Threonine analogue resistant mutants of Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 resistant to a threonine analogue (-amino--hydroxy valeric acid) were predominantly resistant to ethionine and overproduced both threonine and methionine (2 mg/ml each). Novelty of the mutants is discussed.     

Mapping of ilvO loci of Escherichia coli K-12 with bacteriophage lambda dilv.

A set of lambda dilv phage have been used in a deletion mapping procedure to determine the location of two previously characterized ilvO alleles. In contrast to earlier conclusions derived from three-factor crosses and episome-shortening techniques with phage P1, the order found is ilvG-ilvO-ilvEDA. A three-factor cross with phage P1 is described that is not consistent with this location for an ilvO allele. Further analysis of this particular three-factor cross revealed than an artifact attributable to a mutual syntrophism had skewed the apparent frequency of inheritance of the ilvO locus. The role of mutual syntrophism is discussed as a source of mapping errors for the ilvO locus. The value of this set of lambda dilv phage and this mapping procedure for obtaining comparatively unambiguous data on the locations of the ilv structural and regulatory genes is demonstrated.     

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.     

Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.     

New maltose Blu mutations in Escherichia coli K-12.

Mutations in the genes pgi, pfkA, and ptsG resulted in a maltose Blu phenotype in Escherichia coli K-12, bringing the number of known Blu alleles to six. The Blu phenotype, as visualized by staining with iodine vapor, is a convenient mutant isolation technique.     

Suppression by thymidine-requiring mutants of Escherichia coli K-12.

Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations. Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants. Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Assembly-defective OmpC mutants of Escherichia coli K-12.

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.     

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.     

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.     

Recombination of bacteriophage lambda in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.     

Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.