New derivatives of TOL plasmid pWW0

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.