Hiroko Hama

Properties of a Na^+-coupled serine-threonine transport system in Escherichia coli

Based on the following experimental results we conclude that the serine-threonine transport system in Escherichia coli is a Na+-coupled cotransport system. (1) Addition of serine to cell suspensions induced H+ efflux in the presence of Na+. (2) Addition of serine to cell suspensions induced Na+ uptake by cells. (3) Imposition of an artificial electrochemical potential of Na+ in starved cells induced serine uptake. Some of these phenomena were observed when threonine was added instead of serine or inhibited when cells were preincubated with threonine. The Na+/serine (threonine) cotransport system was considerably repressed when cells were grown on a mixture of amino acids. Serine transport in cells grown in the absence of amino acids mixture was stimulated by Na+. The half maximum concentration of Na+ was 21 microM. Sodium ion increased the Vmax of serine transport without affecting the Km.

Target of serine inhibition in Escherichia coli.

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.

Deletion of seven amino acid residues from the γ subunit of Escherichia coli H+-ATPase causes total loss of F1 assembly on membranes

A mutant gene for the gamma subunit of H+-translocating ATPase was cloned from Escherichia coli mutant NR70 isolated by B. P. Rosen [J. Bacteriol. 116, 1124-1129 (1973)]. Determination of its nucleotide sequence revealed a deletion of 21 base pairs between nucleotide residues 64 and 84, resulting in a deletion of seven amino acid residues (LysAlaMetGluMetValAla) from the amino-terminal portion. This deletion resulted in the loss of a hydrophobic domain of the subunit estimated by an analysis of its hydropathic character. Since F1 subunits are reported not to be assembled on the normal F0 portion of NR70, it is concluded that the hydrophobic domain deleted in the mutant subunit is important for assembly of the F1 portion. Introduction of a plasmid pNR70 carrying the mutant allele of NR70 into a wild-type strain gave no recombinants resistant to neomycin. This result suggested that the neomycin-resistant phenotype is not directly related to the defect in the gamma subunit of NR70.