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Development of a System for Discovery of Genetic Interactions for Essential Genes in Escherichia Coli K-12

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Development of a System for Discovery of Genetic Interactions for Essential Genes in Escherichia Coli K-12 Genes Genet. Syst. (2013) 88, p. 233–240 Development of a system for discovery of genetic interactions for essential genes in Escherichia coli K-12 Han Tek Yong1§, Natsuko Yamamoto1‡§, Rikiya Takeuchi1, Yi-Ju Hsieh2#, Tom M. Conrad1, Kirill A. Datsenko2, Toru Nakayashiki1, Barry L. Wanner2† and Hirotada Mori1* 1Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan 2Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907-2054 USA (Received 16 May 2013, accepted 22 July 2013) Genetic interaction networks are especially useful for functional assignment of genes and gaining new insights into the systems-level organization of the cell. While studying interactions of nonessential genes can be relatively straight- forward via use of deletion mutants, different approaches must be used to reveal interactions of essential genes due to their indispensability. One method shown to be useful for revealing interactions of essential genes requires tagging the query protein. However, this approach can be complicated by mutational effects of potential hypomorphic alleles. Here, we describe a pilot study for a new scheme of systematically studying the interactions of essential genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from which the essential gene is conditionally expressed. The essential gene is expressed at lower levels, producing a moderate growth defect in a query host. Secondary mutations are introduced into the query host by conjugation and the resultant exconjugants are scored for growth by imaging them over time. We report results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and yjgP, showing (on average) interactions with nearly 80 nonessential genes. This system should prove useful for genome-wide analyses of other essential genes in E. coli K-12. Key words: complementing F plasmid, epistasis, fitness, gene function processes, studying them can provide useful information INTRODUCTION for elucidation of gene function (Mani et al., 2008). Mod- Genetic interactions occur when two mutations els for genetic interaction networks have been constructed together result in an effect(s) different from either muta- in both eukaryotic and prokaryotic model systems (Byrne tion alone. Interactions are positive (additive) when two et al., 2007; Bakal et al., 2008; Butland et al., 2008; mutations show a synergistic effect that is more extreme Costanzo et al., 2010; Pan et al., 2007; Davierwala et al., than when alone, for example, synthetic lethality; inter- 2005; Roguev et al., 2008; Schuldiner et al., 2005; Tong et actions are negative (diminished) when the phenotype of al., 2001, 2004; Typas et al., 2008). These networks have the double mutant is less severe than either mutation revealed the global modular organization of gene products alone. Since genetic interactions often occur among and functional interaction of bioprocesses in several genes in compensatory pathways or interlinked biological model organisms (Dixon et al., 2009). Analyses of protein-protein interaction networks have Edited by Hisaji Maki revealed that proteins acting as hubs are more likely to * Corresponding author. E-mail: [email protected] be essential than less connected proteins (Jeong et al., † Corresponding author. E-mail: [email protected] 2001). Therefore, genetic interactions of essential genes ‡ Present address: Graduate School of Medicine, Osaka Univer- may unravel key new insights not revealed from studying sity. 2-2 Yamadaoka Suita, Osaka 565-0871, Japan genetic interactions of nonessential genes. Systematic # Present address: Department of Urology, Stanford University School of Medicine, Stanford, CA 94305-3900 efforts to map genetic interactions in E. coli K-12 have § These authors contributed equally. relied on the construction of double mutants by Hfr con- 234 H. T. YONG et al. jugation of donor and recipient cells with deletions and genes present on pFE604T (Ogura and Hiraga, 1983; different antibiotic resistance markers (Butland et al., Mori et al., 1986; Uga et al., 1999). Antibiotic resistance 2008; Typas et al., 2008). While these methods are use- markers (gentamycin and tetracycline) are incorporated ful for examining between nonessential genes, the same into pFE604T for selection. pFE604T has two origins of method cannot be used to study essential genes because replication: ori2 and oriRγ. ori2 is the default origin of such essential gene deletion mutants are non-viable. replication, resulting in a single copy of the plasmid dur- This experimental difficulty has been previously bypassed ing cell division, while oriRγ is a conditional origin of rep- by adding a C-terminal SPA (Sequential Peptide Affinity) lication that requires the trans-acting Π protein (encoded tag to the essential gene (Butland et al., 2008; Babu et al., on the chromosome by pir) for replication and results in 2011). An SPA tag integrated in the C terminal presum- multiple plasmid copies each division. Due to having ably alters the 3’-UTR and hence destabilizes certain these two origins of replication, pFE604T can replicate at transcripts (Babu et al., 2011). However, there are a single or medium plasmid copy number in non-pir or drawbacks to using SPA tags to affect a defect in an pir+ E. coli hosts, respectively. A non-pir host is used for essential gene for genetic interaction studies. The tag genetic interaction experiments, while a pir+ host is used often causes no measurable defect in the absence of sec- for producing additional plasmid. An oriT site is present ondary mutations. Furthermore, the mechanisms of on pFE604T, enabling the one-step transfer of pFE604T- defects are not clearly understood (Butland et al., 2008). ORF from an Hfr host to an array of nonessential gene In order to understand genetic interactions, one needs to deletion mutants en masse via conjugation. Transfer of understand the defect in the query genes. pFE604T-ORF to the nonessential gene mutant is critical Here we describe a pilot study for a new method for before or while transferring the second, essential gene high-throughput genetic interaction analysis in E. coli mutation to the nonessential gene mutant in order for between essential genes and nonessential genes. In this complementation of the essential gene to occur in the dou- method, a chromosomal knockout mutant of the essential ble mutant. gene is crossed with a high-density array of nonessential We began the construction of each chromosomal knock- deletions to construct by conjugation double mutants that out mutant of an essential query gene by transforming E. express knock-down levels of the essential query protein coli K-12 BW25113 with the pFE604T-ORF containing (Fig. 1). The major advantage of the system over the the essential gene. Next, the plasmid-borne essential SPA-tagging system is that the mutational defect of the query gene is expressed from pFE604T-ORF under the chromosomal knockout mutant of an essential gene is induction of IPTG, allowing disruption of the query gene known: the level of essential query gene protein is low- on the chromosome by one-step homologous recombina- ered, causing a moderate growth defect. tion (Datsenko and Wanner, 2000). The behavior of the doubling time of a chromosomal knockout mutant could in principle be modeled as a par- RESULTS abolic curve. Because bacteria tend to be programmed to Construction of low-copy, F-based complementing express proteins at optimal levels (Dekel and Alon, 2005), plasmid pFE604T pFE604T is a single-copy mini-F the minimum of that curve would correspond to an IPTG plasmid (Fig. 2) which was purposefully designed for sys- concentration resulting in expression of the essential gene tematic construction of chromosomal knockout mutants of at optimal, physiological levels. IPTG concentrations essential genes for their high-throughput genetic interac- less than “optimum” result in a subphysiological level of tion analyses. The essential gene is cloned into expression of the essential gene. Under this model, we pFE604T using SfiI restriction sites downstream of a T5 can prove a concentration of IPTG produces subphysiolog- promoter and lacI-O repressor-operator region, where is ical levels of expression of the essential gene by showing inducible by IPTG. Since E. coli genes in the ASKA that a higher concentration of IPTG results in a faster library are flanked by SfiI sites (Kitagawa et al., 2005), doubling time than the original concentration. In fact, any essential E. coli gene can readily be cloned into we observed that for five out of five pFE604T-ORF con- pFE604T. We refer to the pFE604T that can express an structs tested, doubling times were faster when using essential query gene as pFE604T-ORF. The viability of 1 mM IPTG than when using 0.1 mM IPTG (Fig. 3). We the chromosomal knockout mutant is sustained by conclude that a concentration of 0.1 mM IPTG produces expression of the essential query gene from the single- subphysiolical expression levels of the essential gene from copy pFE604T-ORF. Construction of pFE604T-ORF is pFE604T-ORF. Full complementation from pFE604T- shown in Supplementary Fig. S1 and described in detail ORF was not observed even at 1 mM IPTG, likely due to in the Supplementary Methods. the plasmid being present as only a single copy. Because Control of DNA replication, copy number, incompatibil- there are observable growth defects in the chromosomal ity, and partition from pKV713 (Kawasaki et al., 1990) are knockout mutants, they are amenable for genetic interac- stringently controlled by the ori2, incC, repE, and sopABC tion analysis. Discovery of genetic interactions for essential genes 235 Fig. 1. High-throughput method to find essential-nonessential gene interactions. Hfr donor strains are chromosomal knockout mutants with an essential query gene deletion marked with cat (chloramphenicol resistance cassette; blue box) but complemented by the IPTG inducible knock-down level of essential query protein from pFE604T-ORF (green triangle). Recipient strains are nonessential gene deletions marked with kan (kanamycin resistance cassette; pink box).
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