Keio Collection Research Articles

Role of rpoS in the development of cell envelope resilience and pressure resistance in stationary-phase Escherichia coli.

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary-phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure sensitive and took up more PI than the parent strain, with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY, and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment, and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure sensitive, was unaffected in membrane resealing, implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA, and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

Development of an Automated Platform for High-Throughput P1-Phage Transduction of Escherichia Coli

Synthetic biology depends on the ability to rapidly produce strains with improved phenotypes but is limited by the ability to rapidly produce strain collections with directed mutations. Here, we present a system capable of overcoming this limitation through automated P1-phage transductions of Escherichia coli. By combining the Keio collection of single-gene deletion E. coli mutants with P1-phage, it is possible to generate an engineered host-strain collection consisting of every possible gene deletion mutant. This strategy was tested by transducing 355 genetic markers from the Keio collection into five different host strains, and it achieved a 98% success rate. This method offers an improved mechanism for rapidly engineering collections of microbes and provides one method for rapidly deploying a broader synthetic biology effort.

Bacterial Toxin RelE Mediates Frequent Codon-independent mRNA Cleavage from the 5′ End of Coding Regions in Vivo

The enzymatic activity of the RelE bacterial toxin component of the Escherichia coli RelBE toxin-antitoxin system has been extensively studied in vitro and to a lesser extent in vivo. These earlier reports revealed that 1) RelE alone does not exhibit mRNA cleavage activity, 2) RelE mediates mRNA cleavage through its association with the ribosome, 3) RelE-mediated mRNA cleavage occurs at the ribosomal A site and, 4) Cleavage of mRNA by RelE exhibits high codon specificity. More specifically, RelE exhibits a preference for the stop codons UAG and UGA and sense codons CAG and UCG in vitro. In this study, we used a comprehensive primer extension approach to map the frequency and codon specificity of RelE cleavage activity in vivo. We found extensive cleavage at the beginning of the coding region of five transcripts, ompA, lpp, ompF, rpsA, and tufA. We then mapped RelE cleavage sites across one short transcript (lpp) and two long transcripts (ompF and ompA). RelE cut all of these transcripts frequently and efficiently within the first ∼100 codons, only occasionally cut beyond this point, and rarely cut at sites in proximity to the 3' end. Among 196 RelE sites in these five transcripts, there was no preference for CAG or UCG sense codons. In fact, bioinformatic analysis of the RelE cleavage sites failed to identify any sequence preferences. These results suggest a model of RelE function distinct from those proposed previously, because RelE directed frequent codon-independent mRNA cleavage coincident with the commencement of translation elongation.

Single-Gene Deletion Mutants of Escherichia coli with Altered Sensitivity to Bicyclomycin, an Inhibitor of Transcription Termination Factor Rho

We have screened the entire KEIO collection of 3,985 single-gene knockouts in Escherichia coli for increased susceptibility or resistance to the antibiotic bicyclomycin (BCM), a potent inhibitor of the transcription termination factor Rho. We also compared the results to those of a recent study we conducted with a large set of antibiotics (A. Liu et al., Antimicrob. Agents Chemother. 54:1393-1403, 2010). We find that deletions of many different types of genes increase sensitivity to BCM. Some of these are involved in multidrug sensitivity/resistance, whereas others are specific for BCM. Mutations in a number of DNA recombination and repair genes increase BCM sensitivity, indicating that DNA damage leading to single- and double-strand breaks is a downstream effect of Rho inhibition. MDS42, which is deleted for all cryptic prophages and insertion elements (G. Posfai et al., Science 312:1044-1046, 2006), or W3102 deleted for the rac prophage-encoded kil gene, are partially resistant to BCM (C. J. Cardinale et al., Science 230:935-938, 2008). Deletion of cryptic prophages also overcomes the increased BCM sensitivity in some but not all mutants examined here. Deletion of the hns gene renders the cell more sensitive to BCM even in the Δkil or MDS42 background. This suggests that BCM activates additional modes of cell death independent of Kil and that these could provide a target to potentiate BCM killing.

Escherichia coli knock-out mutants with altered electron transfer activity in the Micredox® assay and in microbial fuel cells

Electron transfer from bacteria to external electron acceptors is a biologically important phenomenon that is increasingly being harnessed as useful technology such as in the Micredox® assay and in microbial fuel cells (MFCs). Optimisation of these systems is limited by incomplete knowledge of the underlying genetics of electron transfer. The Keio collection of single gene knock-out Escherichia coli strains is being tested to find genes involved in electron transfer from bacteria to external electron acceptors. Initially, 21 E. coli strains from the Keio collection were selected and tested for altered electro-activity using the Micredox® assay. The Micredox® assay provides a rapid measurement of electron transfer from cells to a soluble electron acceptor (potassium hexacyanoferrate(III)) and was previously developed as a general test for BOD and toxicant measurement. Of the 21 Keio strains, 10 were found to have significantly reduced electron transfer and two were found to have significantly increased electron transfer. The mutant with the lowest electron transfer rate (nuoA) and the highest electron transfer rate (arcA) were then tested for electron transfer in microbial fuel cells (MFCs). The arcA mutant had slightly higher electron transfer rates than the wild type in mediator-less MFC while the nuoA mutant strain had very similar electro-activity to the wild type. However, in a mediated MFC, the mutants were consistently different from the wild type. These results demonstrate that single gene deletion strains of E. coli can have significantly altered electron transfer capabilities, both in the Micredox® assay and in MFCs. Importantly, the Micredox® assay was found to be a rapid and easily scaled-up method to discover genes that are important in electron transfer.

ENVIRONMENTAL DEPENDENCY OF GENE KNOCKOUTS ON PHENOTYPE MICROARRAY ANALYSIS IN ESCHERICHIA COLI

Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray™ (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.

Determination of hypersensitivity to genotoxic agents among Escherichia coli single gene knockout mutants

We have tested the KEIO collection of 3985 different viable single gene knockouts in Escherichia coli to identify genes whose loss increases sensitivity to one or more of six different chemotherapeutic agents and mutagens: Bleomycin (BLM), Cisplatin (CPT), ICR-191 (ICR), 5-azacytidine (5AZ), Zebularine (ZEB), and 5-bromo-2′-deoxyuridine (5BdU). We discovered a set of 156 strains that display a significant increase in sensitivity to at least one of the agents tested. Each genotoxic agent generates a distinct “sensitivity profile” that is characteristic of the agent. Comparison with an independent study of sensitivity profiles for an extensive set of antibiotics pinpoints those effects that are relatively specific for each agent. In some cases engineered double mutants have greatly increased effects. These results provide insight into the mechanism of action of each agent, and define targets for the design of co-drugs that can potentiate these agents. An example is the finding that mutants lacking one of several genes in the folate biosynthetic pathway are hypersensitive to ZEB, leading to a demonstration of synergy between trimethoprim and ZEB.

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.

Enhancement of recombinant enzyme activity in cpxA-deficient mutant of Escherichia coli

We have previously performed a systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) to investigate the effect of cellular components on the activity of whole-cell catalysts (Zhou et al. Appl. Microbiol. Biotechnol. in press (2010)). In the present paper, the cpxA-deficient mutant of E.coli BW25113 was found to be able to enhance the activity of cytochrome P450s, including CYP154A1 of Streptomyces coelicolor, compactin 6β-hydroxylase of Streptomyces flavus A-177, vitamin D3 hydroxylase of Pseudonocardia autotrophica, and a mutant enzyme derived from P450 BM3 of Bacillus megaterium. The cytochrome P450 activities in the cpxA-deficient mutant were 1.8-7.9 times greater than those detected with the parental strain BW25113 (DE3). The cpxA-deficient mutant also exhibited β-galactosidase activity approximately 1.5-fold greater than did the parental strain, showing that the enhancing effect was not unique to the activity of cytochrome P450s. Promoter dependency analysis revealed that the enhanced activity of the enzymes was detectable only when the genes encoding the enzymes were expressed under the control of lactose-inducible promoters. The result of real-time PCR showed that the enhancement of the enzyme activities was attributed to increased transcriptional levels of the genes coding for the enzymes.

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (Delta cpxA, Delta gcvR, Delta glnL, and an unknown-gene-deleted one (Delta uk)) were able to increase the CYP154A1 activity by approximately 1.4-1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (Delta cpxA, Delta gcvR, and Delta glnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Delta uk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of Delta cpxA, Delta gcvR, and Delta glnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.

Antibiotic Sensitivity Profiles Determined with an Escherichia coli Gene Knockout Collection: Generating an Antibiotic Bar Code

We have defined a sensitivity profile for 22 antibiotics by extending previous work testing the entire KEIO collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to 1 of 14 different antibiotics (ciprofloxacin, rifampin [rifampicin], vancomycin, ampicillin, sulfamethoxazole, gentamicin, metronidazole, streptomycin, fusidic acid, tetracycline, chloramphenicol, nitrofurantoin, erythromycin, and triclosan). We screened one or more subinhibitory concentrations of each antibiotic, generating more than 80,000 data points and allowing a reduction of the entire collection to a set of 283 strains that display significantly increased sensitivity to at least one of the antibiotics. We used this reduced set of strains to determine a profile for eight additional antibiotics (spectinomycin, cephradine, aztreonem, colistin, neomycin, enoxacin, tobramycin, and cefoxitin). The profiles for the 22 antibiotics represent a growing catalog of sensitivity fingerprints that can be separated into two components, multidrug-resistant mutants and those mutants that confer relatively specific sensitivity to the antibiotic or type of antibiotic tested. The latter group can be represented by a set of 20 to 60 strains that can be used for the rapid typing of antibiotics by generating a virtual bar code readout of the specific sensitivities. Taken together, these data reveal the complexity of intrinsic resistance and provide additional targets for the design of codrugs (or combinations of drugs) that potentiate existing antibiotics.

Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes

Using the Keio collection of gene-disrupted mutants of Escherichia coli, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism. Among the mutants identified in the study, Delta mgtA, Delta phoP and Delta phoQ cells, all lacking genes that are induced under low extracellular Mg2+ conditions, displayed glycogen-deficient phenotypes. In this work we show that these mutants accumulated normal glycogen levels when the culture medium was supplemented with submillimolar Mg2+ concentrations. Expression analyses conducted in wild-type, Delta phoP and Delta phoQ cells showed that the glgCAP operon is under PhoP-PhoQ control in the submillimolar Mg2+ concentration range. Subsequent screening of the Keio collection under non-limiting Mg2+ allowed the identification of 183 knock-out mutants with altered glycogen levels. The stringent and general stress responses, end-turnover of tRNA, intracellular AMP levels, and metabolism of amino acids, iron, carbon and sulfur were major determinants of glycogen levels. glgC::lacZY expression analyses using mutants representing different functional categories revealed that the glgCAP operon belongs to the RelA regulon. We propose an integrated metabolic model wherein glycogen metabolism is (a) tightly controlled by the energy and nutritional status of the cell and (b) finely regulated by changes in environmental Mg2+ occurring at the submillimolar concentration range.

Genome‐wide screens: novel mechanisms in colicin import and cytotoxicity

Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last 25 years. Genome-wide screening using the 'Keio collection' to test sensitivity to colicins (col) A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the lipopolysaccharide inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of lipopolysaccharide and enterobacterial common antigen. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Peptidoglycan-associated lipoprotein, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene product has the necessary proteolysis function.

The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli

Ribosome biogenesis is coupled to the transcription of ribosomal (r)RNAs, which enables an ordered and hierarchical assembly of the ribosome starting with the 5'-terminal domain. We constructed four circular permutants (CPs) of Escherichia coli rRNAs in which the original termini of 16S or 23S rRNAs were genetically connected, and new termini were created elsewhere within the same rRNAs (in helix 33 of 16S rRNA, or in helices 45, 63, and 78 of 23S rRNA). Unexpectedly, all CPs tested were able to rescue E. coli strain Delta7 prrn, which lacks all chromosomal rRNA operons. This result demonstrates that hierarchical assembly from the 5'-terminal domain of both 16S and 23S rRNAs is not essential for ribosomal formation in the cell. However, severe growth defects of all CPs were found in the absence of the DEAD box RNA helicase deaD, indicating that DeaD assists in the efficient assembly of each subunit in the cell.

GrowMatch: An Automated Method for Reconciling In Silico/In Vivo Growth Predictions

Genome-scale metabolic reconstructions are typically validated by comparing in silico growth predictions across different mutants utilizing different carbon sources with in vivo growth data. This comparison results in two types of model-prediction inconsistencies; either the model predicts growth when no growth is observed in the experiment (GNG inconsistencies) or the model predicts no growth when the experiment reveals growth (NGG inconsistencies). Here we propose an optimization-based framework, GrowMatch, to automatically reconcile GNG predictions (by suppressing functionalities in the model) and NGG predictions (by adding functionalities to the model). We use GrowMatch to resolve inconsistencies between the predictions of the latest in silico Escherichia coli (iAF1260) model and the in vivo data available in the Keio collection and improved the consistency of in silico with in vivo predictions from 90.6% to 96.7%. Specifically, we were able to suggest consistency-restoring hypotheses for 56/72 GNG mutants and 13/38 NGG mutants. GrowMatch resolved 18 GNG inconsistencies by suggesting suppressions in the mutant metabolic networks. Fifteen inconsistencies were resolved by suppressing isozymes in the metabolic network, and the remaining 23 GNG mutants corresponding to blocked genes were resolved by suitably modifying the biomass equation of iAF1260. GrowMatch suggested consistency-restoring hypotheses for five NGG mutants by adding functionalities to the model whereas the remaining eight inconsistencies were resolved by pinpointing possible alternate genes that carry out the function of the deleted gene. For many cases, GrowMatch identified fairly nonintuitive model modification hypotheses that would have been difficult to pinpoint through inspection alone. In addition, GrowMatch can be used during the construction phase of new, as opposed to existing, genome-scale metabolic models, leading to more expedient and accurate reconstructions.

Protein Translocation Across Membranes: Components of Outer Membrane Colicin Translocons

Translocation of the nuclease colicins E2 and E3 across the E. coli outer membrane is initiated by high affinity (Kd < 10−9 M) binding of the receptor-binding (R) domain to the vitamin B12 (BtuB) receptor in the E. coli outer membrane. Based on genetic analysis (1), and crystal structures of BtuB (2), the complex of the R-domain of colicins E2 or E3 bound to BtuB (3, 4), and of the OmpF porin containing the inserted N-terminal disordered segment of the colicin translocation (T) domain (5), a “fishing pole” model for the colicin translocon was inferred (3-5). The T- and C (catalytic) colicin segments must unfold before insertion into OmpF. FRET analysis was employed to study the colicin unfolding upon interaction with BtuB and OmpF (6). A rapid (k1/2 < 1 sec−1) decrease in FRET efficiency between translocation and cytotoxic domains of colicin E3 was observed upon independent and additive colicin binding in vitro to BtuB and OmpF. Colicin interactions with BtuB and OmpF have a major electrostatic component, provided at least partly for BtuB by R-domain Arg399. Thus, free energy for colicin unfolding is provided by binding of the R- domain to BtuB and also by binding/insertion of T-domain to OmpF. Screening the “Keio collection” for cytotoxicity of several group A and B colicins has shown thus far that colicin N binds to the first glucose of the LPS inner core (7).(1) Benedetti et al.,1989.(2) Cherezov et al., J. Mol. Biol, 364,716-, 2006.(3) Kurisu et al., Nat. Struct Biol., 10:948-, 2003.(4) Sharma et al. J. Biol.Chem., 282, 23163, 2007.(5) Yamashita et al., EMBO J., 27, 2171-, 2008.(6) Zakharov et al., Biochemistry, 47: in press, 2008.(7) Sharma et al., in preparation, 2008. [NIH-GM18457(WAC), NIH-GM083296(BLW)].

The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli

L-cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of L-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in L-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in L-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to L-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for L-cysteine tolerance in E. coli cells. However, L-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent L-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of L-cysteine, the transformants exhibited more L-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in L: -cysteine tolerance probably due to its export ability and that TolC overexpression is effective for L-cysteine production in E. coli.

Escherichia coli genes affecting recipient ability in plasmid conjugation: Are there any?

BackgroundHow does the recipient cell contribute to bacterial conjugation? To answer this question we systematically analyzed the individual contribution of each Escherichia coli gene in matings using plasmid R388 as a conjugative plasmid. We used an automated conjugation assay and two sets of E. coli mutant collections: the Keio collection (3,908 E. coli single-gene deletion mutants) and a collection of 20,000 random mini-Tn10::Km insertion mutants in E. coli strain DH5α. The combined use of both collections assured that we screened > 99% of the E. coli non-essential genes in our survey.ResultsResults indicate that no non-essential recipient E. coli genes exist that play an essential role in conjugation. Mutations in the lipopolysaccharide (LPS) synthesis pathway had a modest effect on R388 plasmid transfer (6 – 32% of wild type). The same mutations showed a drastic inhibition effect on F-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative DNA processing or transport per se.ConclusionWe conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. This is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell.

Update on the Keio collection of Escherichia coli single‐gene deletion mutants

Mol Syst Biol. 5: 335 The Keio collection (Baba et al , 2006) has been established as a set of single‐gene deletion mutants of Escherichia coli K‐12. These mutants have a precisely designed deletion from the second codon from the seventh to the last codon of each predicted ORF. Further information is available at http://sal.cs.purdue.edu:8097/GB7/index.jsp or http://ecoli.naist.jp/. The distribution is now being handled by the National Institute of Genetics of Japan (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp). To date more than 4 million samples have been distributed worldwide. As we described earlier (Baba et al , 2006), gene amplification during construction is likely to have led to a small number of mutants with genetic duplications. The design of the Keio deletions was based on annotations that are now outdated. Of 4288 ORFs targeted, mutants were obtained for 3985 (Baba et al , 2006). Re‐annotation based on highly accurate sequencing of E. coli K‐12 (Hayashi et al , 2006) led to changing many coding regions and the total number of ORFs to 4296, including pseudogenes (Riley et al , 2006) (Supplementary Table I). The recent E. coli K‐12 MG1655 GenBank record (U0096, released in December 2008) has an additional 97 ORFs (exclusive of the ORFs in IS elements, Supplementary Table II) that were not targeted. Of these 4214 annotated ORFs, 4186 were targeted for deletion and 28 were not (Supplementary Table III), which resulted in the isolation of two independent mutants for 3864 targeted ORFs. No deletion was found for 299 ORFs, which are candidates for essential genes. Deletions were also isolated for 23 other ORFs; however, re‐annotation led to re‐classification of these ORFs as ‘split ORFs’, because their coding …