Investigation of plasmid-induced growth defect in Pseudomonas putida.
Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant.
DECHEMA Research Institute, Frankfurt am Main, Germany.
Jia
J
Mi
DECHEMA Research Institute, Frankfurt am Main, Germany.
Anne
A
Sydow
DECHEMA Research Institute, Frankfurt am Main, Germany.
Florence
F
Schempp
DECHEMA Research Institute, Frankfurt am Main, Germany.
Daniela
D
Becher
DECHEMA Research Institute, Frankfurt am Main, Germany.
Hendrik
H
Schewe
DECHEMA Research Institute, Frankfurt am Main, Germany.
Jens
J
Schrader
DECHEMA Research Institute, Frankfurt am Main, Germany. Electronic address: buchhaupt@dechema.de.
Markus
M
Buchhaupt
D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*
Kan(R); Metabolic burden; Plasmid copy number; Plasmid induced growth defect; Pseudomonas putida; Rep; pBBR1 replicon