title: Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture of Bacillus subtilis.
abstract: Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.
authors.affiliation: Department of Oral Microbiology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama 700-8525, Japan. shingaki@md.okayama-u.ac.jp
authors.forename: Ryuji
authors.initials: R
authors.lastname: Shingaki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kasahara
authors.forename: Tetsuyoshi
authors.initials: T
authors.lastname: Inoue
authors.forename: Susumu
authors.initials: S
authors.lastname: Kokeguchi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Fukui
mesh_terms: D017103:Bacillus Phages / Q000235:genetics* / Q000502:physiology; D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism / Q000821:virology*; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification; D002876:Chromosomes, Bacterial / Q000378:metabolism*; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D003673:Defective Viruses / Q000235:genetics* / Q000502:physiology; D016521:Electrophoresis, Gel, Pulsed-Field; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D008854:Microscopy, Electron; D039002:Prophages / Q000235:genetics* / Q000502:physiology; D015183:Restriction Mapping; D014771:Virion
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial