Biochemical studies of two Bacillus pumilus plasmids

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Bacillus pumilus strains possess covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecules, referred to as plasmids.
• The NRS 576 strain harbors approximately two copies of the 576 plasmid (MW 28 x 10^6), while strain ATCC 7065 contains at least 10 copies of the 7065 plasmid (MW 6 x 10^6).

Purpose of the Study:

• To investigate the characteristics of plasmids in Bacillus pumilus strains.
• To determine the relationship between plasmid presence and spontaneous mutations affecting spore formation.

Main Methods:

• Buoyant density and molecular weight determination of plasmid DNA.
• Analysis of plasmid copy number in different growth phases (exponential vs. stationary).
• Characterization of spontaneous mutants (W mutants) for the presence of CCC DNA and plasmid homologous sequences.

Main Results:

• Plasmid copy number in B. pumilus NRS 576 and ATCC 7065 remained relatively constant between exponential and stationary phases.
• Spontaneous W mutants of NRS 576, exhibiting enhanced sporulation, lacked detectable CCC DNA and most plasmid homologous sequences.
• An additional, higher molecular weight CCC DNA form was observed in stationary-phase cells of B. pumilus ATCC 7065.

Conclusions:

• The 576 plasmid is likely lost in spontaneous W mutants of B. pumilus NRS 576.
• Loss of the 576 plasmid correlates with an elevated frequency of spore formation in these mutants.
• Plasmids in B. pumilus may play a role in regulating sporulation or other cellular processes.