Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid

Abstract

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.

FIG. 1.

SDS-PAGE of a cell extract prepared from B. cepacia TM1 transformants grown on LB medium. (A) The bands on the SDS-polyacrylamide gel were stained with Coomassie brilliant blue R-250. (B) Immunoblot analysis with anti-HPS polyclonal antibodies. Lane 1, molecular mass standards; lane 2, purified HPS obtained from M. aminofaciens 77a; lane 3, B. cepacia TM1(pBBR122); lane 4, B. cepacia TM1(pBRM1). In panel A, the positions of molecular mass standards (in kilodaltons) are indicated to the left of the gel. The position of HPS is indicated by arrowheads to the right of the gels.

FIG. 2.

Growth profiles of B. cepacia TM1 transformants grown on LB medium containing formaldehyde at a final concentration of 1 mM (A) or 3 mM (B). Growth of B. cepacia TM1(pBRM1) (•) and TM1(pBBR122) (○) and formaldehyde concentration (triangles) in the culture medium of B. cepacia TM1(pBRM1) (▴) and TM1(pBBR122) (▵) are shown. OD₆₁₀, optical density at 610 nm.

FIG. 3.

Formaldehyde incorporation of B. cepacia TM1 transformants grown on LB medium containing [¹⁴C]formaldehyde. (A) X-ray film image for ¹⁴C activities of the extracted proteins (50 μg for each dot). B. cepacia TM1(pBBR122) (row 1) and TM1(pBRM1) (row 2) grown for various times were tested. (B) β-Radiation of the extracted protein (150 μg). Symbols: •, B. cepacia TM1(pBRM1); ○, B. cepacia TM1(pBBR122).

FIG. 4.

Growth profiles of B. cepacia TM1 transformants grown on mineral salt medium containing vanillic acid as the sole carbon source at a final concentration of 60 mM. Growth of B. cepacia TM1(pBRM1) (•) and TM1 [pBBR122] (○) and vanillic acid concentration (triangles) in the culture medium of B. cepacia TM1(pBRM1) (▴) and TM1(pBBR122) (▵) are shown. OD₆₁₀, optical density at 610 nm.

FIG. 5.

Proposed pathway of vanillic acid degradation linked with the RuMP formaldehyde fixation pathway in B. cepacia TM1. Formaldehyde derived from vanillic acid is incorporated into the RuMP pathway. Ribulose-5-phosphate, the acceptor of formaldehyde, may be regenerated from fructose-6-phosphate as in the RuMP pathway of methylotrophs. Enzyme 1, vanillin dehydrogenase; enzyme 2, vanillic acid demethylase.