Genetic manipulation of the restricted facultative methylotroph Hyphomicrobium X by the R-plasmid-mediated introduction of the Escherichia coli pdh genes

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Abstract
The inability of Hyphomicrobium X to grow on compounds such as pyruvate and succinate is most likely due to the absence of a functional pyruvate dehydrogenase (PDH) complex. Further support for this was sought by studying the effect of the introduction of the Escherichia coli pdh genes in Hyphomicrobium X on the pattern of substrate utilization by the latter organism. These genes were cloned by in vivo techniques using the broad-host range conjugative plasmid RP4: :Mucts. Plasmid RP4 derivatives containing pdh genes were selected by their ability to complement a pyruvate dehydrogenase deletion mutant of E. coli, strain JRG746 recA (ace-1pd) Δ18. The plasmids thus obtained could be transferred through an intermediary host (C600 recA), selecting only for an antibiotic resistance coded for by RP4 and back into JRG746 or other E. coli pdh mutants, upon which they still conferred the wild type phenotype. Enzyme assays showed that the latter strains, when carrying plasmid RP4′ pdh1 also possessed PDH complex activity. Conjugation between the auxotrophic E. coli JRG746 (RP4′ pdh1) strain and Hyphomicrobium X on pyruvate minimal agar gave rise to progeny which, on the basis of its morphology (stalked bacteria), their ability to grow on C1-compounds and to denitrify (now also with pyruvate) were identified as hyphomicrobia. This Hyphomicrobium X transconjugant was also able to grow in minimal medium with succinate, but no other novel growth substrates have been identified so far. An analysis of protein extracts with 2-dimensional gel electrophoresis indicated that Hyphomicrobium X and JRG746 only synthesized all three components of the PDH complex when carrying plasmid RP4′ pdh1. These results are compatible with the suggested significance of the lack of a functional PDH complex in wild type Hyphomicrobium X.