Analysis of two formaldehyde oxidation pathways in Methylobacillus flagellatus KT, a ribulose monophosphate cycle methylotroph The GenBank accession numbers for the sequences of 2502 nt containing gndA and part of zwf, and of 2685 nt containing mch, are AF167580 and AF139592, respectively.

Abstract

The roles of cyclic formaldehyde oxidation via 6-phosphogluconate dehydrogenase and linear oxidation via the tetrahydromethanopterin (H 4 MPT)-linked pathway were assessed in an obligate methylotroph, Methylobacillus flagellatus KT, by cloning, sequencing and mutating two chromosomal regions containing genes encoding enzymes specifically involved in these pathways : 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase and methenyl H 4 MPT cyclohydrolase (gndA, zwf and mch). No null mutants were obtained in gndA or zwf, implying that the cyclic oxidation of formaldehyde is required for C 1 metabolism in this obligate methylotroph, probably as the main energy-generating pathway. In contrast, null mutants were generated in mch, indicating that the H 4 MPT-linked pathway is dispensable. These mutants showed enhanced sensitivity to formaldehyde, suggesting that this pathway plays a secondary physiological role in this methylotroph. This function is in contrast to Methylobacterium extorquens AM1, in which the H 4 MPT-linked pathway is essential.

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

“…However, the finding of an alternative system involving N-methylglutamate as an intermediate specifically expressed during growth on methylamine raised a question of whether this additional system contributed to growth on methylamine. The role of the linear pathway for oxidizing formaldehyde has been approached experimentally before (7,18). However, while high activities for the enzymes of the H 4 MPT pathway have been measured, mutants in these enzymes revealed only very subtle phenotypes, suggesting a minor role in formaldehyde oxidation.…”

“…Proteomics revealed the presence of both GndA (Mfla_0918/Mfla_1062) and GndB (Mfla_2599), the latter seeming to be upregulated in the methylamine-grown cells. We have previously failed to obtain GndA mutants (7). However, in light of the presence of GndB, along with the enzymes for the linear pathway, it appeared necessary to retest the relative contributions of these enzymes to formaldehyde oxidation.…”

“…However, this assumption has not been experimentally tested. The relative contributions of each of the redundant pathways encoded in the genome (for example, linear versus cyclic oxidation of formaldehyde) also remained unclear (7). These questions can be assessed through analysis of the expressed portion of the genome under specific growth conditions and especially pre-cisely through a comprehensive analysis of the protein content in the cell (proteome) (3).…”

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

“…In B. methanolicus strain C1, it has been shown that the specific glucose phosphate dehydrogenase activity of the cyclic dissimilatory pathway is three-to fourfold higher in cells growing on methanol than in cells growing on glucose (3). At least four different pathways for bacterial formaldehyde detoxification, including oxidation and fixation, have been described (9,19,20), and it has been shown that some bacteria possess several of these pathways. Interestingly, B. subtilis mutants with yckG or yckF (encoding HPS and PHI activities, respectively) disrupted display only a minor decrease in formaldehyde tolerance, suggesting that the bacterium has alternative biochemical routes for detoxification of formaldehyde (28).…”

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway ofGluconobacter oxydans

“…16) There have been only a few reports on the cloning of the genes encoding NAD W NADP dual specic 6PGDH. 7) It seemed that the 6PGDH in this study had higher activity with NAD than that with NADP (Table 1). Adachi et al reported that 6PGDH from Gluconobacter suboxydans IFO 12528 accepted NAD and NADP at almost the same rate.…”