SUPPRESSION OF INITIATION DEFECTS OF CHROMOSOME-REPLICATION IN BACILLUS-SUBTILIS DNAA AND ORIC-DELETED MUTANTS BY INTEGRATION OF A PLASMID REPLICON INTO THE CHROMOSOMES

We constructed Bacillus subtilis strains in which chromosome replicati on initiates from the minimal replicon of a plasmid isolated from Baci llus natto, independently of oriC. Integration of the replicon in eith er orientation at the proA locus (115 degrees on the genetic map) supp ressed the temperature-sensitive phenotype caused by a mutation in dna A, a gene required for initiation of replication from oriC, In additio n, in a strain with the plasmid replicon integrated into the chromosom e, we were able to delete sequences required for oriC function, These strains were viable but had a slower growth rate than the oriC(+). str ains, Marker frequency analysis revealed that both pyrD and metD, gene s close to proA, showed the highest values among the markers (genes) m easured, and those of other markers decreased symmetrically with dista nce from the site of the integration (proA). These results indicated t hat the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replic ation was bidirectional, Interestingly, these mutants produced anuclea te cells at a high frequency (about 40% in exponential culture), and t he distribution of chromosomes in the cells was irregular, A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination betwe en chromosome replication and chromosome partition or cell division.