Engineering aSinorhizobium melilotichassis with monopartite, single replicon genome configuration

Abstract

While the vast majority of bacterial genomic DNA molecules contain a single origin of replication, some natural isolates and engineered strains were reported to contain chromosomes derived from cointegration events of multiple replicons. We investigated effects of multiple DNA replication origins and terminus regions on spatial DNA organization and spatiotemporal replicon segregation in the alphaproteobacterium Sinorhizobium meliloti. Strains with a bi- and monopartite genome configuration were constructed by Cre/lox-mediated site-specific fusions of the secondary replicons pSymA and pSymB, and of the chromosome, pSymA and pSymB. The design of these strains maintained replichore ratios, GC skew, as well as distribution and orientation of KOPS and coding sequences. Growth of these strains was essentially unaffected, except for high salt conditions. Replication initiation at the three origins as well as key features of spatial organization and spatiotemporal segregation were maintained in the triple-replicon fusion strain. Cell growth was slowed down by deleting, either individually or together, the pSymA- and pSymB-derived replication initiator encoding repC genes with their intrinsic origin of replication from the dual or triple replicon cointegrates, respectively. Replication of the triple cointegrate, characterized by the chromosomal oriC as sole origin and a strongly disbalanced replichore ratio, terminated in the original chromosomal terC region, suggesting a replication trap. Progression of replication of the longer replichore was not blocked but impaired, possibly due to the retained secondary replicon’s terminus regions and reverse alignment of replichore-orienting sequence features following from the deletion of replication origins. Moreover, during the cell cycle of this strain, oriC aberrantly localized and served as replication initiation site in the mid cell area of the cell with the oldest cell pole. Growth deficiency of this strain was attenuated by a suppressor mutation causing amino acid substitution R436H in the cell cycle histidine kinase CckA.