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Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membranebounl Ca2+-and Mg2+-stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).

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Asp-51 and Asp-120 of the Escherichia coli melibiose carrier on plasmid pKKMB were separately replaced by amber codons and transformed into eight amber suppressor strains, producing eight amino acid substitutions for each site. Glu-51 and Glu-120 were the only replacements in the carrier that allowed the cells to ferment melibiose and that showed transport of melibiose against a concentration gradient. Revertants to Glu-51 and Glu-120 show less activity than the wild type. The Asp-51 position is more crucial for Na+-stimulated melibiose accumulation than is the Asp-120 site.The melibiose carrier (meiB) of Escherichia coli is an interesting and versatile example of a cation-substrate cotransport carrier. A remarkable feature of this carrier is the variety of cations that can be used for cotransport. The coupling cation may be the proton (24), Na+ (24), or Li' (23). The sugar substrates cover a wide range of a-galactosides, P-galactosides, and monosaccharides (26), and the configuration of the sugar appears to dictate the cation specificity (25).The melB gene has been cloned (6) and sequenced (28), thus enabling studies of the relationship between the deduced amino acid structure of the carrier and the transport mechanism to be made. Isolation and characterization of spontaneous mutants with altered transport properties is one approach to this problem. Interesting cation recognition mutants have been isolated previously (7,12,19,20); for example, the cell with the mutant carrier Pro-122 --Ser has become dependent on Li' or Na+ for growth on melibiose as

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