Characterization of the chromosome dimer resolution system and analyses of recombination-deficient mutants in Bacillus subtilis.

摘要

Bacteria that have coevolved circular chromosomes and sophisticated recombinational machinery must account for dimeric chromosomes formed by recombination between sister chromosomes during DNA replication. Escherichia coli employs two tyrosine family site-specific recombinases, XerC and XerD, to effect resolution of dimeric chromosomes into monomers at a site near the terminus of DNA replication known as dif. The focus of this work has been to characterize the system of chromosome dimer resolution in the Gram-positive organism, Bacillus subtilis.;Based on partial sequence information suggesting that RipX was a XerD homologue, efforts were made to characterize ripX and establish its relationship to chromosome dimer resolution in B. subtilis. CodV is the XerC homologue in B. subtilis. However, no Xer-like phenotypes were attributed to codV mutants in a report by a separate laboratory. Nevertheless, codV was also investigated in this work.;A ripX mutant and a ripX codV double mutant developed a subpopulation of cells present in chains. The cell length within chains was highly variable. Also, nucleoids in these unusual chains of cells were frequently distorted in comparison to the parent strain nucleoid. Additionally, ripX and ripX codV double mutants were impaired in their ability to sporulate. These phenotypes were also present in codV mutants, but at a reduced frequency compared to either the ripX or ripX codV double mutants. Deletion of the SPbeta bacteriophage substantially elevated the amount of aberrant nucleoid phenotypes seen in codV strains, and to a lesser extent, those seen in ripX strains.;A correlation was found between partition-stalled nucleoids and the polar displacement of FtsZ rings and cell septa during the primary cell cycle of outgrowing germinated ripX spores. This finding suggests that improperly partitioned DNA in ripX cells exerts a negative influence on normal mid-cell division.;The B. subtilis dif site has been located in this work. The site lies at approximately 166° on the chromosome, just ahead of the terminus of replication (172° in B. subtilis). Elimination of the dif site resulted in cell and nucleoid phenotypes similar to those seen in ripX and codV mutants. Additionally, plasmids containing dif were able to integrate into the chromosomes of recA mutants, but not into ripX recA, codV recA, or Deltadif strains. These data suggest that the integration events are independent of homologous recombination and are mediated by RipX and CodV at the newly identified dif locus.