Related Experiment Videos
New derivatives of TOL plasmid pWW0
1Institute of Molecular and Cell Biology, University of Tartu, Republic of Estonia.
Abstract:
Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWW0mut1, isolated from strain PPW1-1. Additional deletions were observed in pWW0mut1 after 'benzoate-curing' (plasmids pWW0mut15, pWW0mut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.
Insights
Two Pseudomonas putida strains, PPW1-1 and PPW161-1, were studied for TOL plasmid gene deletions. Strain PPW161-1 maintained TOL substrate utilization despite gene loss, suggesting chromosomal integration of catabolic genes.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas putida utilizes TOL plasmids for aromatic hydrocarbon degradation.
- TOL plasmids contain operons encoding enzymes for xylene and toluene metabolism.
- Genetic instability and rearrangements in TOL plasmids can affect bacterial catabolic capabilities.
Purpose of the Study:
- To characterize new segregants of Pseudomonas putida with altered TOL plasmids.
- To investigate the genetic basis for changes in substrate utilization in these strains.
- To understand the implications of plasmid gene deletions and potential chromosomal integration.
Main Methods:
- Isolation and characterization of Pseudomonas putida segregants (PPW1-1, PPW161-1).
- Plasmid DNA analysis to identify deletions in transposon Tn4651 and TOL operons.
- Growth studies on various aromatic substrates (m-xylene, m-toluate, benzoate).
- Phenotypic analysis of TOL+ (TOL plasmid) substrate utilization.
Main Results:
- Strain PPW1-1 showed a deletion in the TOL plasmid's upper-operon genes, losing m-xylene growth ability but retaining m-toluate utilization.
- Strain PPW161-1 exhibited deletions in both upper- and meta-operon genes of its TOL plasmid.
- PPW161-1 cells retained the ability to grow on all TOL plasmid substrates, indicating a stable Tol+ phenotype.
- The catabolic genes deleted from the plasmid in PPW161-1 were proposed to have integrated into the bacterial chromosome.
Conclusions:
- Plasmid gene deletions in Pseudomonas putida can lead to altered substrate utilization patterns.
- Chromosomal integration of TOL catabolic genes can compensate for plasmid-borne gene loss.
- This integration mechanism may explain the stable Tol+ phenotype observed in strain PPW161-1 despite plasmid deletions.