Engineering a Sinorhizobium meliloti chassis with monopartite, single replicon genome configuration

Abstract

Multipartite bacterial genomes pose challenges for genome engineering and establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features, such as replichore ratios, GC skew, and KOPS and coding sequence distribution. Under standard culture conditions, growth rates of these strains and the wild type were nearly comparable. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes including the oriVs of pSymA and pSymB from this strain resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth and an aberrant cellular localization of oriC. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth. These strains will facilitate integration of secondary replicons in S. meliloti, and thus be useful for genome engineering applications, such as generating hybrid genomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/493018v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d016caorg.highwire.dtl.DTLVardef@8856ddorg.highwire.dtl.DTLVardef@fee530org.highwire.dtl.DTLVardef@78d9cd_HPS_FORMAT_FIGEXP M_FIG C_FIG