Engineering a Sinorhizobium meliloti Chassis with Monopartite, Single Replicon Genome Configuration

Area of Science:

• Microbiology and Genomics
• Bacterial Genome Engineering

Background:

• Multipartite bacterial genomes, like that of Sinorhizobium meliloti, present significant challenges for genetic manipulation and the integration of new genetic elements.
• The nitrogen-fixing symbiont S. meliloti possesses a complex tripartite genome structure, including a chromosome and two large extrachromosomal replicons (megaplasmid pSymA and chromid pSymB).

Purpose of the Study:

• To simplify the tripartite genome of Sinorhizobium meliloti through targeted replicon fusions, creating bi- and monopartite genome configurations.
• To assess the impact of genome simplification on essential genomic features, growth rates, and symbiotic nitrogen fixation capabilities.
• To develop a platform for enhanced genome engineering applications, including the creation of hybrid genomes.

Main Methods:

• Engineered Sinorhizobium meliloti strains using targeted replicon fusions to create simplified genome architectures (bi- and monopartite).
• Analyzed key genomic features (replichore ratios, GC skew, KOPS, coding sequence distribution) and assessed growth rates and symbiotic nitrogen fixation.
• Investigated the effects of deleting replication origins and introduced suppressor mutations (CckA R436H) or genomic rearrangements (inversion) on genome stability and function.

Main Results:

• Successfully generated bi- and monopartite genome strains while preserving critical genomic features and maintaining comparable growth rates and symbiotic nitrogen fixation to the wild type.
• A monopartite genome strain lacking pSymA and pSymB replication origins exhibited slow growth, aberrant oriC localization, and symbiosis deficiency.
• A suppressor mutation in CckA partially restored growth, but only a large genomic inversion fully recovered symbiotic capacity, highlighting the importance of genomic organization.

Conclusions:

• Genome simplification of S. meliloti via replicon fusion is feasible without compromising essential functions, providing valuable strains for further genome engineering.
• The study demonstrates the utility of these engineered strains for integrating secondary replicons and constructing hybrid genomes.
• Genomic rearrangements play a crucial role in maintaining symbiotic function in simplified bacterial genomes.