Completed chromosomes in thymine-requiring Bacillus subtilis spores

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Investigating Bacillus subtilis spore chromosome replication is crucial for understanding bacterial survival and genetic stability.
• Previous studies suggested potential incomplete chromosome replication in Bacillus subtilis spores based on genetic marker ratios.

Purpose of the Study:

• To determine the actual state of chromosome replication in Bacillus subtilis spores using origin:terminus genetic marker ratios.
• To clarify the cause of abnormal genetic marker ratios observed in Bacillus subtilis 168 thy spores.

Main Methods:

• Quantification of origin:terminus genetic marker ratios (purA:metB, purA:ilvA, purA:leuA) in Bacillus subtilis spore extracts.
• Comparison of marker ratios between different Bacillus subtilis strains (W23 thy his and 168 thy) and their respective controls.
• Analysis of marker ratios under varying extraction efficiencies, media richness, and thymine phenotypes.

Main Results:

• Strain W23 thy his spores exhibited origin:terminus ratios of unity, indicating completed chromosomes.
• Strain 168 thy spores showed abnormal purA:metB (2.26 ± 0.10) and purA:ilvA (0.71 ± 0.06) ratios, but a normal purA:leuA ratio (1.01 ± 0.06).
• The abnormal ratios in 168 thy spores were independent of extraction extent, media, and thymine phenotype.

Conclusions:

• The abnormal origin:terminus marker ratios in Bacillus subtilis 168 thy spores are artifacts, not indicative of incomplete replication.
• These artifacts arise from non-isogenicity in the metB thyB ilvA chromosomal region between strains 168 thy and 168.
• Bacillus subtilis 168 thy spores contain completed chromosomes.