Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Pseudomonas sp. strain AM1 is a bacterium capable of methanol utilization.
• Methanol metabolism in this strain is linked to malyl coenzyme A lyase activity.
• Mutants deficient in this activity cannot metabolize methanol.

Purpose of the Study:

• To genetically identify the gene responsible for malyl coenzyme A lyase activity in Pseudomonas sp. strain AM1.
• To complement a methanol-utilization mutant (PCT57) and isolate the functional gene.

Main Methods:

• Construction of a genomic library from Pseudomonas sp. strain AM1 DNA using the cosmid pVK100.
• Mobilization of the cosmid library into the methanol-deficient mutant PCT57 using the helper plasmid pRK2013.
• Complementation analysis and subcloning to identify the specific gene region.

Main Results:

• Complementation of the PCT57 mutant restored methanol-positive phenotype.
• Six independent isolates contained a recombinant plasmid with a 19.6-kilobase-pair DNA insert from Pseudomonas sp. strain AM1.
• The gene deficient in PCT57 (mcl-1) was localized to a 1.6-kilobase-pair region within a 7.4-kilobase-pair fragment.

Conclusions:

• The gene mcl-1, encoding malyl coenzyme A lyase, was identified in Pseudomonas sp. strain AM1.
• This gene is essential for the methanol utilization pathway in this bacterium.
• The genetic and molecular basis for methanol metabolism in Pseudomonas sp. strain AM1 has been elucidated.