umuDC Operon Research Articles

Mutagenesis Induced by Sub-Lethal Doses of Ciprofloxacin: Genotypic and Phenotypic Differences Between the Pseudomonas aeruginosa Strain PA14 and Clinical Isolates.

Bacterial resistance is a severe threat to global public health. Exposure to sub-lethal concentrations has been considered a major driver of mutagenesis leading to antibiotic resistance in clinical settings. Ciprofloxacin is broadly used to treat infections caused by Pseudomonas aeruginosa, whereas increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin has been reported for the reference strain, PAO1, in vitro. In this study we report increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin for another reference strain, PA14-UCBPP, and lower mutagenesis for clinical isolates when compared to the reference strain. This unexpected result may be associated with missense mutations in imuB and recX, involved in adaptive responses, and the presence of Pyocin S2, which were found in all clinical isolates but not in the reference strain genome. The genetic differences between clinical isolates of P. aeruginosa and the reference PA14-UCBPP, often used to study P. aeruginosa phenotypes in vitro, may be involved in reduced mutagenesis under sub-lethal concentrations of CIP, a scenario that should be further explored for the understanding of bacterial fitness in hospital environments. Moreover, we highlight the presence of a complete umuDC operon in a P. aeruginosa clinical isolate. Even though the presence of umuDC did not contribute to a significant increase in mutagenesis, it highlights the dynamic exchange of genetic material between bacterial species in the hospital environment.

UmuDAb: An Error-Prone Polymerase Accessory Homolog Whose N-Terminal Domain Is Required for Repression of DNA Damage Inducible Gene Expression in Acinetobacter baylyi.

In many bacteria, the DNA damage response induces genes (SOS genes) that were repressed by LexA. LexA represses transcription by binding to SOS promoters via a helix-turn-helix motif in its N-terminal domain (NTD). Upon DNA damage, LexA cleaves itself and allows induction of transcription. In Acinetobacter baumannii and Acinetobacter baylyi, multiple genes are induced by DNA damage, and although the Acinetobacter genus lacks LexA, a homolog of the error-prone polymerase subunit UmuD, called UmuDAb, regulates some DNA damage-induced genes. The mechanism of UmuDAb regulation has not been determined. We constructed UmuDAb mutant strains of A. baylyi to test whether UmuDAb mediates gene regulation through LexA-like repressor actions consisting of relief of repression through self-cleavage after DNA damage. Real-time quantitative PCR experiments in both a null umuDAb mutant and an NTD mutant showed that the DNA damage-inducible, UmuDAb-regulated gene ddrR was highly expressed even in the absence of DNA damage. Protein modeling identified a potential LexA-like helix-turn-helix structure in the UmuDAb NTD, which when disrupted, also relieved ddrR and umuDAb repression under non-inducing conditions. Mutations in a putative SOS box in the shared umuDAb-ddrR promoter region similarly relieved these genes’ repression under non-inducing conditions. Conversely, cells possessing a cleavage-deficient UmuDAb were unable to induce gene expression after MMC-mediated DNA damage. This evidence of a UmuDAb repressor mechanism was contrasted with the failure of umuDAb to complement an Escherichia coli umuD mutant for UmuD error-prone DNA replication activity. Similarly, A. baumannii null umuDAb mutant cells did not have a reduced UmuDˊ2UmuC-mediated mutation rate after DNA damage, suggesting that although this UmuDAb protein may have evolved from a umuDC operon in this genus, it now performs a LexA-like repressor function for a sub-set of DNA damage-induced genes.

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA- mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA- mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA- mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair

Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants.

Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria

The SOS genetic network is responsible for the repair/bypass of DNA damage in bacterial cells. While the initial stages of the response have been well characterized, less is known about the dynamics of the response after induction and its shutoff. To address this, we followed the response of the SOS network in living individual Escherichia coli cells. The promoter activity (PA) of SOS genes was monitored using fluorescent protein-promoter fusions, with high temporal resolution, after ultraviolet irradiation activation. We find a temporal pattern of discrete activity peaks masked in studies of cell populations. The number of peaks increases, while their amplitude reaches saturation, as the damage level is increased. Peak timing is highly precise from cell to cell and is independent of the stage in the cell cycle at the time of damage. Evidence is presented for the involvement of the umuDC operon in maintaining the pattern of PA and its temporal precision, providing further evidence for the role UmuD cleavage plays in effecting a timed pause during the SOS response, as previously proposed. The modulations in PA we observe share many features in common with the oscillatory behavior recently observed in a mammalian DNA damage response. Our results, which reveal a hitherto unknown modulation of the SOS response, underscore the importance of carrying out dynamic measurements at the level of individual living cells in order to unravel how a natural genetic network operates at the systems level.

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49.

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49.

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.

Converting a DNA damage checkpoint effector (UmuD2C) into a lesion bypass polymerase (UmuD'2C).

During the SOS response of Escherichia coli to DNA damage, the umuDC operon is induced, producing the trimeric protein complexes UmuD2C, a DNA damage checkpoint effector, and UmuD'2C (DNA polymerase V), which carries out translesion synthesis, the basis of 'SOS mutagenesis'. UmuD'2, the homodimeric component of DNA pol V, is produced from UmuD by RecA-facilitated self-cleavage, which removes the 24 N-terminal residues of UmuD. We report the solution structure of UmuD'2 (PDB ID 1I4V) and interactions within UmuD'-UmuD, a heterodimer inactive in translesion synthesis. The overall shape of UmuD'2 in solution differs substantially from the previously reported crystal structure, even though the topologies of the two structures are quite similar. Most significantly, the active site residues S60 and K97 do not point directly at one another in solution as they do in the crystal, suggesting that self-cleavage of UmuD might require RecA to assemble the active site. Structural differences between UmuD'2 and UmuD'- UmuD suggest that UmuD'2C and UmuD2C might achieve their different biological activities through distinct interactions with RecA and DNA pol III.

Sequence analysis and distribution in Salmonella enterica serovars of IS 3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS 3-like insertion elements, designated IS 123 0A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS 1230 A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS 1230C was truncated at nucleotide 774 relative to IS 1230B and therefore did not possess the 3′ terminal inverted repeat. The three IS 1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS 1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS 1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS 1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3′ remnant of IS 1230, designated IS 1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS 1230A.

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal R mutants recovered from animals inoculated with either wild-type or MutS − cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model.

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

Intrinsic polymerase activities of UmuD'(2)C and MucA'(2)B are responsible for their different mutagenic properties during bypass of a T-T cis-syn cyclobutane dimer.

In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD'(2)C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA'(2)B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (epsilon subunit) or holE (theta subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of epsilon, or the dnaE antimutator allele spq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD'C, MucAB, or MucA'B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA'(2)B complex is more efficient in promoting translesion replication than the UmuD'(2)C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA'(2)B than by UmuD'(2)C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD' interact physically with the replicative DNA polymerase.

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD'. UmuD', UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD' interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD' promote different events, UmuD and UmuD' interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with beta than it does with alpha, UmuD' interacts more strongly with alpha than it does with beta. We propose that the protein-protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines

The mutagenic actions of many chemicals depend on the activities of bacterial “mutagenesis proteins”, which allow replicative bypass of DNA lesions. Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins. Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101. Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309. This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens. We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo[4,5- f]quinoline (MeIQ), a typical heterocyclic amine mutagen. Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis. Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene.

The role of mutation frequency decline and SOS repair systems in methyl methanesulfonate mutagenesis.

Methyl methanesulfonate (MMS) is an SN2 type alkylating agent which predominantly methylates nitrogen atoms in purines. Among the methylated bases 3meA and 3meG are highly mutagenic and toxic. The excision of these lesions leads to the formation of apurinic (AP) sites and subsequently to AT-->TA or GC-->TA transversions. The in vivo method based on phenotypic analysis of Arg+ revertants of Escherichia coli K12 and sensitivity to T4 nonsense mutants has been used to estimate the specificity of MMS induced mutations. In the E. coli arg-his-thr- (AB1157) strain MMS induces argE3(oc)-->Arg+ revertants of which 70-80% arise by supL suppressor formation as a result of AT-->TA transversions. The remaining 20-30% arise by supB and supE(oc) suppressor formation as a result of GC-->AT transitions. The level of AT-->TA transversions decreases during starvation. This is a consequence of action of the repair mechanism called mutation frequency decline. This system which is a transcription coupled variant of nucleotide excision repair was discovered in UV induced mutations. We describe the mutation frequency decline phenomenon for MMS mutagenesis. MMS is a very efficient inducer of the SOS response and a umuDC dependent mutagen. In MMS treated E. coli cells mutated in umuDC genes the class of AT-->TA transversions dramatically diminishes. A plasmid bearing UmuD(D')C proteins can supplement chromosomal deletion of umuDC operon: a plasmid harbouring umuD'C is more efficient in comparison to that harbouring umuDC. Moreover, plasmids isolated from MMS treated and transiently starved E. coli AB1157 cells harbouring umuD(D')C genes have shown the repair of AP sites by a system which involves the UmuD'C or at least UmuD' protein.

Mutagenesis and more: umuDC and the Escherichia coli SOS response.

The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and post-translational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

Expression of the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold temperature.

Previous studies have shown that several bacterial species exhibit a multigenic response following temperature downshift (cold shock). Evidence for such a response in Salmonella typhimurium is reported, based on the isolation of a range of low-induction-temperature gene fusions containing Mudlux insertions. The fusions exhibited different levels of basal light at 30 degrees C, and were induced at different rates and to different degrees over several hours following a reduction in temperature to 10 degrees C. Of the Mudlux gene fusions isolated, one was found which produced essentially no light when grown at 30 degrees C but exhibited rapid and high-level induction when the temperature was reduced to 10 degrees C. The target of this gene fusion (which was named cspB) was shown to lie adjacent to the umuDC operon and to encode a homologue of the major cold-shock protein of Escherichia coli, CspA. Luminescence studies revealed that substantial light production occurred from the cspB::Mudlux fusion at or below 22 degrees C but not at higher temperatures, even following a temperature drop from 30 degrees C. Moreover, cspB mRNA levels were found to mimic this pattern of luminescence, suggesting that cspB expression occurs below a defined temperature threshold. The cspB mRNA was also found to be very stable at 10 degrees C but to become highly unstable when the temperature was raised towards the threshold temperature, even in the presence of rifampicin. Existing cellular RNases therefore appear to mediate the decay of cspB mRNA at high temperatures, but are incapable of this at low temperatures.

Roles of the mutagenesis proteins SamA′B and MucA′B in chemically induced frameshift mutagenesis in Salmonella typhimurium hisD3052

The mutagenesis induced by ultraviolet light and many chemicals in Escherichia coli is largely dependent upon the proteins encoded by the umuDC operon and their analogs. In Salmonella typhimurium, there are two sets of umuDC-like operons: the umuDC ST operon in the chromosome and the samAB operon located on the 60-MDa cryptic plasmid. The former operon, but not the latter, confers UV mutability on S. typhimurium. Nevertheless, the samAB operon, when carried on high-copy-number plasmids, can efficiently promote UV mutagenesis. In order to characterize the function of samAB in greater detail, we have compared the abilities of MucA′B and a putative activated form of SamAB, i.e. SamA′B, to promote chemically induced frameshift mutagenesis in S. typhimurium hisD3052. MucA′B is an activated form of the products of mucAB, which is the most potent umuDC analog characterized so far. We have used four plasmids, each carrying samA′, samB, mucA′ or mucB with a lac promoter instead of their own promoters. The results indicated that under the conditions of elevated expression, SamA′B can promote chemically induced frameshift mutagenesis by furylfuramide, aflatoxin B 1, 1-nitropyrene, and 1,8-dinitropyrene, with efficiencies comparable to, or even better than, MucA′B. Increase of the levels of expression enhanced the ability of SamA′B to promote the mutagenesis, while it decreased that of MucA′B. Surprisingly, the elevated expression of MucB alone significantly enhanced the frameshift mutagenesis induced by 1-nitropyrene and 1,8-dinitropyrene, whereas the elevated expression of SamB, MucA′ and SamA′ did not enhance it. These results suggest that the abilities of SamA′B and MucA′B to promote mutagenesis strongly depend on their levels of expression. The possible roles of these mutagenesis proteins in chemically induced frameshift mutagenesis are discussed.

Frameshift mutagenesis induced in Escherichia coli after in vitro treatment of double-stranded DNA with methylene blue plus white light: evidence for the involvement of lesion(s) other than 8-oxo-7,8-dihydro-2'-deoxyguanosine.

By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli. The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively. The SOS and umuDC dependences of these mutagenic processes have been investigated. Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced. However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon. In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis. Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E. coli.

The genetic requirements for UmuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis.

The umuDC operon of Escherichia coli, a member of the SOS regulon, is required for SOS mutagenesis. Following the posttranslational processing of UmuD to UmuD' by RecA-mediated cleavage, UmuD' acts in concert with UmuC, RecA, and DNA polymerase III to facilitate the process of translesion synthesis, which results in the introduction of mutations. Constitutive expression of the umuDC operon causes an inhibition of growth at 30 degrees C (cold sensitivity). The umuDC-dependent physiological phenomenon manifested as cold-sensitive growth is shown to differ from SOS mutagenesis in two respects. Intact UmuD, the form inactive in SOS mutagenesis, confers a significantly higher degree of cold sensitivity in combination with UmUC than does UmuD'. In addition, umuDC-mediated cold sensitivity, unlike SOS mutagenesis, does not require recA function. Since the RecA protein mediates the autodigestion of UnmD to UmuD', this finding supports the conclusion that intact UmuD is capable of conferring cold sensitivity in the presence of UmuC. The degree of inhibition of growth at 30 degrees C correlates with the levels of UmuD and UmuC, which are the only two SOS-regulated proteins required to observe cold sensitivity. Analysis of the cellular morphology of strains that exhibit cold sensitivity for growth led to the finding that constitutive expression of the umuDC operon causes a novel form of sulA- and sfiC-independent filamentation at 30 degrees C. This filamentation is observed in a strain constitutively expressing the single, chromosomal copy of umuDC and can be suppressed by overexpression of the ftsQAZ operon.