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.

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function.

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.

Replication of the linear chromosomal DNA from the centrally located oriC of Streptomyces ambofaciens revealed by PFGE gene dosage analysis.

From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously maped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.

An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes.

Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0.93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.

Stimulation of genetic instability and associated large genomic rearrangements in Streptomyces ambofaciens by three fluoroquinolones.

In Streptomyces ambofaciens NSA2002, pigmented wild-type colonies spontaneously give rise to pigment-negative (Pig-) mutants at a frequency of about 0.5%. This genetic instability is related to large deletions which can be associated with amplifications of DNA sequences. The influence of three fluoroquinolones (ciprofloxacin, enoxacin, and norfloxacin) on this property was investigated. At a survival rate higher than 60%, most colonies showed a patchwork phenotype consisting of phenotypically heterogeneous colonies harboring numerous mutant sectors. Moreover, the frequency of Pig- mutants rose to more than 90% at survival rates equal to or higher than 10%. Induced Pig- mutants showed the same phenotypical features as did spontaneous mutants. Most of them also harbored deletions, associated in some cases with DNA amplifications, in two loci of the large unstable region, AUD6 and AUD90 (derived from amplifiable unit of DNA). The size of deletions in induced mutants could rise to 1.5 Mb. These results show that ciprofloxacin, enoxacin, and norfloxacin greatly stimulate genetic instability and the occurrence of DNA rearrangements in S. ambofaciens. Moreover, these three fluoroquinolones had the same rank order for both toxic (i.e., antibacterial) and genotoxic activities. If the antibacterial effect of fluoroquinolones in S. ambofaciens is due to their interference with DNA gyrase, as shown for some other organisms, the genotoxic effect observed could be due to their interaction with this type II topoisomerase. This suggests that DNA gyrase is involved in the process of genetic instability in S. ambofaciens.

Stimulation of genetic instability in Streptomyces ambofaciens ATCC 23877 by antibiotics that interact with DNA gyrase.

In wild-type Streptomyces ambofaciens ATCC 23877, pigment-defective (Pig-) mutants arise at a frequency of about 0.5%; this genetic instability is related to genomic rearrangements such as deletions and/or amplifications of DNA sequences. On media containing oxolinic acid and novobiocin, which interact with the A and B subunits of DNA gyrase, respectively, the frequency of variants increased dramatically. The Pig- mutant frequency was increased to almost 100% on a medium containing oxolinic acid at a concentration allowing 55% survival. On solid medium containing either oxolinic acid or novobiocin at subinhibitory concentrations, most colonies exhibited a 'patchwork' phenotype, characterized by the presence of numerous Pig- sectors. Similar phenomena were not observed on media containing the transcriptional inhibitor rifampicin or the translational inhibitor streptomycin. Many of the Pig- mutants exhibited a pleiotropic phenotype and were affected in aerial mycelium formation, colony growth and/or prototrophy. Moreover, the same kinds of rearrangements (deletions and/or amplifications of DNA sequences) were found in both induced and spontaneous Pig- mutants. The results suggest either that DNA gyrase is directly involved in genetic instability or that an SOS-like system is implicated.

Ultraviolet light, mitomycin C and nitrous acid induce genetic instability in Streptomyces ambofaciens ATCC23877.

In Streptomyces ambofaciens ATCC23877, pigment-negative (Pig-) mutants occur at high frequency (about 0.7 x 10(-2)) in the progenies of wild-type colonies. Furthermore, the offspring of these Pig- mutants can either be phenotypically homogeneous or hypervariable (with no preponderant phenotype). Pig- mutants can also lack antibiotic production and present aerial mycelium deficiency, auxotrophy for arginine, oversensitivity to either ultraviolet (UV) light or mitomycin C and resistance to either novobiocin or nosiheptide. This genetic instability is related to both amplified DNA sequences and deletions. Mutagens such as UV light, mitomycin C and nitrous acid induced genetic instability and increased the Pig- mutant frequency to almost 30% even at a high survival rate. Induced Pig- mutants exhibited the same features as the spontaneous ones at both phenotypic and molecular levels. The frequency of detected genomic rearrangements after treatment was higher than 10%. We postulate that an SOS-like system is involved in the induction of genetic instability in S. ambofaciens.