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Journal of Bacteriology, May 2004, p. 2900-2905, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2900-2905.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Polymerases Leave Fingerprints: Analysis of the Mutational Spectrum in Escherichia coli rpoB To Assess the Role of Polymerase IV in Spontaneous Mutation

Erika Wolff, Mandy Kim, Kaibin Hu, Hanjing Yang, and Jeffrey H. Miller*

Department of Microbiology, Immunology, and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, California 90095

Received 21 October 2003/ Accepted 6 January 2004


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ABSTRACT
 
We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F'128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.


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INTRODUCTION
 
Damage-inducible polymerases (20, 22; for reviews, see references 9 and 16), such as the SOS-induced polymerase IV (Pol IV) and Pol V in Escherichia coli, not only bypass certain noncoding lesions but also increase replication errors across from normal bases (19, 20, 23). Their discovery has led to the suggestion that a significant fraction of spontaneous mutations in growing cells under normal conditions might be due to errors caused by basal levels of error-prone polymerases (18). The dinB-encoded Pol IV is the leading candidate, since the overexpression of dinB on high-copy plasmids leads to increases in base substitutions and frameshifts, particularly –1 frameshifts (11, 12, 23). Moreover, several studies have shown an approximately twofold decrease in spontaneous mutations in strains with an inactivating allele of dinB that also reduces the expression of three genes downstream of dinB-yafNOP (14, 18), although this effect is not present if only dinB is inactivated (14). The expression of dinB and yafNOP is increased after SOS induction by DNA-damaging agents (4), and these four genes have been shown to be part of an operon (14).

We decided to examine the spectra of base substitution mutations in strains with differing levels of dinB expression, since a comparison of detailed genetic fingerprints of these strains might reveal patterns specific to processes involving and not involving Pol IV. We recently characterized a system using mutations in the rpoB gene that yield the rifampin resistance (Rifr) phenotype at 37°C in order to analyze the base substitution profiles of mutagens and mutators (10). We have now characterized 77 mutations in rpoB. Each of the six base substitutions is monitored with a set of 9 to 17 sites. In the study reported here, we looked at cells that carry a single copy of the dinB operon on the chromosome and compared the mutational spectrum of these cells with those of strains with deletions of the dinB gene, strains that carry a second copy of the dinB operon on an F' plasmid, and strains that carry a multicopy plasmid with an insert containing the dinB operon in one case and just the dinB gene in another case. We showed that some mutational hot spots are specific for the overexpression of the dinB operon and that others are found in the spectrum of wild-type strains but not after the amplification of the dinB operon. A comparison of the different spectra leads us to conclude that spontaneous mutations occurring during exponential growth in the absence of SOS induction or a dinB-overexpressing plasmid do not contain a significant contribution from dinB-Pol IV-induced mutations.

Table 1 shows the strains used in this work. We examined the frequencies of Rifr mutants that result from mutations in rpoB in strains P90C, CC107, EW90, EW99, EW100, and EW101. EW90 carries a deletion of dinB (1) that is nonpolar for the expression of the other genes in the dinB operon, and CC107 (5) is a derivative of P90C (15) that has an F'128 with a mutated lac operon, proAB, and the dinB operon (11). The expression of dinB from the F' plasmid has been reported to be three times higher than that from the chromosome (11), so CC107 would have four times the dinB level of P90C. EW100 is a CC107 derivative carrying a multicopy plasmid that overexpresses the dinB operon, and EW101 is the same as EW100 except that the plasmid insert contains only the dinB gene. EW99 carries the plasmid without any insert. We did not detect any difference in mutation frequencies among CC107, P90C, or EW90, as shown in Table 2. EW100 and EW101 have six- to sevenfold higher Rifr frequencies than the EW99 control (Table 2).


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  		TABLE 1. E. coli K-12 strains used in this study


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  		TABLE 2. rpoB frequencies and rates of mutationsa

We analyzed mutations by direct DNA sequencing (for the methods used, see reference 10) and in a number of cases supplemented the sequence analysis with oligonucleotide hybridization (3, 17). In the these cases, colonies were spotted onto LB Omnitrays (Nunc, Rochester, N.Y.), grown overnight at 37°C, and transferred to nylon membranes (Hybond N) from Amersham (Piscataway, N.J.). The membranes were placed in denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 7 min and then in neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl [pH 7.2], 0.001 M EDTA) for 6 min. The membranes were baked at 80°C for 2 h and prehybridized (5x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA {pH 7.7}], 0.5% sodium dodecyl sulfate [SDS], 5x Denhardt's solution, 100 µg of herring sperm DNA/ml) during shaking at 47°C for 1 h. 32P-end-labeled probes (5 pmol of oligonucleotide strand, 10 U of T4 polynucleotide kinase, 5x T4 polynucleotide kinase buffer, 10 µCi of [{gamma}-32P]ATP/µl) were denatured at 100°C for 5 min, and 1 µl was added to hybridization solutions for each oligonucleotide tested. Following hybridization overnight at 47°C, membranes were washed twice in 2x SSPE-0.1% SDS at room temperature for 10 min, once in 1x SSPE-0.1% SDS at 47°C for 15 min, and once in 0.1x SSPE-0.1% SDS at 47°C for 10 min. Autoradiography was carried out with a phosphorimaging screen.

The oligonucleotides were complementary to the mutant and wild-type sequences at the following base mutation sites in E. coli (corresponding base pair mutations from the wild type to the mutant are indicated in boldface type): 437 (T->G), 5'-GTGTTATCGTTTCCCAGCT-3'; 443 (A->T), 5'-ATCGTTTCCCAGCTGCA-3'; 1534 (T->C), 5'-GCCAGCTGTCTCAGTTTAT-3'; 1538 (A->T), 5'-AGCTGTCTCAGTTTATGGA-3'; 1546 (G->A), 5'-TCAGTTTATGGACCAGAA-3'; 1547 (A->G), 5'-GTTTATGGACCAGAACAAC-3'; 1576 (C->A), 5'-CTGAGATTACGCACAAACG-3'; 1576 (C->T), 5'-TGAGATTACGCACAAACG-3'; 1714 (A->C), 5'-TCGGTCTGATCAACTCTCT-3'; 1721 (C->T), 5'-TGATCAACTCTCTGTCCGT-3'.

Table 3 shows the sequence results for 1,057 mutations in rpoB from the strains described above, as well as from EM90 and EC90, mutS (mismatch repair-deficient) derivatives of P90C and EW90, respectively. We can make several comparisons on the basis of Table 3. The sites that are most prominent in the two wild-type spectra, those of CC107 and P90C (with and without F'128, respectively), are well represented in the spectrum of EW90 (which has a deletion of dinB). (Note that the CC107 sample is twice the size of both the P90C and the EW90 samples.) For example, the five most frequent changes in the wild-type (CC107 and P90C) samples are AT->GC at sites 1547 and 1534, GC->AT at site 1576, AT->TA at site 443, and AT->CG at site 1714. The first four of these changes are also prominent in the spectrum of EW90 (which has a deletion of dinB), and the fifth one (AT->CG at site 1714) is still represented in EW90. Therefore, the polymerases operating in the absence of Pol IV are capable of producing the hot spots seen in the wild-type spectra. Only the GC->AT transition at site 1576 is lacking in prominence in the spectrum of P90C (the wild type without the F'128), but this mutation is also very prominent in EW90, the dinB deletion derivative of P90C. Moreover, the distributions of mutations in a mutS background, lacking mismatch repair, are very similar in strains with and without the dinB deletion (Table 3).


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  		TABLE 3. Distribution of mutations in rpoB

We can also compare the mutational profiles of strains CC107, P90C, and EW90 with that of EW100, the strain carrying pdinB-yafNOP and yielding an elevated level of rpoB mutations (Table 2). (It should be noted that the distribution of mutations in CC107 containing the plasmid without an insert [EW99; data not shown] is the same as that for CC107 alone.) A comparison of CC107 and EW100 (with the same sample size of approximately 300 mutations each) shows that although there are marked similarities with regard to some of the hot spots, there are also several clear differences. Most notably, the GC->TA transversion at site 1576 is the most prominent change for EW100 with 62 occurrences, compared with 4 occurrences for CC107. The other transversion at this site, GC->CG, is also more prominent in EW100 than in CC107. In addition, the AT->CG transversion at position 437 is represented by 17 occurrences in the EW100 sample, but it is absent from the spectra of CC107 and P90C (with a sample size of 146). Thus, there are three hot spots present in the Pol IV-overproducing strain (EW100) that are essentially absent from the wild-type (CC107) strain. On the other hand, the AT->TA transversion at 443 appears to occur frequently in all of the strains that lack the Pol IV-overexpressing plasmid but not in the strain (EW100) with the overexpressing plasmid (38 occurrences in CC107 versus 4 occurrences in EW100 for the same sample size).

Figure 1 displays the data from Table 3 by position so that different base substitutions at the same site can be visualized together while the mutation type can still be distinguished. It can be seen that position 1576 is the most prominent hot spot in the EW100 (pdinB-yafNOP) spectrum, with the total number of mutations there accounting for 30% of all the base substitutions in the EW100 profile. In contrast to the predominance of GC->TA and GC->CG transversions at site 1576 in the pdinB-yafNOP (EW100) spectrum, mutations at this site in the wild-type (CC107) spectrum are mostly GC->AT transitions. Although there are many similarities between these two spectra, they clearly differ at positions 437 and 443, in addition to the pronounced difference in transversions at 1576. We also found no difference in the distributions of mutations in EW100 (carrying pdinB-yafNOP) and EW101 (carrying pdinB) (Fig. 2).



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  		FIG. 1. Distribution of mutations in rpoB that lead to Rifr in the wild-type strain (CC107) and in a derivative (EW100) carrying dinB-yafNOP on a multicopy plasmid. Similar sample sizes (294 for CC107 and 289 for EW100) were used. Different base substitutions at the same site are indicated by different patterns. The positions of the sites are indicated with numbers above and below (see Table 3) and are not to scale.



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  		FIG. 2. Distribution of mutations in rpoB that lead to Rifr in the wild-type strain (CC107) and a derivative (EW101) carrying the dinB gene on a multicopy plasmid. Because different sample sizes are used, the peak heights here represent percentages of the sample size (140 for EW101 and 294 for CC107, with the heights of the EW101 peaks being effectively normalized [by multiplying by 2.1] to fit the CC107 sample size). The vertical axis indicates actual numbers, but the scales of the peak heights differ. This approach allows a visual comparison with Fig. 1. Note that the transversions at site 1576 are prominent in EW101, as they are in EW100 (Fig. 1). In EW101, at site 1576, GC->CG transversions occur in 17 of 140 instances, compared with 5 of 294 instances for CC107, and GC->TA transversions occur in 15 of 140 instances, compared with 4 of 294 instances for CC107.

Taken together, the data in Table 3 and Fig. 1 and 2 argue that spontaneous base substitutions in wild-type strains such as P90C and CC107 do not contain significant contributions from the dinB-encoded Pol IV or products of the other dinB operon genes (yafNOP). The underrepresentation of the prominent hot spots in rpoB that are specific to the pdinB-yafNOP-containing strain (EW100) in the spectrum of mutations from CC107 allows us to place an upper limit on the contribution from Pol IV and putative products of the yafNOP genes at no more than 10%. The data also indicate that there is no difference in the base substitution mutational spectra of strains with and without the F'128 plasmid, even though there is a fourfold difference in Pol IV levels. It should be noted that all of these determinations are for mutations occurring in growing cells. For adaptive mutations occurring in the stationary phase (2, 8), a requirement for Pol IV has been reported (13, 21).


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ACKNOWLEDGMENTS
 
We thank Roger Woodgate and Pat Foster for bacterial strains.

J.H.M. was supported by a grant from the National Institutes of Health (grant no. ES0110875).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 609 Charles E. Young #1602, Los Angeles, CA 90095. Phone: (310) 825-8460. Fax: (310) 206-3088. E-mail: jhmiller{at}mbi.ucla.edu. Back


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Journal of Bacteriology, May 2004, p. 2900-2905, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2900-2905.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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