Article21 February 2006Open Access Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection Tomoya Baba Tomoya Baba Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Takeshi Ara Takeshi Ara Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Miki Hasegawa Miki Hasegawa Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan Search for more papers by this author Yuki Takai Yuki Takai Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan Search for more papers by this author Yoshiko Okumura Yoshiko Okumura Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Miki Baba Miki Baba Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Kirill A Datsenko Kirill A Datsenko Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Masaru Tomita Masaru Tomita Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Barry L Wanner Corresponding Author Barry L Wanner Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Hirotada Mori Corresponding Author Hirotada Mori Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Tomoya Baba Tomoya Baba Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Takeshi Ara Takeshi Ara Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Miki Hasegawa Miki Hasegawa Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan Search for more papers by this author Yuki Takai Yuki Takai Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan Search for more papers by this author Yoshiko Okumura Yoshiko Okumura Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Miki Baba Miki Baba Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Kirill A Datsenko Kirill A Datsenko Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Masaru Tomita Masaru Tomita Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Search for more papers by this author Barry L Wanner Corresponding Author Barry L Wanner Department of Biological Sciences, Purdue University, West Lafayette, IN, USA Search for more papers by this author Hirotada Mori Corresponding Author Hirotada Mori Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Author Information Tomoya Baba1,2, Takeshi Ara1, Miki Hasegawa1,3, Yuki Takai1,3, Yoshiko Okumura1, Miki Baba1, Kirill A Datsenko4, Masaru Tomita1, Barry L Wanner 4 and Hirotada Mori 1,2 1Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan 2Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan 3CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan 4Department of Biological Sciences, Purdue University, West Lafayette, IN, USA *Corresponding authors. Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, USA. Tel.: +1 765 494 8034; Fax: +1 765 494 0876; E-mail: [email protected] School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: +81 743 72 5660; Fax: +81 743 72 5669; E-mail: [email protected] Molecular Systems Biology (2006)2:2006.0008https://doi.org/10.1038/msb4100050 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/). Synopsis The long-term goal of biomedical research has always been the complete understanding of biological systems. In the last century, reductionist approaches proved immensely powerful in elucidating many biochemical, genetic, and molecular mechanisms. In this century, we are entering a more synthetic phase in which we will accomplish the goal of completely understanding biological systems in their incredible living complexity. This understanding will be expressed in a number of models, ranging from traditional biological understanding (where individuals construct models in their heads) to formal mathematical models. In any case, reaching a complete understanding requires an unprecedented standardization and completeness of data, greatly improved methods of accessing and linking information, and improved techniques and approaches for mathematical modeling. E. coli K-12 is the best-characterized organism at the molecular level. In the accompanying report, we describe its highly accurate sequence (Hayashi et al, 2006), perhaps more accurate than of any genome of similar size, maybe even error free. Determination of a highly accurate sequence provided the impetus for re-annotation of its genome (Riley et al, 2006), which is of fundamental importance to studies not only of E. coli biology but also of other organisms because properties of more than half of its gene products have been experimentally determined. More than a half-century of experimental investigation has led to the identification of nearly all the metabolic reactions and the small molecule metabolites involved therein. Many of the regulatory circuits have been identified and computational methods for the predication of many regulatory sites are available. It is thus a truism that ‘… all cell biologists have two cells of interest, the one they are studying and Escherichia coli’ (Neidhardt, 1996). E. coli has the further advantage of being a simple unicellular organism without as extensive an elaboration of compartments and transport mechanisms as are present even in simple eukaryotes such as yeast (Figure 7, Holden, 2002). The completeness of our knowledge and the relative simplicity of E. coli provide compelling reasons for choosing it as the first cellular system to be targeted for complete understanding. This was clearly seen by Francis Crick when in 1973 (Crick, 1973) he proposed ‘Project K: the complete solution of E. coli.’ Of course, his suggestion was hopelessly premature, being before many key technologies, rapid computation, and the web (Crick, 2002). With a goal towards complete understanding of E. coli as a simple cellular system, we have begun the construction of uniformly designed and comprehensively prepared resources. Here, we describe a complete set of precisely defined, single-gene deletions of nonessential E. coli K-12 genes. These mutants were constructed by using a PCR gene replacement method similar to the one used to create a nearly complete set of yeast gene mutants (Giaever et al, 2002), except by using E. coli cells carrying a plasmid expressing the highly efficient λ Red recombinase (Datsenko and Wanner, 2000) (Figure 8). Deletions were obtained for 3985 of 4288 targeted genes. Based on finding mutants with the predicted structures, the majority of these 3985 genes are probably nonessential. Because a small fraction (ca. 0.2%) of cells are predicted to contain genetic duplications (Anderson and Roth, 1977), a small number of these 3985 genes may in fact be essential. The majority of the 303 genes for which no mutants were obtained are candidates for essential genes, at least under our selection conditions (aerobic growth on a complex medium at 37°C). In bacteria, genes are often arranged in operons that are transcribed as a unit and in which neighboring genes frequently overlap a few to several nucleotides. In such arrangements, mutation of a single gene can simultaneously affect function of neighboring or downstream genes. To circumvent these kinds of problems, mutants were designed taking into account gene organization to avoid affecting properties of more than one gene simultaneously. All mutants contain a kanamycin resistance cassette in place of the gene coding region. In most cases, the coding region from the 2nd through the 7th codon from the C-terminus has been deleted. The kanamycin resistance gene is oriented for expression of downstream genes (Figure 8A). Further, the mutants were constructed by use of a resistance cassette that can be easily eliminated (Datsenko and Wanner, 2000). The resultant kanamycin-sensitive derivatives are predicted to encode a small in-frame peptide in place of the mutated gene, in order to reduce effects on expression of downstream genes (Figure 8B). Results of profiling the mutants for growth on synthetic and rich media are described in the manuscript. These mutants provide a new basic resource not only for systematic functional genomics studies but also experimental data source for systems biology approaches. By providing this resource openly to the research community, the authors hope to contribute to worldwide efforts directed towards a comprehensive understanding of the E. coli K-12 model cell. Accordingly, we are making the entire mutant collection freely available for nonprofit, noncommercial use via GenoBase (http://ecoli.aist-nara.ac.jp) for cost of duplication and shipping fees. Commercial and for-profit investigators should contact one of the corresponding authors directly. Introduction The increased availability of genome sequences has provided the basis for comprehensive understanding of organisms at the molecular level. Besides sequence data, a large number of experimental and computational resources are required for genome-scale analyses. Escherichia coli K-12 has been one of the best-characterized organisms in molecular biology. Yet, many key resources for functional genomics and systems biology studies of E. coli are still lacking. Whole genome sequences are now available for two closely related K-12 strains, MG1655 (Blattner et al, 1997) and W3110 (Hayashi et al, 2006). Whole-genome comparative sequencing and reconciliation of differences by re-sequencing selected regions from both strains have recently provided the most accurate genome of any organism (accompanying manuscript; Hayashi et al, 2006). Of 267 regions that were initially found to have short insertion or deletion (indel) and nucleotide (nt) disparities, only eight sites were found to be true differences. The vast majority (243) were due to errors in the original 4.5-Mb E. coli K-12 MG1655 genome (an error rate of less than 1 per 13 000 nt 8 years later); 16 were due to errors in the 2.6 Mb of the W3110 genome reported from 1992 to 1997. Sequence corrections resulted in major changes in the translation of 111 MG1655 open-reading frames (ORFs), mostly due to frame shifting (85), but also due to gene fissions (2), gene fusions (23), and inversion (1; Hayashi et al, 2006). The availability of highly accurate E. coli K-12 genomes (Hayashi et al, 2006) provided an impetus for the cooperative re-annotation of both MG1655 and W3110 (Riley et al, 2006). Sequence corrections also changed many gene boundaries, which led to dropping 31 previously annotated genes and adding 66 new ones. The composite K-12 genome has 4453 genes, encoding 4296 ORFs (including 74 pseudogenes), 156 RNAs, and one annotated feature (oriC). Major differences between the MG1655 and W3110 genomes are the 12 additional sites of an insertion sequence (IS) in W3110, and one additional IS site and the defective CPZ-55 phage (seven prophage genes) only in MG1655. Consequently, MG1655 and W3110 have two and 17 extra copies of IS genes, respectively, and MG1655 has 11 and W3110 has 21 unique genes (including seven additional pseudogenes). Thus, on the basis of the 2005 annotation snapshot, MG1655 has a total of 4464 genes and W3110 has 4474 (Hayashi et al, 2006). In addition to updating annotations of gene functions, start sites were changed for 682 MG1655 ORFs (Riley et al, 2006). An additional 76 ORFs that have been predicted in W3110 have been targeted, for a total of 4550 genes encoding 4390 ORFs (Hayashi et al, 2006), although these have not been recognized as ORFs in the recent K-12 annotation workshops. An E. coli K-12 functional genomics project was initiated in Japan to (1) create new experimental resources, (2) establish new analysis methods, (3) develop new computational approaches, (4) improve databases, and (5) analyze gene function through experimentation by using these resources, methods, approaches, and databases (Mori et al, 2000). Newly created experimental resources now include: (a) two E. coli K-12 ORFeome plasmid banks of nearly all predicted ORFs, the ASKA clone sets (Kitagawa et al, 2005), (b) a large collection of transposon-generated gene-disruption mutants (Mori et al, 2000), and (c) mutants individually deleted of all nonessential E. coli K-12 genes (this study). Newly established analysis methods have included DNA microarrays (Oshima et al, 2002b), proteome analysis tools (Katayama et al, 2002), and tagged genes for detecting protein–protein interactions (Arifuzzaman et al, 2006). Newly developed computational approaches have included tools for gene clustering and codon usage diversity (Kanaya et al, 2001). An improved E. coli K-12 GenoBase (version 5.0; http://ecoli.aist-nara.ac.jp/) supports data analysis based on these new resources, analysis methods, and computational approaches. These resources and methods have been helpful for assignment of new cellular roles to many genes of unknown or poorly described function (e.g. Oshima et al, 2002a). In a Saccharomyces cerevisiae functional genomics project, a nearly complete set of single-gene deletions covering 96% of yeast annotated ORFs was constructed by using a PCR gene replacement method (Giaever et al, 2002). The yeast mutants were isolated by direct transformation with PCR products encoding kanamycin resistance and containing 45-nt flanking homologous sequences for adjacent chromosomal regions. Genome-scale disruption of Bacillus subtilis genes (Kobayashi et al, 2003) was carried out by inactivating each gene with a gene-specific plasmid clone. Comprehensive transposon mutagenesis of Pseudomonas aeruginosa was carried out by generating a large set (30 100) of sequence-defined mutants (Jacobs et al, 2003). Two groups began projects to construct comprehensive transposon mutant libraries of E. coli K-12. In Japan, chromosomal segments in a phage λ library (Kohara et al, 1987) were subjected to transposon mutagenesis, after which the mutations were recombined onto the chromosome by homologous recombination (Mori et al, 2000; T Miki, personal communication). The other group subjected PCR products encoding ORFs to in vitro Tn5 transposition (Goryshin et al, 2000), and then recombined the mutations onto the chromosome by λ Red-mediated recombination (Datsenko and Wanner, 2000), which led to the creation of insertion alleles for 1976 ORFs (Kang et al, 2004). Although transposon mutagenesis has yielded large unique collections of valuable mutants, the methodologies for building a comprehensive library are laborious. First, it is necessary to define the insertion sites by PCR or DNA sequencing. Second, rearrangements or genetic duplications can result when recombining mutations onto the chromosome, compounding results, and requiring additional testing. Third, complications resulting from transposon mutagenesis, such as incomplete disruption of the targeted gene and polarity effects on downstream genes, are unavoidable. While the project for building a transposon library was underway in Japan, a highly efficient method for direct inactivation of chromosomal genes in E. coli K-12 was reported (Datsenko and Wanner, 2000). This breakthrough provided a simple and efficient method for gene deletion analogous to the one that has been used in yeast (Baudin et al, 1993), except by use of cells carrying an easily curable, low-copy-number plasmid expressing the λ Red recombinase. Advantages are being able to target genes for complete deletion, to design deletions arbitrarily and precisely, and to easily eliminate the antibiotic resistance marker subsequently. Here, we used the λ Red system for the systematic construction of a set E. coli K-12 mutants with precisely defined single-gene deletions, called the Keio collection, which upon release of the resistance marker will leave behind an in-frame deletion. For convenience of gene transfer, the Keio collection retains the resistance marker. Results and discussion Keio collection mutants The Keio collection is comprised of 3985 deletions in duplicate (7970 total) of E. coli K-12 strain BW25113 (Datsenko and Wanner, 2000), a strain with a well-defined pedigree that has not been subjected to mutagens (Figure 1; Supplementary Table 1). Mutants were directly selected as kanamycin-resistant (KmR) colonies after electroporation of BW25113 carrying the λ Red expression plasmid pKD46 (Datsenko and Wanner, 2000). To alleviate problems that can arise in high-throughput experiments, resulting from handling errors, crosscontamination, and accumulation of secondary mutations, two independent mutants were saved for each deletion. Figure 1.Derivation of E. coli K-12 BW25113. Strain BD792, like MG1655, is a two-step descendent of ancestral E. coli K-12, EMG2, originally called WG1 (Bachmann, 1996; Hayashi et al, 2006; late BJ Bachmann, personal communication). Like its predecessor W1485F+ (Hayashi et al, 2006), BD792 has the rpoS396(Am) allele (codon 33, TAG (Am); unpublished data). Strain BW25113 was derived from BD792 in a series of steps involving generalized transduction and allele replacements, which included introducing the pseudoreversion rpoS (Q33) allele from MG1655 into a predecessor of BW25113 (Supplementary Table 1). The derivation of W3110 is shown in Figure 1 of accompanying manuscript (Hayashi et al, 2006). Download figure Download PowerPoint Design of in-frame, single-gene deletion mutants Chromosomal genes were targeted for mutagenesis with PCR products containing a resistance cassette flanked by FLP recognition target (FRT) sites and 50-bp homologies to adjacent chromosomal sequences (Figure 2). To reduce polar effects on downstream gene expression, primers were designed so that excision of the resistance cassette with the FLP recombinase would create an in-frame deletion of the respective chromosomal gene (Figure 3). Primer sequences were based on the highly accurate E. coli K-12 genome (Hayashi et al, 2006), in which the majority of the corrections to coding regions and start codon re-assignments had been made in accordance with the November 2003 E. coli K-12 annotation workshop (Riley et al, 2006). Figure 2.Primer design and construction of single-gene deletion mutants. Gene knockout primers have 20-nt 3′ ends for priming upstream (P1) and downstream (P2) of the FRT sites flanking the kanamycin resistance gene in pKD13 and 50-nt 5′ ends homologous to upstream (H1) and downstream (H2) chromosomal sequences for targeting the gene deletion (Supplementary Table 2). H1 includes the gene B (target) initiation codon. H2 includes codons for the six C-terminal residues, the stop codon, and 29-nt downstream. The same primer design with respect to gene B was used to target deletions regardless of whether gene B lies in an operon with genes A and C, as shown, or in different chromosomal arrangements. Novel junctions created between the resistance cassette and neighboring upstream (gene A) and downstream (gene C) sequences were verified by PCR with kanamycin (k1 or k2) and locus-specific (U or D) primers. Structures created after excision of the resistance gene are verified by PCR with neighboring gene-specific primers and by direct DNA sequencing of the region encompassing the H1-P1-FRT-P2-H2 scar to verify correct ones, as described elsewhere (Datsenko and Wanner, 2000). SD, Shine–Dalgarno ribosome binding sequence. Download figure Download PowerPoint Figure 3.Structure of in-frame deletions. FLP-mediated excision of the FRT-flanked resistance gene is predicted to create a translatable scar sequence in-frame with the gene B target initiation codon and its C-terminal 18-nt coding region. Translation from the authentic gene B SD and start codon is expected to produce a 34-residue scar peptide with an N-terminal Met, 27 scar-specific residues, and six C-terminal, gene B-specific residues. Download figure Download PowerPoint The targeting PCR products were designed to create in-frame deletions of the 2nd through the 7th codon from the C-terminus, leaving the ORF start codon and translational signal for a downstream gene intact (Figure 2). However, according to its latest genome annotation, E. coli K-12 has 742 overlapping genes, ranging in length from 1 to 260 nt, with the longest being for ytfP and yzfA. Although the majority are short (1–8 nt), 191 genes overlap by at least 9 nt. Thus, our standard design for construction of in-frame deletions can in some cases simultaneously affect the coding of two overlapping ORFs, which can be especially important when evaluating gene essentiality. For example, folC encodes bifunctional folylpolyglutamate and dihydrofolate synthases and has an 11-nt overlap with the downstream dedD, encoding a conserved protein of unknown function. In agreement with an earlier study (Pyne and Bognar, 1992), we found folC to be essential (on the basis of the criteria below). Preliminary results suggested that dedD was also essential. However, due to the folC-dedD gene overlap, it was conceivable that the lethality of a dedD deletion was due to alteration of the folC C-terminus. To address these kinds of issues, a small number of primers were redesigned to avoid altering two genes simultaneously, by taking into account gene overlaps. Indeed, dedD was successfully deleted with a PCR product that was synthesized with an N-terminal primer that was redesigned to prevent altering the folC coding region. Primer extensions are given in Supplementary Table 2. Construction and verification of deletion mutants Our standard protocol usually yielded 10–1000 KmR colonies when cells were incubated aerobically at 37°C on Luria broth (LB) agar containing 30 μg/ml kanamycin. The most critical step was preparation of highly electrocompetent cells (>109 transformants per 1 μg of plasmid DNA under standard conditions). Mutants were isolated in batches, in which each batch included a PCR product for disruption of ydhQ as a positive control as well as a no PCR product negative control. The latter usually gave only 10–100 tiny colonies. From every gene deletion experiment, four or eight KmR colonies were chosen and checked for ones with the correct structure by PCR using a combination of locus- and kanamycin-specific primers (Figure 2), as described elsewhere (Datsenko and Wanner, 2000). Mutants were scored as correct if two or more colonies had the expected structure based on PCR tests for both junction fragments. Keio collection deletions Of 4288 genes targeted, deletions were obtained for 3985 ORFs (Supplementary Table 3). Based on finding mutants with the predicted structure, these 3985 genes are (probably) nonessential, while the 303 genes (including 37 genes of unknown function), for which no mutants were found, are candidates for essential genes (Figure 4; Table I). Our ORF deletions include 3912 genes annotated in both E. coli K-12 MG1655 and W3110 and 73 previously annotated genes (Supplementary Table 4). The 3912 composite K-12 ORF deletions include 2157 characterized genes and 1755 genes of uncharacterized or unknown function. ORFs not targeted include 79 IS genes, four genes for small toxic polypeptides (ldrA, ldrB, ldrC, and ldrD), and seven genes already disrupted in BW25113 (araBAD, lacZ, and rhaBAD; Datsenko and Wanner, 2000). No in-frame deletion was targeted to 12 ORFs whose coding region was changed at the March 2005 annotation workshop (Riley et al, 2006) after completion of the Keio collection (Supplementary Table 5). RNA genes were also not targeted. Figure 4.Mutagenesis of E. coli K-12 ORFs. See text. Download figure Download PowerPoint Table 1. Mutant summary Class ORFs Total 4390 Targeteda 4288 Nonessential 3985 Essential 303 Not targetedb 102 ORF=open-reading frame. a All targeted ORFs are in given Supplementary Table 2. b ORFs not targeted are given in the text. Evaluation of gene essentiality Several causes can contribute to finding too many or too few nonessential genes. One way to evaluate gene essentiality is to examine our knockout efficiency (Table II), that is, the percent of the KmR colonies with the correct structure. For nearly 50% of the targeted ORFs, all KmR colonies had the expected structure for the correct deletion; for 93% of the ORFs, at least 50% were correct; and, with one exception, for all Keio mutants, at least 25% were correct. The exceptional case, secM, has a translational arrest sequence within its C-terminus that is required for expression of the downstream secA, encoding an essential preprotein translocase SecA subunit (Murakami et al, 2004; Nakatogawa et al, 2005). Thus, it is reasonable to suggest that the sole secM mutant arose because it acquired a suppressor allowing secA expression. Essential gene candidates are given in Supplementary Table 6. Table 2. Knockout efficiencya Percent correctb ORFs Essentiality scorec <−1 −1 to +1 ⩾+1 100 1946 1916 30 0 87.5 729 719 10 0 75 499 487 12 0 62.5 316 307 9 0 50 219 211 8 0 37.5 116 112 4 0 25 160 149 11 0 12.5 1 0 1 0 0 302 0 88 214 Total 4288 3901 173 214 ORF=open-reading frame. a Data are in given Supplementary Table 3. b Percent of the four or eight KmR colonies shown to have the correct structure by PCR as described in text is given. c The number of ORFs with different essentiality scores is given. Scores less than −1 or greater than +1 mean that the gene is nonessential (<−1) or essential (>+1) with no inconsistency with previous studies. Scores between −1 and +1 mean some inconsistency exists. The ability to select directly for knockout mutants may have led to other mutants with suppressors. For example, the same mutagenesis strategy has been used elsewhere to create a deletion of mreB (Kruse et al, 2003), an essential gene, in which case, the mutant was later shown to carry a suppressor (Kruse et al, 2005). Yet, we repeatedly failed to recover a ΔmreB mutant, even when using the identical primers and host. We also confirmed the absence of mreB coding sequences in their ΔmreB mutant, thus ruling out the possibility of a duplicate mreB sequence (data not shown). Clearly, secM and mreB are examples of ‘quasi-essential’ genes, for suppressors allow viability of mutants with the respective deletions. By definition, deletions of truly essential genes cannot be mutationally suppressed. In addition to suppressors, a functional redundancy or duplication can hide gene essentiality. It is difficult to assess functional redundancy without further experimentation. However, gene duplications can explain why we recovered mutants with deletions of some genes, like ileS and glyS, encoding isoleucyl-tRNA and glycine (β-subunit) tRNA synthetase
