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Laboratory of Genetics, University of Wisconsin Madison, Madison, Wisconsin 53706
Received 24 October 2003/ Accepted 22 January 2004
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Essential genes are of particular interest because they include the core functions upon which basic cellular processes are based, they tend to be widely conserved, and they can serve as a starting point for minimal genome designs (24, 27). The number of essential genes in an organism depends on the conditions used; in this case, we defined them broadly as genes required for growth on rich medium. Different experimental methods have been used to determine the number of essential genes. In E. coli, at least 620 ORFs are essential (11); in Bacillus subtilis, 271 out of 
4,100 ORFs are essential (21); in Haemophilus influenza, 670 out of 1,850 ORFs are essential (1); and in Staphylococcus aureus, 658 out of 2,630 ORFs are essential (10). Only about 15 essential gene products are targeted by antibiotics. Identification of new targets may help combat the problem of increasingly prevalent antibiotic resistance. Essential gene mutants are important in genomics-based drug discovery for target validation and prioritization and can be used to screen compound libraries and determine the mode of action. Unfortunately, difficulties in mutating essential genes have impeded their genetic study.
Several approaches to conditional mutagenesis can be considered. Temperature-sensitive (TS) mutations offer direct and sometimes reversible control of protein function, but it is extremely difficult to design proteins with a TS phenotype. In some cases, TS alleles may not be possible (33) or activity may be suboptimal even at permissive temperature. Another approach is to place the target gene under the control of an experimentally controllable promoter either on a plasmid (12) or in the genome (17). This allows turning gene expression on and off over a large dynamic range, but the natural expression level and regulation of the gene are overridden by the inducing promoter. Another approach uses antisense RNA in which short pieces of RNA are produced that interfere with translation (10, 15). Antisense mutants are made by using a random whole-genome approach, and it is not clear if a high enough percentage of genes is amenable to this approach to allow disruption of the complete set of essential genes. A fourth approach uses a TS suppressor tRNA to convert amber mutations into a TS phenotype (31) but suffers from a low dynamic range and the relative inefficiency of the TS amber suppressors.
The method we developed uses conditional suppression of amber mutants through inducible expression of the Ala2 amber suppressor tRNA from the arabinose promoter on expression plasmid pBAD/sup2 (see Fig. 1A) (13). The amber codon was chosen because it is the least common of the three stop codons in E. coli, terminating only 326 out of 4,290 annotated ORFs. The Ala2 suppressor was selected because of its high suppression efficiency and its specificity in introducing only the correct amino acid (29). We also have found that expression of this suppressor causes minimal perturbation of global transcription in E. coli (C. D. Herring and F. R. Blattner, unpublished data).
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The genes chosen for this study represent a broad group of essential functions but are restricted to genes at the distal ends of operons. The gene frr encodes ribosome release factor, which is required for the disassembly of ribosomes and mRNA following translational termination (14). FtsZ is a tubulin-like GTPase that polymerizes into a ring that girdles the middle of the cell and leads to cell division. The gene gcpE has been recently reported to encode a terminal step in the non-mevalonate pathway of isoprenoid biosynthesis, which is necessary for the production of diverse metabolites such as quinones and carotenoids (23). The gene lpxC encodes the first committed step in the synthesis of lipid A, which acts as an anchor for lipopolysaccharide in the outer membrane (7). Methionine aminopeptidase is encoded by the gene map and is required for removal of the N-formylmethionine present at the amino terminus of proteins following translation. The gene murA encodes the first committed step in the cytoplasmic synthesis of peptidoglycan, the main component of the cell wall (6). Pi is a central metabolite involved in nearly all areas of cell physiology. The gene ppa encodes inorganic pyrophosphatase, which generates Pi from PPi, a product of nucleic acid polymerization, aminoacylation of tRNAs, and other reactions. The gene rpsA encodes protein S1, the largest ribosomal protein, which is known to have high affinity for mRNA and has been implicated in translation initiation and elongation (34). The new method worked efficiently for all but one of these essential genes of diverse function, arguing that it will be applicable to many of the essential genes of E. coli.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of pK-HT. pK-HT (Fig. 1) was derived from pKD46 (8) by inserting the I-SceI gene under tetracycline control, switching the lambda red genes from arabinose to rhamnose control, and replacing the TS replicator with a temperature-stable allele. First we amplified I-SceI, the tetA promoter, and the tetR gene from pST98-AS (32) by using NcoI adapter primers OF428 and OF429 and digested and then ligated the fragment into the NcoI site of pKD46, resulting in pKDTS. Next, the rhaBAD promoter and rhaSR genes were amplified from E. coli genomic DNA with primers OF434 and OF435 and then digested with SacI/BssHII and ligated into the SacI/BssHII site of pKDTS, resulting in pKDTSR. Finally, the single nucleotide mutation responsible for the TS ori phenotype (3) was replaced with the wild-type (WT) allele by megaprimer PCR and ligation as follows. A megaprimer with the desired modification was amplified from pKDTSR with primers OF436 and OF438 and then extended in a second reaction by adding primer OF439 and a small amount of the full-length OF438-OF439 product as the template. It was then reamplified with OF438-OF439. This PCR product was digested with SpeI/PciI and ligated into the SpeI/PciI site of pKDTSR, resulting in pK-HT.
Generation of mutagenic DNAs. An alanine codon (GCX) was selected near the middle of the coding region of the targeted gene and changed to amber (TAG). If possible, an alanine codon preceded by a C residue was chosen so that each mutant would be marked by CTAG, the relatively rare restriction site for BfaI, so that mutagenesis could be easily confirmed by PCR and restriction digestion. If no codon with an upstream C residue was available, one was selected that could be changed to a C without altering the amino acid sequence.
Four individual pieces were amplified separately with primers designed to overlap one another (Fig. 1B). The ORF was amplified in two halves with primers at the amino and carboxyl termini (called A- and C-term primers in Table S3), paired with overlapping divergent primers in the middle of the gene, both containing the desired amber mutation (mut1 and mut2). The gene for CAM resistance (Camr) was amplified with primers OF105 and OF375, each containing the recognition site for I-SceI. A small part at the end of the ORF and some of the downstream sequence were amplified as a fourth "tail" fragment, with one primer 100 nucleotides (nt) before the C terminus (tail start) and another located approximately 500 nt downstream (tail end). The overlapping PCR products were joined in a two-round overlap extension PCR. In the first round, the four primary pieces were joined to a neighboring fragment in three pairwise combinations. In the second round, the pairwise reactions were combined and the complete fusion was amplified. Details of the PCR fusions were as follows. All amplifications were performed with Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.). In the first round, the four primary pieces were purified with QIAquick (Qiagen, Valencia, Calif.) and then 4 µl of each neighboring pair was combined with Pfu buffer, deoxynucleoside triphosphates (2.5 mM each), and Pfu Turbo in a 25-µl reaction mixture and subjected to 20 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. In the second round of fusion, 5 µl of each unpurified product from the three pairwise combinations was combined with 3.5 µl of Pfu buffer, 5 µl of deoxynucleoside triphosphates, 1 µl of Pfu Turbo, and 20.5 µl of H2O and subjected to five cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 6 min. A 2.5-µl volume of each of the A-term and tail end primers (5 µM) was then added, and the reactions were cycled 20 to 25 more times by the same regimen. Smears and multiple bands were often visible on agarose gels, but a strong primary product was the correct size for all of the genes attempted except one, which was not subsequently used. Mutagenic fragments were made for the genes murA and rpsA by this method. For the genes frr, gcpE, map, ppa, and ycfB, amber alleles had previously been generated by megaprimer PCR and cloned (13). These were amplified with the A-term primers and OF169 and then fused to the gene for Camr and the tail by a similar two-round overlap extension PCR. The mutagenic fragments for ftsZ and lpxC were generated by a much less rapid method as follows. The amber stop codons were introduced by megaprimer PCR and cloned adjacent to Camr, and then the amber allele and the gene for Camr were amplified as one unit with the A-term and p14 primers. This PCR product and the tail piece were digested with BamHI, ligated, and then PCR amplified with the A-term and tail end primers. The ftsZ product was further gel purified, cloned, and then amplified again with OF205 and OF364.
Electroporation and integration.
Electrocompetent recombinogenic cells were made by growing E. coli carrying pK-HT in shake flasks in Luria-Bertani medium containing AMP at 37°C. The plasmid-encoded lambda red functions were induced by adding Rha 2 h before harvesting. When the optical density at 600 nm (OD600) reached 0.5, cells were centrifuged, washed twice in chilled water and once in chilled 10% glycerol, resuspended in 10% glycerol at approximately 1/100 of the original volume, and then frozen. Approximately 100 to 200 ng of overlap extension PCR product, purified with QIAquick and G50 spin columns, was electroporated together with 70 to 90 ng of pBAD/sup2 (13) plasmid DNA. After electroporation, cells were incubated for 1 h at 37°C in SOC/Ara (SOC made with arabinose rather than glucose) (32a) and grown on RDM/Ara/KAN/CAM/AMP plates for 16 to 24 h at 37°C. Colonies were patched on RDM/KAN/CAM/AMP/Glu and RDM/KAN/CAM/AMP/Ara plates to identify conditional lethal mutants that could grow on Ara but not on Glu.
Conditional lethal mutants were not recovered in ppa except when the growth medium was RDM/Ara or a 1:1 mixture of SOC/Ara and RDM. The medium may be important for full expression of the arabinose-inducible suppressor. For all genes, the conditional lethal clones tended to be small colonies on the transformation plates. For the gene rpsA, conditional lethal colonies were only identified after 24 h of growth. These very small colonies were too small to be detected after only 16 h. The small size may be due to slow growth from incomplete suppression but may also be due to lag time before the suppressor is fully expressed. Very few conditional lethal mutants were obtained when plates were grown at 30°C. For this reason, the plasmid origin from pKD46 was corrected during construction of pK-HT so that it would replicate at 37°C.
Counterselection. Conditional lethal mutants were resuspended from the Ara patch and plated on RDM/KAN/AMP/ATC/Ara plates at 30°C to induce I-SceI (without inducing lambda red recombinase). Survivors were patched on RDM/KAN/AMP/Ara plates with and without CAM to confirm loss of the Camr marker. Counterselection with I-SceI was more effective at 30°C than at 37°C. The I-SceI allele used here came from pST98-AS and is missing four amino acids near the N terminus (32). Other alleles of I-SceI under control of the Ara promoter used in this laboratory work efficiently at 37°C, so the effect may be due to either the small deletion or the level of I-SceI expressed from the tetA promoter. One gene (gcpE) was also mutated by an alternate counterselection method with the tetracycline resistance gene rather than I-SceI, as described in the supplemental material.
Plasmid curing and sequence verification. pK-HT was cured from the conditional mutants by streaking and then plating on RDM/KAN/Ara/Rha. Clones that no longer carried the plasmid were identified by patching on RDM/KAN/Ara plates with and without AMP. The targeted essential genes were amplified from one isolate of each mutant with primers located adjacent to the ORF so that only the chromosomal locus could possibly be amplified. Sequencing revealed intact ORFs identical to the WT except for the introduced amber mutation. The primers used for sequencing were OF210, OF215, OF218, OF219, OF242, OF243, OF304, OF305, OF306, OF307, OF308, OF376, OF377, OF378, OF379, OF380, OF381, OF382, OF383, OF410, OF411, OF412, OF413, OF423, OF425, OF443, OF444, OF447, OF448, and OF450.
Growth curves. The lpxC amber mutant used to determine growth curves was the one mutant made by gene gorging, while all of the others were those made as tagalong mutations as described here. Frozen glycerol stocks of each strain were streaked on RDM/KAN/Ara plates, and single colonies were inoculated into tubes containing 1 ml of RDM/KAN/Ara and grown overnight at 37°C. The overnight cultures were inoculated at ratios of 1:50 to 1:6,400 into 200 µl of RDM/KAN plus either Glu or Ara in 96-well plates with lids (Falcon no. 3072; Becton Dickinson, Franklin Lakes, N.J.). Each lid was taped open about 1 mm to allow aeration, and unused wells were filled with water. The cultures were then grown at 37°C with shaking in a Spectramax 384 plus (Molecular Devices, Sunnyvale, Calif.) and read every 15 min, subtracting the reading for the uninoculated plate as a blank. The plate was removed three times to allow sampling. For light microscopy, bacterial smears were heat fixed, stained with crystal violet, and observed at a magnification of x1,000 with a Zeiss Axiovert 35 microscope. Viable cells were counted on RDM/KAN/Ara plates incubated at 37°C. The validity of using a small culture volume (200 µl) in these experiments was verified by using the strains with amber mutations in map and lpxC. Very similar growth curves were observed for these mutants in 150-ml cultures. Additionally, WT strain MG1655 grew essentially the same in 200-µl and 100-ml cultures (29- and 28-min doubling times, respectively, in RDM/Glu medium).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutagenesis strategy.
Conditional amber mutations were introduced into essential genes as outlined in Fig. 1.
PCR primers were designed to amplify a target ORF and a region downstream with WT genomic DNA as the template. Four individual PCR fragments with overlapping sequences at the ends were joined by overlap extension PCR, resulting in a DNA fragment containing an amber stop codon in the target gene, a Camr gene flanked by I-SceI sites, a small duplication of the terminus of the target gene, and some of the downstream sequence.
These linear DNAs were then electroporated along with arabinose-inducible suppressor plasmid pBAD/sup2 into competent cells of E. coli MG1655 expressing lambda red from plasmid pK-HT (Fig. 1A). Double recombination spanning the Camr gene resulted in integration of the linear DNA into the targeted gene in the presence of the induced suppressor tRNA. Clones with the amber mutation incorporated along with the selected marker as a tagalong mutation were identified by their dependence on the suppressor with replicate patching on glucose and arabinose media.
Next, the chloramphenicol resistance gene downstream of the essential gene was precisely removed by counterselection with the I-SceI enzyme (32), induced from plasmid pK-HT with ATC. The recognition sequence for I-SceI does not occur in the WT E. coli genome, so the mutant chromosome was cut on both sides of the gene for Camr but nowhere else. Such a double-strand break is lethal, and the majority of the surviving cells were those that had undergone Rec-mediated recombination between the end of the gene and the 100-nt identical region on the other side of the gene for Camr. The essential-gene ORFs were then sequenced and shown to be completely WT except for the introduced mutation.
Mutagenesis results. Linear DNA fragments were generated for nine genes (Table 1). Camr integrants were obtained for all of the genes, and the percentage that carried the tagalong amber mutation ranged from 2.8 to 53%. Similar integration results were obtained by expressing lambda red from plasmid pKD46 (8), with conditional lethal mutants ranging from 2.5 to 38%. No conditional lethal mutants were recovered with ftsZ. The large variation in the percent amber may be due to incomplete suppression shortly after integration and differing sensitivity to loss of each gene. The highest percentage of amber mutations was observed with the gcpE mutation, which gives rise to reversible bacteriostasis when the suppressor is withdrawn (see below). Conversely, the lowest occurred with the murA mutation, which results in very rapid lysis upon withdrawal of the suppressor.
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10 colonies each, and for most genes the majority of colonies were sensitive. The mutants were cured of plasmid pK-HT by taking advantage of the toxic effects of lambda red (35) by inducing lambda red in the absence of selection for the plasmid. After induction, 10 to 100% of the screened colonies were Amps, indicating loss of pK-HT. Characterization of mutants. To characterize the behavior of the conditional mutants, growth was monitored by OD under both permissive and nonpermissive conditions (Fig. 2). First, the growth rate was measured in arabinose medium, which permits maximal suppression of the amber mutations. Table 2 shows that the doubling time of most strains was only slightly longer than that of the WT, indicating good suppression. The mutants with amber mutations in rpsA and murA showed longer doubling times, suggesting incomplete suppression. The final culture density of the map mutant in arabinose was reproducibly lower than that of the others.
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The mutants were observed by light microscopy at various points during growth arrest (Table 2; Fig. S4 in the supplemental material). Abnormally shaped cells were more common in the mutants than in the WT control, but for strains with amber mutations in frr, gcpE, ppa, and rpsA, normal cells were predominant. In contrast, most cells were abnormal in lpxC and murA amber mutants, and the majority were elongated or filamentous in the strain with a map amber mutation. Notably, lpxC and murA encode enzymes required for synthesis of the cell wall. Debris was observed for many strains, probably indicating lysis.
To determine what effect, if any, the gene encoding Camr might have on the growth phenotypes of these mutants, strains were compared before and after the counterselection step. All of them grew the same except for those with mutations in ppa and rpsA, which stopped growing at a lower OD in glucose when the gene for Camr was present (data not shown). Thus, the downstream Camr marker may in some cases have unintended effects and should be removed, as demonstrated here.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Potential disadvantages of the method include the failure to mutate all of the genes targeted, incomplete suppression of some genes, and the presence of a high-copy-number suppressor plasmid, leading to the spontaneous occurrence of constitutive suppressor mutants. Such mutants are infrequent enough to allow growth experiments, as shown here, but the problem might be greatly reduced by deleting one of the redundant Ala2 tRNA genes (alaX or alaW). Also, single-copy suppressors have been used to achieve complete suppression of some genes (30), although such efficiency may not be possible for genes encoding highly abundant proteins. Regardless, this method could be improved by the development of a better inducible suppressor, especially one at single copy. Additionally, it should be noted that an amber mutation in the middle of the gene could result in a partially active product if the truncated protein is still functional. A systematic-mutagenesis project under way in our laboratory has isolated transposon insertions in known essential genes and should identify those where position effects may be a problem (Y. Kang, T. Durfee, J. D. Glasner, Y. Qiu, D. Frisch, K. M. Winterberg, and F. R. Blattner, submitted for publication). Such situations may be resolved by positioning the amber mutation near the beginning of the gene and integrating the selectable marker upstream rather than downstream. Other methods might avoid this problem by complete deletion of the gene and plasmid complementation, but then the native copy number and regulation would be lost, as with a promoter insertion.
A recent study with pathogenic strains of E. coli (26) reported a 10-fold increase in spontaneous rifampin-resistant mutants following overnight expression of lambda red but no increase following a 1-h induction (2 h was used in this study). More study is needed since it is conceivable that rifampin-resistant mutations in RNA polymerase might have been selected by reducing the transcription of toxic lambda red. In any case, the induction time used in future work to make competent cells and for plasmid curing should probably be reduced to minimize the possibility of introducing undesired mutations. The use of I-SceI does not increase the occurrence of spontaneous rifampin mutants (32), and recombinational repair of double-stranded breaks appears to be error free (22).
Polarity effects on the expression of downstream genes can lead to difficulty in interpreting the results of experiments with conditional mutations. Antisense mutations may affect the expression of entire operons, while promoter insertions affect all of the genes downstream of the insertion site. Amber mutations are not expected to have polar effects except in cases of rho-mediated termination. Our difficulty mutating ftsZ may have been due to the presence of lpxC immediately downstream (under the control of its own promoter). Integration of the Camr drug marker and subsequent transcription originating from the Camr promoter might impact genes located downstream. The genes targeted here were at the distal end of transcriptional units or in single-gene operons. On the basis of known or predicted operon structure (5), 468 out of the 684 genes determined to be essential by either Gerdes et al. (11) or the PEC database (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp) occur without other essential genes downstream. We did not test whether this method is limited to such situations. Regardless, it is still widely applicable to most essential genes.
Conflicting results exist for whether ycfB (also named mnmA, asuE, or trmU) is truly essential. Arigoni et al. (2) could not obtain a null mutant, but when the gene was under direct arabinose promoter control, it was insensitive to the absence of arabinose. Kambampati and Lauhon (20) used a different putative translation start site to make an in-frame deletion of this gene, but Gerdes et al. have recently identified it as an essential gene (11). We identified a glucose-sensitive mutation in ycfB on one occasion, but after removal of the gene for Camr and plasmid curing, it was found to grow normally on glucose. Sequencing showed that it was in fact amber, suggesting that ycfB is not essential.
The seven essential gene mutants made here displayed a wide range of behaviors under nonpermissive conditions. For example, growth of the mutant with a murA amber mutation arrested almost immediately, whereas the map amber mutant was relatively unaffected for many generations. When cells are transferred to nonpermissive medium, a number of events must occur before a phenotype is observed: residual arabinose must be metabolized, transcription of the suppressor tRNA must be halted, residual levels of suppressor must degrade or be diluted by cell growth, residual levels of the targeted gene product must turn over, and finally the loss of that product then has a physiological effect that stops growth. Since all but the last two events ought to be the same for all mutants, the wide variety of behaviors that we observed must be due to gene-specific factors, such as protein degradation rates or how closely a protein is expressed relative to the critical threshold necessary for survival.
The protein encoded by murA is the target for the antibiotic fosfomycin (18), and antibacterial agents have been developed against the lpxC gene product (7). The ribosomal protein S1 from rpsA is an essential component of the ribosome that is targeted by many antibiotics, while the products of frr, gcpE, and map are potential targets (16, 19, 36). The behavior of these mutants may give an important indication as to their efficacy as drug targets. If loss of an essential gene product takes many generations to impede cellular functions, then even the most potent drug may be unusable for the treatment of infection. In assessing this, it is important to remember that while a drug may directly inactivate a protein, withdrawal of the suppressor blocks new synthesis.
Strains can be engineered for the titration of arabinose-controlled expression to intermediate levels (25). RNA isolated from conditional lethal mutants at suboptimal suppression levels can be analyzed for expression patterns diagnostic of loss of that essential function. By comparing these patterns with those from cells subjected to an uncharacterized antibacterial compound, the mode of action of that compound may be investigated. Attenuated essential gene mutants are often more sensitive to compounds that target the mutated gene product and can be used to screen for new drugs and to establish the targets of existing drugs (9, 10). The wide variety of behaviors observed here in nonpermissive medium indicates that conditional amber mutants will also be useful in basic research to characterize essential genes of unknown or poorly understood function, such as those identified by Gerdes et al. (11).
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant GM35682.
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FOOTNOTES |
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Supplemental material for this article may be found at http://jb.asm.org/.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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