A Novel Operon Encoding Formaldehyde Fixation: the Ribulose Monophosphate Pathway in the Gram-Positive Facultative Methylotrophic Bacterium Mycobacterium gastri MB19

Abstract

A 4.2-kb PstI fragment harboring the gene cluster of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation was identified in the chromosome of a gram-positive, facultative methylotroph, Mycobacterium gastri MB19, by using the coding region of 3-hexulose-6-phosphate synthase (HPS) as the hybridization probe. The PstI fragment contained three complete open reading frames (ORFs) which encoded from the 5 end, a DNA-binding regulatory protein (rmpR), 6-phospho-3-hexuloisomerase (PHI; rmpB), and HPS (rmpA). Sequence analysis suggested that rmpA and rmpB constitute an operon, and Northern blot analysis of RNA extracted from bacteria grown under various conditions suggested that the expression of the two genes is similarly regulated at the transcriptional level. A similarity search revealed that the proteins encoded by rmpA and rmpB in M. gastri MB19 show high similarity to the unidentified proteins of nonmethylotrophic prokaryotes, including bacteria and anaerobic archaea. The clusters in the phylogenetic tree of the HPS protein of M. gastri MB19 and those in the phylogenetic tree of the PHI protein were nearly identical, which implies that these two formaldehyde-fixing genes evolved as a pair. These findings give new insight into the acquisition of the formaldehyde fixation pathway during the evolution of diverse microorganisms.Many C 1 compounds, including formaldehyde, methane, and formic acid, were present in the interstellar dust clouds of the primitive earth and were important precursors of more complex organic compounds. Among them, formaldehyde is the key intermediate for biological fixation of C 1 compounds.The ribulose monophosphate (RuMP) pathway of formaldehyde fixation exists in a wide range of methylotrophic bacteria that can grow on C 1 compounds (14). The RuMP pathway can be divided into three phases. The first, the fixation phase, involves two reactions that are unique to the RuMP pathway: the condensation of formaldehyde with ribulose 5-phosphate and the subsequent isomerization of the product, D-arabino-3-hexulose 6-phosphate, to yield fructose 6-phosphate. These reactions are catalyzed by 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The second phase involves cleavage of the hexose phosphate to form two triose phosphates; glyceraldehyde 3-phosphate (GAP) enters the third phase of formaldehyde fixation, which consists of rearrangement reactions, while dihydroxyacetone phosphate (DHAP) enters the central pathway for synthesis of cell constituents. The third, the rearrangement phase, contains the reactions that are necessary to regenerate the acceptor of formaldehyde fixation, ribulose 5-phosphate.The lack of genetic studies on the RuMP pathway had prevented further insights into the physiological and evolutionary aspects of this pathway. We recently reported the gene cluster of the RuMP pathway of a methylotrophic bacterium, Methylomonas aminofaciens 77a (16). The rmpA and rmpB genes which encode HPS and PHI, respectively, are adjacent to rmpI...

The Physiological Role of the Ribulose Monophosphate Pathway in Bacteria and Archaea

“…3A). [3][4][5][6] Although the primary amino acid sequence of HPS and PHI showed a high degree of similarity among these organisms, the gene organization and regulatory mechanism were quite different for the obligate methylotrophs. In the obligate methylotroph, Methylomonas aminofaciens 77a, a transposase (IS10-R), located between hps and phi, is involved in the regulation of the adjacent gene expression.…”

“…5,6) Induction by a key intermediate of methylotrophic metabolism is indispensable for facultative methylotrophs that grow on diverse carbon sources including C 1 compounds. In Mycobacterium gastri MB19, a putative GntR-family transcriptional regulator, rmpR, is located upstream of the operon (Fig.…”

“…6) Initially the RuMP [10][11][12][13][14][15][16][17][18][19][20][21]2006 Review pathway was thought to be confined to methylotrophs, 7) but recent data show that the formaldehyde-fixing reaction is more widely distributed. 5,8) As well as methylotrophic bacteria, genes homologous to HPS and PHI have been found in a variety of non-methylotrophic organisms, including bacteria and archaea.…”

“…The genes encoding these two enzymes were first cloned from an obligate methylotroph, Methylomonas aminofaciens 77a. 3,4) These genes have also been cloned from a facultative methylotroph, Mycobacterium gastri MB19, 5) and a thermotolerant methylotroph, Bacillus brevis S1. 6) Initially the RuMP [10][11][12][13][14][15][16][17][18][19][20][21]2006 Review pathway was thought to be confined to methylotrophs, 7) but recent data show that the formaldehyde-fixing reaction is more widely distributed.…”

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…1A) (7,13), D-arabino-3-hexulose 6-phosphate (6HP) is first produced by a condensation reaction between formaldehyde and ribulose 5-phosphate (Ru5P) by 3-hexulose-6-phosphate synthase (Hps) (6,13,14). This is followed by the isomerization of heluxose 6-phosphate (H6P) by 6-phospho-3-hexuloseisomerase (Phi) (6,13,14) into fructose 6-phosphate (F6P), which can either enter central metabolism Significance Methane, the major natural-gas component, can be converted industrially to syngas (a mixture of CO 2 , CO, and H 2 ), which is used to produce methanol, an important industrial precursor for producing commodity and specialty chemicals. Unlike methanol's chemical conversion, which requires high temperature and pressure, its biological conversion, proceeding through its oxidation to formaldehyde, takes place at ambient conditions but suffers from relative low rates.…”