A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli.

Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. Methylorubrum extorquens AM1 is an important platform organism of a future C1-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested repABC regions to maintain extrachromosomal DNA in M. extorquens. We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between Escherichia coli and M. extorquens. These mini-chromosomes are compatible among each other and with high-copy number plasmids of M. extorquens. We also developed a set of inducible promoters of wide expression range, reaching levels exceeding those currently available, notably the PmxaF-promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in M. extorquens, which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.

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.