Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth

Abstract

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C(1) transfers, the tetrahydrofolate (H(4)F) pathway and the tetrahydromethanopterin (H(4)MPT) pathway, and both are required for growth on C(1) compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H(4)F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C(1) compounds. Unlike mutants defective for the H(4)MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [(14)C]methanol to [(14)C]CO(2). Collectively, these data confirm our previous presumptions that the H(4)F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H(4)F pathway in this organism in which it functions to convert formate to methylene H(4)F for assimilatory metabolism.

FIG. 1.

SDS-PAGE analysis of purified FtfL from M. extorquens AM1. Proteins were separated in a 14% polyacrylamide gel and stained with Coomassie brilliant blue R250. Lane A, molecular mass standards; lane B, 2.2 μg of purified FtfL.

FIG. 2.

Growth of wild-type M. extorquens AM1 (filled symbols) and the ftfL mutant CM216K.1 (open symbols) pregrown in succinate, harvested, and resuspended in media containing succinate (squares), succinate with methanol added to 125 mM at 2 h (triangles), or methanol (diamonds).

FIG. 3.

Whole-cell production of [¹⁴C]CO₂ from [¹⁴C]methanol. Strains examined are wild type (filled squares), the dmrA mutant CM212K.1 (filled diamonds), the ftfL mutant CM216K.1 (open squares), the ftfL dmrA double mutant CM216-212K.1 (open diamonds), and the mxaF mutant CM194K.1 (filled triangles).