Intragenic suppression in the uvrD gene of Escherichia coli. I. Temperature-sensitive uvrD mutations

A temperature-sensitive uvrD mutant, HD323 uvrD4, was isolated from the uvrD mutant HD4 uvrD3. The temperature sensitivity of the uvrD4 gene product was reversible. The suppressor mutation uvrD44 which rendered the uvrD3 mutant temperature-sensitive could be separated from the uvrD3 mutation by replacing the PstI fragment, which encodes the C-terminal half of the UvrD protein. The uvrD44 mutation was found to make host bacteria lethal at non-permissive temperatures only when cloned on a low copy vector pMF3. The nucleotide sequence of the uvrD3 and uvrD4 mutant genes was determined. The nucleotide change found in the uvrD3 at +1235, GAA to AAA, only alters the amino acid sequence from Glu at 387 to Lys. The uvrD44 has another nucleotide change at +1859, GAA to AAA (Glu at 595 to Lys), which is considered to be the suppressor mutation uvrD44.     

Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus.

The influenza A/Alaska/77 (H3N2) virus mutant 143-1 is temperature sensitive (ts) due to a spontaneous in-frame 36-nucleotide deletion in the nonstructural (NS) gene segment, which leads to a 12-amino-acid deletion in the NS1 protein. In addition, it has a small-plaque phenotype on MDCK cell monolayers. However, phenotypically revertant (i.e., ts+) viruses were isolated readily following replication of the 143-1 virus both in vitro and in vivo. In order to determine the genetic mechanism by which escape from the ts phenotype occurred, we performed segregational analysis and found that an intrasegmental suppressor mutation caused the loss of the ts phenotype. Nucleotide sequence analysis revealed the presence of an intragenic mutation in each of the ts+ phenotypic revertant viruses, involving a substitution of valine for alanine at amino acid 23 of the NS1 protein. This mutation resulted in acquisition of the ts+ phenotype and also in the large-plaque phenotype on MDCK cells, characteristic of the wild-type A/Alaska/77 parent virus. This amino acid substitution is predicted to generate an area of alpha helix in the secondary structure of the amino-terminal portion of the NS1 protein of the revertant viruses which may compensate for loss of an alpha-helical region due to the deletion of amino acids 66 to 77 in the NS1 protein of the 143-1 virus.     

Partial suppression of an Escherichia coli TonB transmembrane domain mutation (ΔV17) by a missense mutation in ExbB

Active transport of vitamin B₁₂ and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the amino-terminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The ΔV17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonBΔV17. Molecular modeling suggested that the ΔV17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonBΔV17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the ΔV17 mutation interrupted productive TonB–ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonBΔV17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonBΔV17 export. Unlike the tonBΔV17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonBδV17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (≅ 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.     

Phenotypic suppression of a fructose-1,6-diphosphate aldolase mutation in Escherichia coli

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.     

Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.     

Suppression of the Escherichia coli dnaA46 mutation by a mutation in trxA, the gene for thioredoxin

Summary The dasC mutation, an extragenic suppressor of dnaA46, was mapped by P1 transduction near the rep, trxA, rho region of the Escherichia coli chromosome. The dasC mutation could not be separated from trxA by P1 transduction indicating that dasC and trxA are allelic. Multicopy plasmids containing an intact trxA gene were able to reverse the suppressive effect of the dasC mutation on the dnaA46 mutation. Introduction of a frameshift mutation into the cloned trxA coding region abolished the ability of these recombinant plasmids to reverse the suppressive effect. These results indicate that dasC is allelic with trxA, the gene encoding thioredoxin.     

Deletion mutation analysis of the mutS gene in Escherichia coli

The MutS protein is part of the dam-directed MutHLS mismatch repair pathway in Escherichia coli. We have constructed deletion derivatives in the mutS gene, which retain the P-loop coding region for ATP binding. The mutant proteins were assayed for ATP hydrolysis, heteroduplex DNA binding, heterodimer MutS formation, and the ability to interact with MutL. Dimerization was assayed by expressing His6-tagged wild-type and non-tagged deletion mutant proteins in the same cell and isolating the His6-tagged protein followed by MutS immunoblotting after SDS-polyacrylamide gel electrophoresis. MutS-MutL interaction was measured using the same technique except that the MutL protein carried the His6 tag. Our results indicate that DNA binding ability resides in the N-terminal end of MutS, and dimerization and MutL interactions are located in the C-terminal end. Given the extensive amino acid homology in the MutS family our results with E. coli should be applicable to MutS homologues in other prokaryotes and eukaryotes.     

Second-site suppression of RNase E essentiality by mutation of the deaD RNA helicase in Escherichia coli

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.     

Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli.

We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218. The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser. A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly. Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly. In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV. DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3'. Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC. The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid. This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome. We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.     

Base pair substitutions caused by the uvr502 mutation affecting mutation rates and UV-sensitivity of Escherichia coli

Summary The types of base pair substitutions induced by the uvr502 mutator activity were studied using the isogenic uvr ⁺ and uvr502 strains bearing an ochre or missense mutations in the trp operon. It was found that the uvr502 mutation increased the frequency of both structural gene (true) reversions and suppressor mutations in the trp _oc ^– mutant. The trpA58 missense mutation was also reverted by the uvr502 allele and 5-methyl tryptophane resistant as well as 5-methyl tryptophane sensitive Trp⁺ revertants were formed. However the uvr502 mutation was unable to increase significantly the frequency of Trp⁺ revertants in the rpA78 mutant. With the help of key of Yanofsky et al. (1966b) and codon catalogue it could be concluded that the uvr502 mutation induces transitions in both directions but not A:TC:G and probably not G:CT:A transversions. Incubation of the uvr502 mutant with either of four deoxyribonucleosides has no effect on its spontaneous mutability while deoxyguanosine and deoxyadenosine reduce the mutagenic effect of 2-aminopurine in the uvr ⁺ strain, suggesting that the mutator effect of the uvr502 mutation has nothing to do with the formation of mutagenic base analogue or insufficient synthesis of bases.     

Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli.

A chloramphenicol resistance gene was cloned into a plasmid-borne dut gene, producing an insertion mutation that was then transferred to the chromosome by allelic exchange. The mutation could not be acquired by haploid strains through substitutive recombination, even when two flanking markers were simultaneously transduced. The insertion was easily transferred, via generalized transduction, into the chromosomal dut region of strains harboring a lambda dut + transducing phage; however, the resulting dut mutant/lambda dut + merodiploid could not then be cured of the prophage. This apparent lethality of the mutation could not be explained by effects on adjacent genes; the dfp gene retained complementing activity, and a ttk insertion mutant was viable. The dut gene product, deoxyuridine triphosphatase, is known to reduce incorporation of uracil into DNA and to be required in the de novo synthesis of thymidylate. Therefore, an attempt was made to determine whether the dut insertion would be tolerated in strains carrying the following compensatory mutations: dcd (dCTP deaminase) and cdd (deoxycytidine deaminase), which should reduce dUTP formation; ung (uracil-DNA glycosylase), which should reduce fatally excessive excision repair; deoA (thymidine phosphorylase), which should enhance the utilization of exogenous thymidine; and sulA, which should reduce the lethal side effects of SOS regulon induction. These mutations, either alone or in various combinations, did not permit the survival of a haploid dut insertion mutant, suggesting that the dut gene product might have an essential function apart from its deoxyuridine triphosphatase activity.