Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli.

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.     

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.     

Isolation of zidovudine resistant Escherichia coli from AIDS patients

Zidovudine-resistant Escherichia coli were isolated from faecal samples from 6 out of 11 AIDS patients receiving zidovudine. Resistance appeared to be due to the loss of thymidine kinase activity which is required for the phosphorylation of zidovudine to its active form. No zidovudine resistant enterobacteria were isolated from 30 control faecal samples. Hence, clinically, there appeared to be a high correlation between the development of zidovudine-resistance in E. coli and exposure to zidovudine (chi 2: 11.77, P less than 0.001). However the development of zidovudine resistance does not appear to be associated with cross-resistance to other antimicrobial agents as the zidovudine-resistant E. coli did not display a high degree of resistance to other antibacterials.     

Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi.

Four mutants of Escherichia coli that are resistant to the flagellotropic phage chi, but are motile, were isolated. When they were observed in liquid culture bylight microscopy, one mutant exhibited circular movement and another tumbled at high frequency on the surface of a glass slide. The remaining two mutants moved normally. None of these mutants adsorbed the wild-type strain of chi. P1 transduction revealed that the mutation sites of these four mutants were more than 97% contransducible with a site in hag, the structural gene for flagellin. When flagellins of these mutants were chromatographed on a diethylaminoethyl-cellulose column, two eluted slower and one eluted slightly faster than the flagellin of the parental strain. The other flagellin eluted at the same position as that of the parent. Host range mutants of phage chi, which could infect these bacterial mutants, were isolated.     

Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli.

We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.     

Isolation of a new division altered mutant of Escherichia coli 15T

A Mutant of $\text{Escherichia coli}$ 15T^? (555-7) has been isolated which grows at a rate equal to that of the wild type at division times of 40 min or less, but grows faster than normal at division times greater than 40 min. At division times greater than 40 min the division time of the mutant is identical to the chromosome synthesis time of the wild type in the same medium. In one slow-growth medium (M9-aspartic acid) chromosome synthesis and gap times of the mutant were measured and the time required for synthesis of a chromosome was approximately the same as that of the wild type, but the gap in DNA synthesis observed in the mutant was only about 12% of that observed in wild type.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.     

Isolation and characterization of an Escherichia coli mutant tolerant to colicins Ia and Ib

Two classes of spontaneous colicin I insensitive mutants of Escherichia coli have been isolated. The first class (called cir) has lost its ability to adsorb either colicin Ia or Ib, maps at 41 min on the E. coli genetic map, and retains sensitivity to all other colicins tested. The cir phenotype is probably due to an alteration in the colicin I receptor. The second class of mutant (called tolI) retains full capacity to adsorb [(125)I]colicin I and, therefore, represents the isolation of a mutant tolerant to colicin I. The tolI mutant is sensitive to all other colicins tested and has a map location of 89-1 min. The tolI mutant grows with a reduced mass yield when glucose is used as a carbon source and cannot utilize succinate or acetate for growth. The tolI mutant shows a reduced sensitivity to sodium azide and phenethylalcohol. It is suggested that tolI is deficient in some aspect of aerobic metabolism which must be operative for colicin I sensitivity.