Related Experiment Videos
Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus
Abstract:
A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.
Insights
A soil bacterium, Bacillus pumilus, can be induced to form spores by infection with a specific bacteriophage, PMB1. This phage-mediated sporulation is maintained as long as the phage is present in the bacterial cells.
Area of Science:
- Microbiology
- Bacteriology
- Virology
Background:
- Oligosporogenic Bacillus pumilus strain NRS 576 exhibits very low sporulation frequency (<1%).
- Spore-negative mutants with even lower sporulation frequencies (<10-8) were derived from strain NRS 576.
Purpose of the Study:
- To investigate the ability of isolated bacteriophage PMB1 to restore sporulation in oligosporogenic Bacillus pumilus.
- To characterize the mechanism and stability of phage-induced sporulation.
Main Methods:
- Isolation of pseudolysogenic phage PMB1 from soil.
- Infection of spore-negative Bacillus pumilus mutants with PMB1.
- Assessment of sporulation frequency in liquid and semi-solid media.
- Analysis of spore characteristics (heat sensitivity, morphology, composition).
Main Results:
- PMB1 infection converted over 50% of isolated spore-negative Bacillus pumilus mutants to a spore-positive phenotype.
- Phage-induced sporulation was stable as long as PMB1 was maintained within the bacterial cells.
- PMB1 infection increased sporulation frequency in mutant GW2 by over 106-fold.
- PMB1-induced spores showed slightly increased heat sensitivity but were otherwise comparable to wild-type spores.
Conclusions:
- The pseudolysogenic phage PMB1 can restore sporulation capability to specific spore-negative mutants of Bacillus pumilus.
- Phage-mediated sporulation is dependent on the presence of the phage within the host bacterium.
- PMB1 represents a novel tool for studying and potentially manipulating bacterial sporulation.