In vivo cloning of genes determining lithoautotrophy (Aut) on a plasmid from Alcaligenes hydrogenophilus

Abstract

Hydrogenase ( hox) genes on the megaplasmid pHG21-a from Alcaligenes hydrogenophilus, whose lithoautotrophic growth (Aut) is supported by H 2-oxidation (Hox) and CO 2-fixation (Cfx), were cloned in vivo using a broad host range IncP1 plasmid R68.45. The recombinant plasmid was detected by the characteristic that it was transferred at a frequency 10 6-fold higher than pHG21-a in intrastrain mating of the Hox − Cfx + bacterium Pseudomonas oxalaticus OX1. All of six recombinant plasmids designated pFUs inherited all three resistance markers of R68.45. Four plasmids (pFU3, pFU8, pFU11, and pFU15) with a molecular size of 69 Md had only membrane-bound hydrogenase ( hoxP) genes, and two plasmids (pFU7 and pFU9) of 85 Md had both hoxP and soluble hydrogenase ( hoxS) genes. The Hox − Cfx − bacteria P. oxalaticus OX4 and OX6 gained Aut − phenotype by the possession of pHG21-a, pFU7 or pFU15. These results showed that Hox plasmid pHG21-a was an Aut plasmid and pFU7 and pFU15 inherited this phenotype, pFU7 was maintained stably in P. oxalaticus OX1 and had all of the lithoautotrophic phenotypes of pHG21-a. pFU7, rather than pHG21-a, is useful for further studies on the transfer of the Aut phenotype to a broad range of bacteria.