A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.

3139Two methylotrophic bacteria having similar cell morphologies have been isolated from cultures of 'Methylobacterium ethanolicum' grown on methane. One of these, strain POC, is an obligate methanotroph containing the serine pathway and Type I1 intracytoplasmic membranes, which appears to be a 'Methylocystis' species. The second organism, strain H4-14, can fix N, and grows on a variety of substrates, including methanol, formate, ethanol, succinate, fructose and H2 + CO,; during growth on methanol, carbon is assimilated via the Calvin-Benson cycle.Strain H4-14 appears to be a Xanthobacter species. Mixtures of the two organisms formed stable associations during growth on methane, consisting of approximately 5-20% H4-14 and 80-95 % POC. Strain POC excreted biotin, which was required by strain H4-14. Our results suggest that 'Methylobacterium ethanolicum' cultures grown on methane consist of a syntrophic association of two methylotrophs.