Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12.

Summary A ColE1 hybrid plasmid, pNU1, carrying the amp operon coding for chromsomal -lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning.By reciprocal recombination between pNU1 and chromosome of two unstable -lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the -lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.     

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.     

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).     

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.     

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.     

Selection of Escherichia coli K-12 chromosomal mutants that prevent expression of F-plasmid functions.

Chromosomal mutants of Escherichia coli deficient in the expression of F-plasmid functions were selected by mutagenizing F- cells, introducing an F' plasmid into the mutagenized cells by conjugation, and identifying transconjugants resistant to the donor-specific bacteriophage Q beta by a simple spray test. All but 1 of 25 mutants were defective in an extracellular stage of Q beta infection, suggesting that they fail to elaborate F-pili. At least six of these were also deficient as deoxyribonucleic acid donors. More than half of the mutants appear to be altered in peviously undetected chromosomal genes required for the expression of F-related cellular functions.     

Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12.

Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.     

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.     

Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase.

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Actinomycin sensitive mutants of Escherichia coli K-12

Summary Actinomycin sensitive mutants of E. coli K12 have been isolated and shown to have pleiotropic defects in the fermentation of sugars. The locus of a gene controlling actinomycin resistance is very close to that of the lactose gene.     

Recipient ability of bacteriophage-resistant mutants of Escherichia coli K-12.

The ability of a wide range of bacteriophage-resistant mutants to act as recipients in conjugation with F'lac pro and R100-1 donors has been studied. A number of mutant types defective in recipient ability with F'lac pro, as well as mutants which were hyperrecptive with R100-1, have been detected.     

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis.

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E. coli which are conditionally defective in AdoMet synthesis. Temperature-sensitive mutants in metK, the structural gene for the S-adenosylmethionine synthetase (AdoMet synthetase) expressed in minimal medium, were constructed by in vitro mutagenesis of a plasmid-borne copy of metK. By homologous recombination, the chromosomal copy was replaced with the mutated metK gene. Both heat- and cold-sensitive mutants were examined. At the nonpermissive temperature, two such mutants had 200-fold-reduced intracellular AdoMet levels and required either methionine or vitamin B12 for growth. In the presence of methionine or vitamin B12, the mutants grew at normal rates even though the AdoMet levels remained 0.5% of wild type. A third mutant when placed at nonpermissive temperature had less than 0.2% of the normal AdoMet level and did not grow on minimal medium even in the presence of methionine or vitamin B12. All of these mutants grew normally on yeast-extract-based medium in which an alternate form of S-adenosylmethionine synthetase was expressed.