Evidence that a linear megaplasmid encodes enzymes of aliphatic alkene and epoxide metabolism and coenzyme M (2-mercaptoethanesulfonate) biosynthesis in Xanthobacter strain Py2

Abstract

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C(3) unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862-4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis.

FIG. 1

Induction of alkene monooxygenase and epoxide carboxylation activities in acetate-grown Xanthobacter strain Py2 by propylene or epoxypropane. (A) Propylene remaining; (B) epoxypropane remaining. Symbols: □, wild-type strain Py2; ■ wild-type strain Py2 with rifampin and chloramphenicol; ○, propylene-negative strain Py2.101.

FIG. 2

Identification of a linear DNA molecule in Xanthobacter strain Py2 by CHEFE. Lane 1, fractionated high-molecular-weight DNA in propylene-negative strain Py2.101; lane 2, fractionated high-molecular-weight DNA in wild-type strain Py2; lane 3, molecular size markers (lambda concatemer).

FIG. 3

Genetic map of alkene monooxygenase (xamo) and epoxide carboxylase (xec) genes of Xanthobacter strain Py2. Representative genes are drawn to scale. xamoA, xamoB, xamoE, genes encoding the alpha, gamma, and beta subunits, respectively, of the alkene monooxygenase epoxygenase component; xamoC, gene for the alkene monooxygenase ferredoxin component; xamoF, gene for the alkene monooxygenase reductase component; xecA, gene for epoxyalkane:CoM transferase; xecC, gene for NADPH:2-ketopropyl-CoM carboxylase/oxidoreductase; xecD, gene for 2-R-hydroxypropyl-CoM dehydrogenase; xecE, gene for 2-S-hydroxypropyl-CoM dehydrogenase. xecB, xecF, and xecG encode hypothetical proteins of unknown function.

FIG. 4

PCR amplification of xamoA (A), xecC (B), and cfxL (C). Lanes 1, total high-molecular-weight DNA from wild-type strain Py2; lanes 2, CHEFE-purified genomic DNA from wild-type strain Py2; lanes 3, CHEFE-purified linear megaplasmid pEK1; lanes 4, total high-molecular-weight DNA from propylene-negative strain Py2.101; lanes 5, CHEFE-purified genomic DNA from propylene-negative strain Py2.101. The positions of the amplified products, which were verified by total sequence analysis, are indicated by the arrows.

FIG. 5

Multiple sequence alignment with Xanthobacter strain Py2 (Xanthobacter) hypothetical protein XecG, M. thermoautotrophicum (strain Delta H) (M. thermoauto) hypothetical protein MTH1674, M. jannaschii hypothetical protein MJ0255, and B. subtilis hypothetical protein YitD. Residues in white typeface are identical in all four proteins, and residues highlighted in gray are 75% identical or similar in properties. GenBank accession numbers for the genes encoding the protein sequences of XecG, MTH1674, MJ0255, and YitD are AY024334, G69090, Q57703, and E69839, respectively.