Positive regulation of the Escherichia coli glycine cleavage enzyme system

Abstract

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...

DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli

“…GcvA plays a dual role in the regulation of gcv, since it is also required for six-to sevenfold activation of gcv when glycine is included in the growth media (37). Whether GcvA responds directly to two different cellular metabolites or whether GcvA interacts with another protein is unknown but is under investigation.…”

Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT:: lacZ expression in Escherichia coli

“…Effects on gcvT: :/at2 expression of separating GcvAbinding sites 3 and 2 from the gcv control region by integral turns of DNA Both Lrp and GcvA are required for expression of the gcv operon Wilson et al, 1993b;Lin et al, 1992). One possible model for regulation of the operon is that one of these two proteins interacts with RNAP and the other is required either for co-operative binding of the two proteins to the DNA, or to induce an altered conformation of the DNA to allow for an appropriate DNA-protein interaction to occur.…”

“…The 1rp::TnlO allele is from strain CV1008 (Platko et al, 1990). The gcvAl (Wilson et al, 1993b) (Urbanowski & Stauffer, 1986). All the lysogens carry the cI857 mutation, resulting in a temperaturesensitive repressor, and were grown at 30 "C. After singlecolony purification, all the lysogens were verified to carry a single copy of the 2 phage by testing for their ability to support lytic infection by 2 cI90c17 (Shimada et al, 1972).…”

“…To confirm that the repressed levels of P-galactosidase observed in the GS852 + 15-21, and +25-21, lysogens were mediated through the GcvA protein, the control 9981gcuT: : lac2 lysogen and the mutant GS998 + 15-213 and +25-21, lysogens were each transformed with the single-copy gcuA+ plasmid pGS341 (Wilson et al, 1993b), the transformants were grown in G M or G M + glycine medium and the P-galactosidase levels were measured. The presence of pGS341 restored GcvAmediated activation in the 998AgcvT: : f a d control lysogen (Table 3, compare rows 1 and 2).…”

GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation

“…1) (Wilson et al, 1995). GcvA acts as an activator of gcv expression when glycine is present in the growth medium, causing a six-to sevenfold induction (Wilson et al, 1993b). GcvA also represses the gcv operon over a fivefold range when the purine inosine is added to the growth medium (Wilson et al, 1993a).…”