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. Author manuscript; available in PMC: 2009 Mar 1.
Published in final edited form as: DNA Repair (Amst). 2008 Jan 31;7(3):507–514. doi: 10.1016/j.dnarep.2007.10.003

Deletion of dnaN1 generates a mutator phenotype in Bacillus anthracis

Hanjing Yang 1, Jeffrey H Miller 1
PMCID: PMC2367095  NIHMSID: NIHMS41895  PMID: 18242150

Abstract

The dnaN gene in eubacteria is an essential gene that encodes the β subunit of replicative DNA polymerase. Nearly all eubacterial genomes sequenced to date predict a single copy of the dnaN gene in a well-conserved neighboring gene context. However, 19 genomes out of 348 scanned, including Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus weihenstephanensis, predict more than one dnaN gene. In most cases, these genomes appear to maintain a copy of the dnaN homolog in its usual neighboring gene context (designated as dnaN1) in addition to a second copy (designated as dnaN2) in an entirely different gene context. We used B. anthracis as our model system to investigate the role of these DnaNs. We constructed a single knockout mutant of dnaN1 and of dnaN2; however, we could not make a viable double knockout mutant of dnaN1 and dnaN2. The dnaN1 knockout mutant displays a markedly reduced colony size. It also displays a significantly increased mutation rate, which is similar to that of a mismatch repair deficient strain and to a strain deficient both in dnaN1 and mismatch repair. The dnaN2 knockout mutant, however, has a similar growth rate and a comparable mutation rate to that of the wild type. This is the first study demonstrating the existence of two functional DnaN homologs in the B. anthracis genome, with DnaN1 appearing to be more crucial than DnaN2. Our results also suggest the direct involvement of DnaN1 in the DNA mismatch repair process, which is consistent with previous findings.

1. Introduction

The dnaN gene of Escherichia coli encodes the β subunit of E. coli DNA polymerase III [1–4]. The β subunit dimerizes to form a ring-shaped sliding clamp, which encircles an intact duplex DNA and moves freely on it [5,6]. This ring-shaped topology is also observed in the homotrimer (or heterotrimer) of proliferating cell nuclear antigen (PCNA), a eukaryotic and archaeal counterpart of the β subunit, despite their unrelated sequences [7–11]. Owing to this unique topology, the sliding clamp acts as a processivity factor for the replicative DNA polymerase by tethering the core polymerase to its respective DNA template [5,12]. For example, in the presence of the β sliding clamp the DNA synthesis rate and the processivity of the E. coli core polymerase increases from less than 20 nucleotides s−1 to 1kb s−1 and less than 10 base pairs to over 50 kb, respectively [2]. The high processivity of the replicative DNA polymerase is vital for survival.

Recent studies have shown, however, that the function of the sliding clamp is not limited to serving as a subunit of the replicative DNA polymerase [1–4,13]. In E. coli, the β sliding clamp is found to also play a role in okazaki fragment maturation by interacting with DNA ligase and DNA polymerase I [14]. Additionally, it is also involved in various DNA repair processes by interacting with DNA polymerase II, translesion DNA polymerase IV and V [3,15–17], and DNA mismatch repair proteins MutS and MutL [14,15,18]. Studies also show that PCNA from eukaryotic replisome interacts with many protein components that are involved in DNA replication, repair and recombination, and cell cycle control [13,19]. In summary, the sliding clamp plays a significant role in the protein interaction network associated with various DNA processing activities.

DNA mismatch repair machinery is one of several important DNA repair systems that encounter errors arising during DNA replication. MutS and MutL are the two central protein components in mismatch repair in bacteria. MutS directly recognizes and binds to the mismatched base pair, which then recruits MutL to trigger the subsequent repair events [20]. López de Saro and O’Donnell first reported the involvement of β clamp in mismatch repair by demonstrating protein-protein interactions between β clamp and MutS [14]. Later, Dalrymple et al identified a pentapeptide motif (QL[SD]LF) that enables MutS and several other proteins to interact directly with the β clamp [15]. Recently, López de Saro et al examined in detail the interactions between MutS (MutL) and the β clamp. They showed that MutS and MutL with mutated β clamp binding sites are defective in both &beta clamp binding and mismatch repair [18]. They proposed that the β clamp is an essential player in orchestrating the sequence of events in this multi-step repair pathway in the cell.

The β sliding clamp is essential for cell viability. With the exception of several genomes that will be described below, only one gene encoding the β subunit homolog, dnaN, is predicted in nearly all eubacterial genomes that have been sequenced thus far [21]. Thus far, a viable dnaN knockout mutant has not been reported in the genomes with only one dnaN gene. However, point mutation mutants as well as temperature sensitive mutants of the β clamp have been constructed and studied, and have provided insights into β clamp structure and function [22–25]. In this work, we report that 19 genomes, including Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus weihenstephanensis, predict more than one dnaN gene. We used B. anthracis as a model system to investigate the role of two annotated dnaNs, dnaN1 and dnaN2. We were able to construct the dnaN1 and dnaN2 single knockout mutants in B. anthracis. We also determined the mutation rates and mutational specificities in the dnaN1 and dnaN2 knockout mutants. We discuss the relationship of the β clamp with regard to the DNA mismatch repair machinery.

2. Materials and methods

2.1 Phylogenetic study of DnaN homologs

The protein sequences of DnaN homologs were obtained from the BLAST database [26,27]. A single complete genome of each species was chosen in the study, although there are frequently multiple genomes from different subspecies available within the same species. A total of 348 genomes were analyzed in this study and 75 genomes within the phylum of Firmicutes were used in the phylogenetic analysis. The phylogenetic distribution of the predicted DnaN homologs within the phylum of Firmicutes was constructed using a multiple sequence alignment program ClustalW at the website www.ebi.ac.uk. To investigate the possible conservation of the location “context” for the additional copy (or copies) of the predicted dnaN genes from 19 bacterial genomes, the protein sequences encoded by the two open reading frames immediately upstream and the two open reading frames immediately downstream of the predicted dnaN gene were compared among 19 bacterial genomes.

2.2 Media and growth conditions

B. anthracis strains were grown non-selectively in LB medium. Erythromycin-resistant transformants were selected on LB agar plates supplemented with 5 μg/ml erythromycin. Spectinomycin or kanamycin resistant colonies were selected on LB agar plates supplemented with 100 μg/ml spectinomycin or 100 μg/ml kanamycin. All growth occurred at 37°C.

To measure the growth rate in liquid culture, a culture containing 2 ml LB medium with inoculation of a single colony was grown overnight in a 37°C incubator. Half milliliter of the overnight culture was used to inoculate 50 ml LB medium in a 500 ml flask. The culture was first warmed up for 30 min in a 37°C waterbath without agitation followed by 30 min on a 37°C shaker with agitation before any measurement was taken place. The optical density of the culture at 600 nm was measured at a 15-min interval for a period of four and half hours. The growth curve was generated by plotting the duration vs. the optical density at 600 nm on a semi-log scale. The doubling time was estimated within the linear range of the plot, which was typically expanded over 1.5 hour for the wild type and dnaN2 knockout mutant, and 2.5 hour for the dnaN1 knockout mutant. The doubling time of each strain was obtained as an average value calculated from two to seven independent experiments.

2.3 B. anthracis strain constructions

The protocols used for constructing single and double knockout mutants were as previously published [28–31]. The primer sequences are available upon request.

2.4 Detection of Rifr mutants and base substitution spectrum in the rpoB gene

Mutation frequencies (f) were determined using the protocols as described previously [31]. Mutation rate (μ) per replication was calculated by the method of Drake [32]. The 95% confidence limits were determined following the guidelines dictated by the method of Dixon and Massey [33]. The procedures used for the detection of Rifr mutants and determination of the base substitution spectrum in the rpoB gene were as previously published [31, 34].

3. Results and discussion

3.1 Two β subunit homologs (dnaN1 and dnaN2) in the B. anthracis genome

Nearly all eubacterial genomes sequenced to date predict only one gene (dnaN) that encodes the β subunit homolog of DNA polymerase. The dnaN locus is strongly linked to several other genes such as dnaA, recF, and gyrB [21]. This conservation of location “context” throughout many bacterial species infers the functional linkage between DnaN, DnaA, RecF and GyrB, all of which are involved in DNA replication process [35–37]. Interestingly, nineteen genomes out of 348 scanned predict more than one dnaN gene: a copy of a dnaN homolog in its usual gene context (in most cases), and a second copy of a dnaN homolog flanked by genes without any obvious conservation among most of the 19 genomes analyzed. The exceptions are two cyanobacteria genomes including Anabaena variabilis and Nostoc sp., which do have a similar neighboring gene context surrounding the second predicted dnaN gene. Also, four bacillus genomes including Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus weihenstephanensis, have a different conserved set of flanking genes. Examples of the gene-neighboring context of single dnaN genes from genomes of Escherichia coli and Streptococcus pyogenes, and multiple dnaN genes predicted from 19 genomes are shown in Figure 1. The four bacillus genomes, B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis, predict two chromosomal copies of dnaN genes. They were designated as dnaN1 and dnaN2, where dnaN1 is linked to dnaA, recF, gyrA and gyrB (B. cereus genome predicts an additional dnaN gene, dnaN3, originated from a plasmid). We used B. anthracis as our model system to investigate the role of these DnaNs. We speculate that in B. anthracis DnaN1 may behave similarly as DnaNs from E. coli and S. pyogenes, since DnaN1 is in the conserved dnaN gene surroundings.

Figure 1.

Figure 1

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(A) Annotated gene context of dnaN from Escherichia coli and Streptococcus pyogenes, both of which have a resolved crystal structure of the DnaN protein. The arrow indicates the annotated transcription direction of the gene. The number of genomes out of 348 genomes scanned that predict one dnaN gene is indicated in the parentheses. (B) A list of 19 genomes that predict more than one dnaN. Each phylum is labeled in bold. In each phylum, the number of genomes that have more than one dnaN gene vs. the total number of genomes scanned is indicated in parentheses. The arrow indicates the annotated transcription direction of the gene. The boxed sequences are identified on plasmids from their corresponding bacterial hosts. The asterisks mark the genomes, in which the predicted dnaN genes appear to be not linked to dnaA, recF, gyrA, or gyrB.

We analyzed the sequence divergence between B. anthracis DnaN1 and DnaN2 through a phylogenetic analysis of the DnaN homologs of 75 complete genomes (with the exception of the Bacillus weihenstephanensis) that are in the order of Bacillales, Lactobacillales, Clostridia, and Mollicutes within the phylum of Firmicutes. It can be seen that DnaN homologs within each order are clustered together, including those DnaN1 homologs from the genomes of B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis (The only exception being a DnaN homolog from the Symbiobacterium thermophilum genome). However, DnaN2 homologs are not clustered with their corresponding DnaN1 homologs, but instead, are clustered together to form a separate clade (Figure 2).

Figure 2.

Figure 2

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Phylogenetic distribution of DnaN protein sequences from 75 genomes within the phylum of Firmicutes. The clades of DnaN1 and DnaN2, and DnaN3 (originated from a plasmid) are indicated. The number of genomes analyzed in each order is indicated in the parentheses. The asterisk marks the genome that belongs to the order of Lactobacillales.

The protein sequences of B. anthracis DnaN1 and DnaN2 were analyzed in detail with respect to that of Streptococcus pyogenes DnaN, and of Escherichia coli DnaN (both of which have a resolved crystal structure [6,39], Figure 3). B. anthracis DnaN1 and DnaN2 share 40% protein sequence identity amongst themselves. Both homologs also share a similar extent of sequence identity with S. pyogenes DnaN (39% and 33%, respectively). However, they are less homologous to DnaNs from gram-negative bacteria (25% and 24%, respectively, with E. coli DnaN). Highly conserved amino acid residues [39,40] are present in both B. anthracis DnaN1 and DnaN2 (Figure 3), suggesting that both proteins may be functional in the cell.

Figure 3.

Figure 3

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Alignment of DnaN protein sequences from E. coli (EC), S. pyogenes (SP), and B. anthracis (BAS0002 and BAS2499) using the program ClustalW [42]. The secondary structure elements are labeled according to ref.39. The conserved amino acid residues proposed by Neuwald are shaded [40].

3.2 B. anthracis dnaN1 and dnaN2 single knockout mutants are viable

We asked whether DnaN1 or DnaN2 could function independently in B. anthracis. Since having at least one dnaN gene is essential, we investigated whether a single knockout mutant of dnaN1 or dnaN2 is viable in B. anthracis. We engineered two knockout constructs aimed at replacing the open reading frame of dnaN1 or dnaN2 with an antibiotic resistance encoding cassette for spectinomycin or kanamycin, respectively (Figure 4A and 4B). Using previously published protocols [31] we were able to obtain dnaN1 and dnaN2 single knockout mutants. The gene replacement in each knockout mutant was verified by PCR, indicating the absence of a dnaN1 coding region in the dnaN1 knockout mutant and the absence of a dnaN2 coding region in the dnaN2 knockout mutant (Figure 4C and 4D). The fact that both dnaN1 and dnaN2 single knockout mutants are viable indicates that DnaN1 and DnaN2 are capable of forming a functional β-sliding clamp in the absence of the other homolog in B. anthracis.

Figure 4.

Figure 4

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B. anthracis dnaN1 and dnaN2 knockout mutants. A. Strategy for generating the dnaN1 null allele. The location for each primer E1, F1, K, L, and SP3 is as marked. B. Strategy for generating the dnaN2 null allele. The location for each primer E2, F2, M, N, and Kan2 is as marked. C. PCR analysis of genomic DNA isolated from wild type, dnaN1 and dnaN2 knockout mutants using primer set E1-F1 (for wild-type dnaN1), K-L (for wild-type dnaN1), and E1-SP3 (for Omega;-sp cassette). D. PCR analysis of genomic DNA isolated from wild type, dnaN1 and dnaN2 knockout mutants using primer set E2-F2 (for wild-type dnaN2), M-N (for wild-type dnaN2), and E2-Kan2 (for Ω-km cassette). E. Photograph of LB agar plates streaked with wild type, dnaN1 knockout, and dnaN2 knockout mutant, after an overnight growth at 37°C.

The colony size of the dnaN2 knockout mutant is similar to that of the wild-type strain on a LB agar plate; however, a significantly reduced colony size was observed in the dnaN1 knockout mutant (Figure 4E). The growth rate of dnaN1 and dnaN2 knockout mutants in the liquid culture was also assessed. The doubling time for wild type and dnaN2 knockout mutant was 32.0 min and 30.5 min, respectively. However, the doubling time of dnaN1 knockout mutant was 41.8 min, which was about 1/3 slower than that of the wild type and dnaN2 (Figure 4F). Thus, dnaN1 appears to be more crucial than dnaN2.

We failed to construct a dnaN1 and dnaN2 double mutant, despite the fact that we were able to make double knockout mutants with dnaN1 and other genes such as mutM or mutS (data not shown). Failure to construct the dnaN1 and dnaN2 double knockout mutant strongly infers that the deletion of both dnaN1 and dnaN2 is lethal for B. anthracis.

3.3 DNA mismatch repair defect in dnaN1 knockout mutant

Recent studies show that the β sliding clamp not only serves as a part of the DNA replication machinery to offer processitivity to DNA replicative polymerases, but also physically interacts with many other proteins that are involved in different processes of DNA metabolism [13]. We focused on the role of the β sliding clamp in the DNA mismatch repair process. It has been proposed that the β sliding clamp orchestrates the sequence of events that leads to mismatch repair in the cell [18]. We asked whether deletion of dnaN1 or dnaN2 affects mutation rate in B. anthracis. Cultures of the two knockout mutant strains and a wild-type strain were grown in LB liquid medium and were determined for the mutation levels in the rpoB gene leading to the Rifr phenotype. Our results indicate that the dnaN2 knockout strain and the wild-type strain have similar levels of spontaneous rpoB mutations (frequencies of 3.8 × 10−9 and 3.0 × 10−9, respectively; and rates per replication of 2.4 × 10−9 and 2.0 × 10−9, respectively), which is similar to our previously published work (31). The dnaN1 knockout strain, however, has a spontaneous rpoB mutation frequency of 95 times that of the wild type and a mutation rate of 29 times that of the wild type (Table 1).

Table 1.

B. anthracis rpoB mutation frequencies (f) and rates (μ)

Genotype f (×109)a μ (×109)b
wild type 3.8 (1.1 – 7.7) 2.4 (1.2–3.8)
dnaN1 360 (290 – 480) 70 (59–90)
dnaN2 3.0 (0.92 – 19) 2.0 (1.0–7.0)
mutS 740 (670 – 810) 140 (130–150)
dnaN1 mutS 700 (590 – 820) 120 (110–140)
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a

Values in parentheses are 95% confidence limits [33].

b

Mutation rates, μ, are calculated by the method of Drake [32].

To eliminate the possibility that the mutator phenotype observed in the dnaN1 knockout mutant resulted from an unrelated mutation introduced into the genome during the process of constructing the dnaN1 knockout mutant, we constructed an independent dnaN1 knockout mutant in which the dnaN1 open reading frame was replaced by a kanamycin resistance encoding cassette. In this dnaN1 knockout mutant, a similar growth phenotype and mutation rate was observed (data not shown), which supports the idea that the mutator phenotype observed in the dnaN1 knockout mutant must indeed be attributed to the absence of dnaN1.

We asked whether (1) an increased level of replication errors generated by DNA polymerase or (2) reduced efficiency of DNA mismatch repair system causes this apparent mismatch repair deficiency phenotype observed in the dnaN1 knockout mutant. To distinguish between these two possibilities, we constructed a double-knockout mutant of dnaN1 and mutS, and measured the mutation rate in this double knockout mutant. If the first possibility were correct, a significantly increase in mutation rate would be observed in the dnaN1, mutS double knockout strain. On the other hand, if the second possibility were correct, a mutational rate similar to that of the mismatch repair deficient strain would be observed. The results are shown in Table 1. The level of the mutation rate in the double knockout strain was closely to that of the mismatch repair deficient strain. Our results indicate that in the absence of dnaN1, the mismatch repair efficiency is significantly reduced. This finding suggests that DnaN1 plays an essential role in DNA mismatch repair in B. anthracis.

Our previous work has shown that the base substitution spectrum in the rpoB gene leading to the Rifr phenotype contains distinct hotspots for each mutator [31] and is therefore a useful diagnostic tool in the analysis of mutational pathways responsible for the apparent mutator phenotypes. We investigated the base substitution spectrum of the rpoB gene in the dnaN1 knockout mutant background. The results are shown in Table 2. It can be observed that the mutations in the rpoB gene are dominated by two A→G hotspots at 1403 bp and 1442 bp, which is similar to the fingerprint of a mismatch repair deficient strain [31].

Table 2.

Distribution of mutations leading to Rifr in B. anthracis rpoB gene

B. anthracis site (bp) Amino acid change Base-pair change Spontaneousa dnaN1 mutSb
1403 Q454R AT→GC 20 13 19
1442 H467R AT→GC 30 30 20
1441 H467Y GC→AT 33 2 1
1391 S450F GC→AT 0 0 0
1457 S472F GC→AT 1 0 0
1457 S472Y GC→TA 1 0 0
1402 Q454K GC→TA 4 1 0
1403 Q454L AT→TA 0 0 0
1417 T459S AT→TA 0 0 0
1444 K468Q AT→CG 0 0 0
1442 H467P AT→CG 1 0 0
1391 S450C GC→CG 0 0 0
1441 H467D GC→CG 0 1 0
1600 S453C GC→CG 0 0 0
Total 14 90 47 40
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a

Includes data from ref. 31.

b

From ref. 31.

This is the first study demonstrating the existence of two functional DnaN homologs in the B. anthracis genome, with DnaN1 appearing to be more crucial than DnaN2. Our results suggest the direct involvement of DnaN1 in the DNA mismatch repair process, which is consistent with previous findings [18]. A possible explanation for the growth and mismatch repair deficiency in dnaN1 knockout strain is that DnaN2 homolog functions much less efficiently than DnaN1. We speculate that the rate of DNA replication and the rate of DNA mismatch repair process are reduced in the dnaN1 mutant, which contribute to decreased growth and increased mutation rate. However, the fidelity of the replicative DNA polymerase apparently remains unchanged. Further biochemical analysis is necessary to address the phenotypical difference between DnaN1 and DnaN2 at the molecular level. The B. anthracis dnaN1 knockout mutant can serve as a valuable model system for studying other β clamp related DNA processing pathways.

Nineteen genomes predict more than one dnaN gene (Figure 1). It is unclear whether these additional dnaN genes are vestigial remnants from the past or the landmarks of specific horizontal gene transfer events. Whether these dnaN genes behave similarly to the B. anthracis dnaN1 and dnaN2 remains to be studied. The presence of the dnaN2 gene in the four bacillus genomes, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus weihenstephanensis, suggests that these species were closely related during evolution [41].

Acknowledgments

This research was supported by a grant from the National Institutes of Health (ES010875).

Footnotes

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