaves like a leaky mutation.The results are discussed in relation to the recA gene product and its control.