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  • Title: Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex.
    Author: Fillingame RH, Knoebel K, Wopat AE.
    Journal: J Bacteriol; 1978 Nov; 136(2):570-81. PubMed ID: 152309.
    Abstract:
    A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.
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