Establishment of Tn5096-based transposon mutagenesis in Gordonia polyisoprenivorans

Abstract

The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 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 Tn5096-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 Tn5096 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 alpha-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.

FIG. 1.

Molecular organization of transposable plasmid pMA5096 comprising Tn5096. Unique restriction sites and relevant structural genes: Ap^r, ampicillin resistance gene; Apra^r, apramycin resistance gene; ORFA, unknown function; ORFB, transposase encoded by Tn5095; ColE1 ori, plasmid origin of replication for E. coli used in the absence of helper phage; f1(−) ori, f1 filamentous phage origin of replication. Arrows indicate the 5′ to 3′ direction of the coding regions.

FIG. 2.

Proposed β-oxidation pathway for the degradation of intermediates resulting from enzymatic cleavage of poly(cis-1,4-isoprene), such as 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al. One molecule of acetyl-CoA and one molecule of propionyl-CoA are released alternately during β-oxidation. Mcr is supposed to catalyze the conversion of the (R) isomer of a hypothetical 2-methyl-branched intermediate (marked with a frame) to the (S) isomer to enable desaturation during β-oxidation (modified according to Sakai et al. [43]).