Genetics of the serine cycle in Methylobacterium extorquens AM1: cloning, sequence, mutation, and physiological effect of glyA, the gene for serine hydroxymethyltransferase

Abstract

The gene (glyA) ofMethylobacterium extorquens AM1 encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the serine cycle for C1 assimilation, was isolated by using a synthetic oligonucleotide with a sequence based on amino acid sequence conserved in SHMTs from different sources. The amino acid sequence deduced from the gene revealed high similarity to those of known SHMTs. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced an SHMT null mutant. Surprisingly, this mutant had lost its ability to grow on C1 as well as on C2 compounds but was still able to grow on succinate. The DNA fragment containing glyA was shown not to be linked with fragments carrying serine cycle genes identified earlier, making it the fourth chromosomal region of M. extorquens AMI to be indicated as being involved in C1 assimilation.Methylobacterium extorquens AM1 is a pink-pigmented serine cycle methylotroph able to grow on methanol and methylamine as well as on a variety of multicarbon substrates (26,34). This organism has been successfully used as a model for genetic studies of methanol and methylamine oxidation (14,17,18). Recently, progress has also been achieved in studying the genetics of the serine cycle in this strain, with three regions coding for serine cycle enzymes having been identified. One of these regions has been shown to encode genes for serine glyoxylate aminotransferase (sgaA), hydroxypyruvate reductase (hprA), methylenetetrahydrofolate dehydrogenase (mtdA4 [7]), malate thiokinase (mtkA and mtkB [8]), phosphoenolpyruvate carboxylase (ppcA [2]), and malyl coenzyme (CoA)-lyase (mcA [12]). Mutations in all of these genes have been obtained to confirm that they are required for the operation of the serine cycle (2, 6-8). Two other regions of the M. extorquens AM1 chromosome that contain serine cycle genes have been less extensively studied. One of these complements glycerate kinase mutants, and another complements mutants with lesion(s) in the unknown acetyl-CoA oxidation pathway portion of the serine cycle (33). Neither of these fragments overlaps the region containing sgaA, hprA, mtdA, mtkAB, ppcA, and mcL4 (7). The gene (glyA) for another serine cycle enzyme, serine hydroxymethyltransferase (SHMT), has been recently cloned and sequenced from an obligate methylotroph, Hyphomicrobium methylovorum GM2 (24); however, its location relative to other methylotrophy genes in that bacterium is unknown.We were interested in cloning glyA from M. extorquens AM1 in order to clarify its role in this organism. It has been suggested that during growth of M. extorquens AM1 on succinate and other multicarbon compounds, the serine necessary for cell biosynthesis is produced by the phosphorylated pathway (15). During growth on C1 compounds, the phosphorylated pathway plays a minor role, since the serine cycle is the major source of serine under these growth conditions (Fig. 1) (1, 13) 395-2940. glycine from serine...

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

“…It has been reported previously that mutants lacking serine hydroxymethyltransferase (glyA) and phosphoserine phosphatase (serB) activities both had defects in growth on C 2 compounds (10,28,29). The phenotypes of these mutants suggest that interconversions of glycine and serine play a role in C 2 metabolism.…”

Methylotrophy in Methylobacterium extorquens AM1 from a Genomic Point of View

“…Later, mutants with defects in methanol oxidation were isolated via a specific allyl alcohol selection (32,33). The availability of these C 1 -negative mutants allowed identification and isolation of the corresponding DNA regions encoding key methylotrophy genes, and it became evident that some of these genes are clustered together (2,4,5,6,8,9,11). As sequencing became a routine technique, these clusters were analyzed and expanded via chromosomal walking (5,12,15,27).…”

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

“…Another gene, gbA, is located on a separate chromosomal fragment and encodes serine hydroxymethyltransferase. This enzyme apparently plays a dual role in M. extorpens AM1, firstly in the serine cycle and secondly in the oxidation of acetylCoA (Chistoserdova & Lidstrom, 1994b). The third chromosomal fragment not linked to the two fragments mentioned above has been shown to complement a number of chemically induced mutants such as PCT48 that are unable to grow on C, and C, compounds and are complemented by the addition of glycolate or glyoxylate (Stone & Goodwin, 1989;Smith & Goodwin, 1992;Smith et a/, 1995).…”

“…decades, this organism has been used as a model for studying both the biochemistry and the genetics of the serine cycle utilized for assimilation of 6, compounds at the level of formaldehyde (Salem & Quayle, 1971;Dunstan e t al., 1972a, b;Dunstan & Anthony, 1973;Anthony, 1982 ;Chistoserdova & Lidstrom, 1992, 1994a. In the serine cycle, formaldehyde in the form of methylenetetrahydrofolate condenses with glycine to produce serine, which then undergoes transamination with glyoxylate, producing hydroxypyruvate, which in turn is reduced to D-glycerate.…”