Novel Alterations in Plasmid DNA Associated with Aromatic Hydrocarbon Utilization by Pseudomonas putida R5-3

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Pseudomonas putida R5-3 exhibits dynamic plasmid DNA content.
• Aromatic hydrocarbon utilization influences bacterial genetic material.
• Plasmid dynamics are crucial for microbial adaptation to environmental substrates.

Purpose of the Study:

• To investigate how Pseudomonas putida R5-3 alters its plasmid DNA in response to different aromatic hydrocarbon carbon sources.
• To characterize the changes in plasmid size and content under varying growth conditions.
• To determine the fate of plasmid DNA during adaptation to toluene and m-xylene.

Main Methods:

• Culturing Pseudomonas putida R5-3 on minimal media with specific aromatic hydrocarbons (p-methylbenzoate, m-xylene, toluene).
• Plasmid DNA isolation and analysis using restriction enzyme digestion.
• Southern blot hybridizations to compare DNA fragments and identify homologies.
• Analysis of chromosomal DNA integration.

Main Results:

• Growth on p-methylbenzoate yielded strains with 115-kb and 95-kb plasmids.
• Transfer to m-xylene or toluene resulted in the loss of the 95-kb plasmid and appearance of 50-kb or 60-kb plasmids.
• Reversion to p-methylbenzoate restored the original plasmid profile.
• Restriction analysis revealed deletions and rearrangements in derivatives grown on m-xylene/toluene.
• Southern blots showed partial integration of lost plasmid DNA into the chromosome of xylene-grown cells.
• The 95-kb plasmid showed homology to meta-fission pathway genes of the TOL plasmid pWWO.

Conclusions:

• Pseudomonas putida R5-3 demonstrates significant genomic plasticity in response to aromatic hydrocarbon utilization.
• Plasmid DNA content and structure are actively modulated, including loss, gain, and chromosomal integration.
• These alterations are linked to the metabolism of specific aromatic compounds, suggesting adaptive genetic mechanisms.