A new mutation in Escherichia coli, designated gevAl, that results in noninducible expression of both gcv and a gcvT-kacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv.In enteric bacteria, the conversion of serine to glycine and 5,10-methylenetetrahydrofolate occurs through the action of the enzyme serine hydroxymethyltransferase, the glyA gene product (10). This reaction is an important contributor of one-carbon units in cell metabolism. The oxidative cleavage of glycine by the glycine cleavage (GCV) enzyme system provides a second pathway for one-carbon biosynthesis (14). A glycine-inducible GCV enzyme system has been demonstrated in both Eschenchia coli (8,11,12) and Salmonella typhimurium (16).E. coli mutants blocked simultaneously in the GCV enzyme system and in the serine biosynthetic pathway are unable to use glycine as a serine source and require an exogenous source of serine (the GCV-phenotype). At present, six classes of mutations have been shown to result in the GCV-phenotype under the appropriate growth conditions. The first class maps at min 62.6 on the E. coli chromosome and presumably affects the gcv structural genes (12). The second class maps at min 14.8 and disrupts the lipoic acid biosynthetic pathway (18). The third class maps at min 2.7 and alters the lpd gene, encoding the L protein of the GCV enzyme complex (17). The fourth class maps at min 54.8 and partially inactivates serine hydroxymethyltransferase, the glyA gene product (13). The fifth class maps at min 95.6 and disrupts the cycA gene involved in glycine transport (2, 4); this class results in the GCV-phenotype because of altered glycine uptake (4a). The sixth class maps at min 20 and inactivates the lIp gene (7). We report here a seventh locus that results in a GCV-phenotype because of the cell's inability to induce gcv expression.Isolation of a new class ofgcv mutations. Using a penicillin counterselection previously described (1, 11, 12), we isolated several mutants defective in the GCV enzyme pathway. One of the mutants isolated that displayed the GCVphenotype and that had a very low level of GCV enzyme activity did not map with the known structural genes encoding GCV. This strain was designated GS786 (the complete genotypes of all strains used in this study are listed in Table 1). A [2-14C]glycine uptake assay was performed to determine whether transport was altered in strain GS786. This strain was found to have normal transport of glycine (data not shown). Mutations in lipoic acid biosynthesis also result in a GCV-phenotype in serine auxotrophs, and growth of these mutants on glucose minimal medium (GM) containing glycine can be restored if lipoic acid is added to the medium (18). However, the addition of exogenous lipoic acid did not restore the GCV+ phenotype in strain GS786. These resul...

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.

The nucleotide sequence of an Escherichia coli gene which presumably encodes the H-protein of the glycine cleavage (GCV) enzyme complex is presented. The gene, designated gcvH, encodes a polypeptide of 128 amino acids with a calculated molecular weight of 13,665 daltons. The translation start site was determined by N-terminal amino acid sequence analysis of a gcvH-lacZ encoded fusion protein. The E. coli H-protein shows extensive homology with the H-proteins from the pea (Pisum sativum) and the chicken liver GCV enzyme complexes. 85 of 128 amino acid residues are identical or chemically similar between the E. coli and the pea H-proteins, and 74 of 128 amino acid residues are identical or chemically similar between the E. coli and the chicken liver H-proteins. All three proteins have identical amino acid sequences from residues 61-65. This sequence contains the lysyl residue involved in lipoic acid attachment in the chicken liver H-protein.