DNA methylation patterns differ between free-living Rhizobium leguminosarum RCAM1026 and bacteroids formed in symbiosis with pea (Pisum sativum L.)

Rhizobium leguminosarum (Rl) is a common name for several genospecies of rhizobia able to form nitrogen-fixing nodules on the roots of pea (Pisum sativum L.) and undergo terminal differentiation into a symbiotic form called bacteroids. In this work, we compared the genomes of the free-living and differentiated forms of the Rl strain RCAM1026 using Oxford Nanopore long reads. No significant genome rearrangements were observed, but the relative abundances of replicons were different between the cell states. GANTC, GGCGCC and GATC methylated motifs have been found in the genome, along with genes for methyltransferases with matching predicted targets. Methylation patterns for the GANTC and GATC motives differed significantly depending on the cell state, which indicates their possible connection to the regulation of symbiotic differentiation. The GGCGCC motif was completely methylated in both bacteria states, and, apparently, is a target for the modification-restriction system. Currently, the methylation patterns in symbiotic bacteria are not extensively studied, so a further investigation of the topic coupled with gene expression data is needed to elucidate the function of differential methylation in terminal differentiation of R. leguminosarum and other rhizobia.

Improving mobilization of foreign DNA into Zymomonas mobilis ZM4 by removal of multiple restriction systems

Zymomonas mobilis has emerged as a promising candidate for production of high value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr -like Type IV Restriction Modification (RM) system, a Type I-F CRISPR system, a chromosomal Type I RM ( hsdMS c ) and a previously uncharacterized Type I RM system, located on an endogenous plasmid ( hsdRMS p ). The DNA recognition motif of HsdRMS p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS c and hsdRMS p systems to the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS c and HsdRMS p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction - strains that we constructed, and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. Importance Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study we have developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identified a previously unknown Type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis .

Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.