Genes Genet. Syst. (2016) 91, p. 183–188 Systematic identification of synthetic lethal mutations with reduced-genome : synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure

Keisuke Watanabe, Kento Tominaga, Maiko Kitamura and Jun-ichi Kato* Department of Biological Sciences, Graduate Schools of Science and Engineering, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan

(Received 22 October 2015, accepted 25 January 2016; J-STAGE Advance published date: 2 May 2016)

Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interac- tion between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA III, and yoaA, encoding a putative DNA . Addition of the yoaA disruption increased the methyl methanesulfonate (MMS) sensitivity of xthA single-deletion or xthA nfo double-deletion mutants. This increased MMS sensitivity was not suppressed by the presence of multi-copy holC. These results indicate that yoaA is involved in MMS sensitivity and suggest that YoaA functions together with HolC.

Key words: Escherichia coli, MMS sensitivity, DNA helicase, DNA polymerase III, AP endonuclease

A minimal set contains only those genes that are a series of reduced-genome Escherichia coli strains by essential and sufficient to sustain a functioning cell. The combining large-scale chromosomal deletion mutations minimal gene set includes not only a set of all essential (Hashimoto et al., 2005; Kato and Hashimoto, 2007; genes but also functionally redundant nonessential genes Iwadate et al., 2011). In the present work, using these involved in essential processes. It is difficult to identify reduced-genome strains, we identified a synthetic lethal- such cryptic essential genes because the phenotypes ity involving the deletion of genes encoding AP endonu- resulting from their individual disruption are not detect- cleases. In addition, we identified a novel DNA repair able in the wild-type strain. However, reduced-genome gene, yoaA, as a multi-copy suppressor of this synthetic strains provide useful tools for systematically identifying lethality. YoaA may function together with the chi sub- cryptic essential genes by taking advantage of their syn- unit of DNA polymerase III. thetic lethal interactions. To identify such genes, and First, we constructed a large-scale deletion mutation, ultimately a minimal gene set, we previously constructed LD3-20-1, using a red+ derivative of the wild-type strain MG1655 (Fig. 1A). Next, we tried to introduce this dele- Edited by Hisaji Maki tion by P1 phage-mediated transduction into a reduced- * Corresponding author. E-mail: [email protected] genome strain, Δ33b, a fast-growing derivative of the DOI: http://doi.org/10.1266/ggs.15-00068 strain with the largest reduction in genome size, Δ33a 184 K. WATANABE et al.

Fig. 1. Large-scale chromosome deletion mutations responsible for synthetic lethality. Large-scale chro- mosome deletion mutations were constructed by introducing DNA fragments into the E. coli strain MG1655 red, which was constructed using KM22 (Murphy, 1998). ApR and SmR colonies were isolated, and the dele- tion mutations were confirmed by PCR. Thick lines represent the E. coli chromosome, and numbers above the lines indicate map positions (bp). Thin lines below the thick lines show the deleted regions of mutant chromosomes. Names of deletion mutations are shown to the right of the thin lines. (A) A large-scale chromosome deletion mutation, LD3-20-2, and its partial deletion derivatives. DNA fragments used to con- struct the deletion mutations were prepared by PCR using the ApR gene from plasmid pJK286 (Kato and Ikeda, 1996) as a template and oligonucleotides described in Supplementary Table S2 as primers. (+) to the right of the deletion name indicates that the deletion mutation was successfully introduced by transduc- tion into the reduced-genome strain Δ33b. (–) to the right of the deletion name indicates that the deletion mutation was not introduced into the reduced-genome strain, suggesting that it contains a synthetic lethal mutation. The map position of the nfo gene is indicated by an arrow below the thin lines. (B) The large- scale chromosome deletion mutation LD3-5-1 and its partial deletion derivatives. DNA fragments used to construct the deletion mutations were prepared by PCR using the SmR gene from plasmid mini-F (which carries the SmR gene within the ApR gene) as a template and oligonucleotides described in Supplementary Table S2 as primers. To the right of the deletion mutation names, (+)(+) indicates that the deletion muta- tion was successfully introduced by transduction into the strains MG1655 Δnfo ΔxthA and MG1655 Δnfo ΔxthA LD20; (–)(–) indicates that the deletion mutation was not introduced into either of those strains; and (+)(–) indicates that LD3-5-4 was successfully introduced into MG1655 Δnfo ΔxthA but not into MG1655 Δnfo ΔxthA LD20. The map positions of the uvrC, uvrY and vsr genes are indicated by arrows below the thin lines.

(Iwadate et al., 2011, Supplementary Table S1). How- deletion LD3-20-1 is synthetically lethal with other large- ever, no transductants appeared, suggesting that a gene scale deletion mutation(s) present in Δ33b (Supplemen- within the chromosomal region affected by the large-scale tary Tables S1 and S4). E. coli yoaA and holC function in MMS sensitivity 185

To identify the responsible gene, we constructed a obtained no transductants containing LD20, strongly sug- series of partial deletions of LD3-20-1 and tried to intro- gesting that the synthetic lethality was caused by these duce them into Δ33b (Fig. 1A). The results revealed that four deletions. To identify the responsible gene within nfo is responsible for the observed synthetic lethality. the region of LD3-5-1, we constructed a series of partial The nfo gene encodes DNA endonuclease IV, which is deletions of LD3-5-1 and tried to introduce them into involved in DNA repair. This enzyme cleaves phospho- MG1655 harboring two (Δnfo and ΔxthA) or all three diester bonds at apurinic or apyrimidinic sites (AP sites) (Δnfo, ΔxthA and LD20) of the other deletions (Fig. to produce new 5’-ends that are base-free deoxyribose 5- 1B). The results suggested that deletions of uvrC and/or phosphate residues, and preferentially attacks modified uvrY were responsible for the synthetic lethality of the AP sites created by bleomycin and neocarzinostatin four deletions (Δnfo, ΔxthA, LD3-5-1 and LD20), but nei- (Levin et al., 1991; Hosfield et al., 1999). ther of these deletions alone were sufficient for a growth To identify other responsible genes, we introduced the defect in the presence of the Δnfo and ΔxthA deletions. nfo disruption into other reduced-genome strains. nfo The responsible gene may be uvrC, because the uvrC gene disruptants were obtained with Δ20a but not with Δ21a is involved in DNA repair and uvrY codes for a member (Supplementary Tables S1 and S4). The Δ21a strain was of the two-component regulatory system involved in the constructed by introducing a large-scale chromosome regulation of carbon metabolism. deletion, LD3-17-1 (deletion location is 1,821,337– The synthetic lethality of the four deletions (Δnfo, 1,860,378 bp and the length is 39,042 bp; for further infor- ΔxthA, LD3-5-1 and LD20) was also confirmed by con- mation, see the PEC database, http://www.shigen.nig.ac.jp/ structing two plasmids, miniFts-nfo+ and miniFts-xthA+, ecoli/pec/), into Δ20a, suggesting that a second gene that could not replicate at high temperature. In the responsible for synthetic lethality is present in the chro- presence of either plasmid, quadruple-deletion mutants mosomal region affected by this deletion (Supplementary were obtained at 30 °C but not at 42 °C, indicating that Tables S1 and S4). We suspected xthA as the responsi- the synthetic lethality was caused by the four deletions. ble gene in this region: it encodes DNA endonuclease VI/ To identify other responsible genes in the deleted DNA exonuclease III, and is also involved in DNA repair regions of LD3-5-1 and LD20, as well as multi-copy sup- (Mol et al., 1995; Shida et al., 1996). XthA is a major AP pressors, we obtained temperature-resistant (tr) colonies endonuclease that removes damaged DNA at cytosines by introducing an E. coli chromosome library constructed and guanines by cleaving on the 3’-side of an AP site via in the multi-copy plasmid pACYC184 (Chang and Cohen, a beta-elimination reaction. It also exhibits 3’-5’-exonu- 1978). Plasmids were extracted from 150 tr colonies, clease, 3’-phosphomonoesterase, 3’-repair diesterase and and each of them was re-introduced into the ts quadruple- activities. We tried to introduce the xthA deletion mutants to confirm multi-copy suppression. deletion in addition to the nfo deletion into Δ20a by trans- Among the 150 plasmids, 89 were confirmed to suppress duction, but did not obtain any transductants, suggesting the synthetic lethality. After elimination of three large that xthA was responsible for the synthetic lethality. plasmids, sequencing and PCR analyses of the resultant To search for further responsible genes, we introduced 86 plasmids revealed that 34 plasmids carried nfo, 34 con- the nfo and xthA disruptions into other reduced-genome tained xthA, 17 carried a chromosomal region including strains. Anfo xth double disruptants were obtained with holC, and one contained the region containing yoaA. To Δ16aK, but not with Δ17aK (Supplementary Tables S1 identify the responsible multi-copy suppressor gene and S4). The Δ17a strain was constructed by introduc- within the chromosomal region including holC, we con- ing a large-scale chromosome deletion, LD3-5-1, into structed deletion-derivative plasmids and examined their Δ16a, suggesting that a responsible gene was present in suppression activity (Supplementary Fig. S1A). The this chromosomal region. When we introduced the nfo results revealed that holC was indeed responsible for and xthA disruptions into other reduced-genome strains, suppression. Likewise, to identify the responsible multi- we observed that many colonies appeared with Δ13a, but copy suppressor gene within the chromosomal region con- few with Δ14a. Because the Δ14a strain was constructed taining yoaA, we constructed deletion-derivative plasmids by introducing a large-scale chromosome deletion, LD20 and examined their suppression activity (Supplementary (deletion location is 1,349,195–1,650,779 bp and the Fig. S1B). The results confirmed that yoaA was respon- length is 301,585 bp; for further information, see the dele- sible. tion ‘20’ in the PEC database), into Δ13a, this deletion holC and yoaA were identified as multi-copy suppres- also appeared to be responsible for the synthetic lethality. sors of the synthetic lethality of the four deletions (Δnfo, To confirm that these four deletions were sufficient for ΔxthA, LD3-5-1 and LD20), and their multi-copy plasmids the synthetic lethality, we tried to introduce all four dele- also suppressed the MMS sensitivity of the ΔxthA mutant tions (Δnfo, ΔxthA, LD3-5-1 and LD20) into MG1655. (Fig. 2A). MMS is an alkylating agent and produces When we attempted to introduce LD20 into MG1655 har- DNA damage (Lundin et al., 2005). To confirm the boring Δnfo, ΔxthA and LD3-5-1 by transduction, we involvement of yoaA in MMS sensitivity, we introduced a 186 K. WATANABE et al.

Fig. 3. MMS sensitivity due to the yoaA mutation. The paren- tal strain MG1655 (WT) and its mutants were grown overnight at 37 °C in LB medium; 0.1 ml of each of these cultures was added to 2 ml of LB medium containing 0.1% glucose and incu- bated at 37 °C for 5 h. The cultures were diluted and grown at 37 °C on Antibiotic Medium 3 (DIFCO) containing 0, 20, 30 or 40 μl/100 ml MMS.

complemented by the yoaA plasmid, indicating that YoaA is involved in determining MMS sensitivity. To investigate the possibility that yoaA, like nfo and xthA, is involved in DNA repair, we examined the MMS sensitivity of the ΔyoaA derivatives of the Δnfo and ΔxthA single-deletion mutants and the Δnfo ΔxthA double- deletion mutant. As shown in Fig. 3, the ΔxthA and Δnfo ΔxthA mutants became more sensitive to MMS upon addi- tion of the ΔyoaA mutation. However, acquisition of the ΔyoaA mutation did not affect the sensitivity of the paren- tal strain or the Δnfo single mutant (Fig. 3). YoaA is a putative DNA helicase (UniProt, http:// Fig. 2. Multi-copy suppression and complementation of MMS www.uniprot.org/uniprot/P76257), and HolC is the chi sensitivity. MG1655 ΔxthA and MG1655 ΔxthA ΔyoaA mutants subunit of DNA polymerase III (Carter et al., 1993; Xiao carrying pACYC184-yoaA+, pACYC184-holC+ or pACYC184 were et al., 1993a, 1993b). An interaction between YoaA and grown overnight at 37 °C in LB medium containing Cm; 0.1 ml HolC was previously revealed by interactome analysis of each of these cultures was added to 2 ml of LB medium con- (DIP (DIP-12786N), http://dip.doe-mbi.ucla.edu/dip/Browse. taining 0.1% glucose and incubated at 37 °C for 5 h. The cul- tures were diluted and grown at 37 °C on Antibiotic Medium 3 cgi?ID=DIP-12786N; IntAct (P76257), http://www.uniprot. (DIFCO) plates containing 3.54 mM MMS. (yoaA), (holC) and org/uniprot/P76257#interaction). To investigate func- (vec) represent the plasmids pACYC184-yoaA+, pACYC184-holC+ tional interaction between these two proteins, we exam- and pACYC184, respectively. The results of multi-copy sup- ined multi-copy suppression by holC in the yoaA pression (A) and complementation (B) of MMS sensitivity are disruptant. In addition, we introduced ΔxthA into the shown. +Cm and –Cm in (B) represent the presence and Δ Δ absence of Cm in the first culture. Δlac indicates Δlac::Cm, quadruple-deletion mutant ( nfo, yoaA, LD3-5-1 and used as a control. cfu, colony forming units. LD20) and examined the effects of the holC multi-copy plasmid on the growth of the transductants (Fig. 4). Multi-copy suppression by pACYC184-holC+ was observed yoaA-complementing plasmid (pACYC184-yoaA+) into the when the yoaA gene was present, but not in cells harbor- ΔxthA ΔyoaA double mutant and the Δnfo ΔxthA ΔyoaA ing ΔyoaA. In addition, we examined the multi-copy sup- triple mutant, and examined the MMS sensitivity of the pression against MMS sensitivity of the ΔxthA mutant. resultant cells (Fig. 2B). Although MMS sensitivity was As shown in Fig. 2A, MMS sensitivity was suppressed by influenced by plasmid and antibiotic (Cm), ΔyoaA was the holC multi-copy plasmid when the yoaA gene was E. coli yoaA and holC function in MMS sensitivity 187

Fig. 4. Multi-copy suppression of synthetic lethality. The ΔxthA:: ApR mutation was introduced by transduction into the strains MG1655 Δnfo LD3-5-1 LD20 or MG1655 Δnfo ΔyoaA LD3-5-1 LD20 carrying pACYC184-yoaA+, pACYC184-holC+ or pACYC184, and incubated at 37 °C on Antibiotic Medium 3 (DIFCO) plates containing Ap. The Δ(yjaA-yjaB):: ApR muta- tion, which did not affect the cell growth of the strains MG1655 Δnfo LD3-5-1 LD20 or MG1655 Δnfo ΔyoaA LD3-5-1 LD20, was used as a control. present, but not in cells harboring ΔyoaA. These results sis of synthetic lethality using reduced-genome strains suggest that YoaA functions in conjunction with HolC. should be a useful approach for elucidating many func- In this work, we systematically discovered the basis of tional interactions. the synthetic lethality of four deletion mutations, Δnfo, ΔxthA, LD3-5-1 and LD20, using a series of reduced- This study was supported by research funds from the Institute genome strains. We also identified two multi-copy sup- for Fermentation, Osaka, and a Grant-in Aid for Scientific pressors, holC and yoaA, using temperature-sensitive Research (C) from the Japan Society for the Promotion of Sci- mutants and a chromosome library. Furthermore, we ence. showed that the ΔxthA single mutant and the Δnfo ΔxthA double mutant became more sensitive to MMS upon addi- REFERENCES tion of ΔyoaA. Thus, we revealed synthetic genetic inter- Carter, J. R., Franden, M. A., Lippincott, J., and McHenry, C. S. actions between YoaA and XthA related to survival from (1993) Identification, molecular cloning and characteriza- MMS exposure. A recent study reported a synthetic tion of the gene encoding the chi subunit of DNA poly- genetic interaction between YoaA and RadA, a RecA- merase III holoenzyme of Escherichia coli. Mol. Gen. related protein, involved in survival from azidothymidine Genet. 241, 399–408. (AZT) exposure (Cooper et al., 2015). Here, we describe Chang, A. C., and Cohen, S. N. (1978) Construction and charac- terization of amplifiable multicopy DNA cloning vehicles additional synthetic genetic interactions between YoaA derived from the P15A cryptic miniplasmid. J. Bacteriol. and HolC. YoaA is predicted to be a DNA helicase and 134, 1141–1156. may function together with DNA polymerase III. Alter- Cooper, D. L., Boyle, D. C., and Lovett, S. T. (2015) Genetic anal- natively, YoaA may form a complex with a free chi sub- ysis of Escherichia coli RadA: functional motifs and genetic unit and single-strand binding protein. Further interactions. Mol. Microbiol. 95, 769–779. Hashimoto, M., Ichimura, T., Mizoguchi, H., Tanaka, K., biochemical analysis is needed to understand the mecha- Fujimitsu, K., Keyamura, K., Ote, T., Yamakawa, T., nism by which YoaA is involved in DNA repair. Yamazaki, Y., Mori, H., Katayama, T., and Kato, J. (2005) This study demonstrates that identification and analy- Cell size and nucleoid organization of engineered Escherichia 188 K. WATANABE et al.

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