Table 1.

Strains and plasmids used in this study

Strain or plasmidDescriptionReference or source
R. eutropha
 Ae H16Wild type, gentamicin resistant, also termed DSM 428 and ATCC 17699ATCC 17699
 H2262phaP::Tn5 strain derived from HF39, partial Tn5 approx. 0.1 kb upstream ofphaP ORF18
 H2271phaP::Tn5 strain derived from HF39, Tn5 insertion 26 bp upstream of phaPORF18
 H2275phaP::Tn5strain derived from HF39, Tn5 insertion 156 bp upstream ofphaP ORF18
 Re1052phaP precise deletion gene replacement strain, derived from Ae H16/pGY63This study
E. coli
 BL21(DE3)Strain used for pET expression system16
 S17-1Strain for conjugative transfer of plasmids into R. eutropha for gene replacement13
Plasmids
 pET5aT7 expression system plasmid, confers ampicillin resistancePromega
 pGY63phaP precise deletion fragment cloned into pJQ200mp18KmThis study
 pGY101phaP expression plasmid, corresponding to phaP ORF cloned into pET5aThis study
 pJQ200mp18Gene replacement vector, carries sacB, oriV, oriT, traJ, confers gentamicin resistance11
 pJQ200mp18KmDerivative of pJQ200mp18, gentamicin resistance gene disrupted, confers kanamycin resistanceThis study
 pUT-miniTn5-KmSource of kanamycin resistance gene for pJQ200mp18Km1