ABSTRACT
The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.
Subject(s)
Bacillus subtilis/physiology , Exodeoxyribonucleases , Rec A Recombinases/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacterial Proteins/genetics , Cell Nucleus , Colony Count, Microbial , DNA-Binding Proteins/genetics , Fluorescent Dyes , Indoles , Mutation , Rec A Recombinases/genetics , Spores, Bacterial/physiologyABSTRACT
Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6 degrees counterclockwise from the B. subtilis terminus of replication (172 degrees ), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPbeta-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains.
Subject(s)
Bacillus subtilis/genetics , Chromosomes, Bacterial/genetics , DNA Nucleotidyltransferases/metabolism , Escherichia coli Proteins , Integrases , Recombination, Genetic , Sigma Factor , Bacillus subtilis/ultrastructure , Bacterial Proteins/metabolism , Chromosomes, Bacterial/ultrastructure , DNA-Binding Proteins/metabolism , Dimerization , Models, Genetic , Plasmids/genetics , Protein Binding , Rec A Recombinases/genetics , Recombinases , Spores, Bacterial , TATA-Box Binding Protein , Transcription Factors/metabolismABSTRACT
Dimeric chromosomes can be formed during replication of circular bacterial chromosomes by an odd number of homologous recombination events between sister chromosomes. In the absence of a compensating recombination reaction such dimers cannot be segregated from each other as the cell divides. This review highlights the shared and divergent mechanisms employed by Escherichia coli and Bacillus subtilis in their effort to resolve and partition dimeric chromosomes safely. In particular, we discuss the Xer-type recombinases, RecA, FtsK/SpoIIIE, and dif.
Subject(s)
Bacillus subtilis/genetics , Chromosomes, Bacterial/physiology , Escherichia coli/genetics , Genome, Bacterial , Integrases , Sigma Factor , Transcription Factors , Bacterial Proteins/metabolism , Cell Division , DNA Nucleotidyltransferases/metabolism , Dimerization , Escherichia coli Proteins , Membrane Proteins/metabolism , Recombinases , Recombination, GeneticABSTRACT
The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.
