Establishment of Tn 5096 -Based Transposon Mutagenesis in Gordonia polyisoprenivorans

Abstract

ABSTRACT The transposons Tn 5 , Tn 10 , Tn 611 , and Tn 5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn 5 or Tn 10 . The thermosensitive plasmid pCG79 harboring Tn 611 integrated into the chromosome of G. polyisoprenivorans ; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn 5096 -mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn 5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly( cis -1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly( cis -1,4-isoprene). One rubber-negative mutant was disrupted in mcr , encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly( cis -1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.