Complementation of Cobalamin Auxotrophy in Synechococcus sp. Strain PCC 7002 and Validation of a Putative Cobalamin Riboswitch In Vivo

The euryhaline cyanobacteriumSynechococcus sp. strain PCC 7002 has an obligate requirement for exogenous vitamin B12 (cobalamin), but little is known about the roles of this compound in cyanobacteria. Bioinformatic analyses suggest that only the terminal enzyme in methionine biosynthesis, methionine synthase, requires cobalamin as a coenzyme inSynechococcus sp. strain PCC 7002. Methionine synthase (MetH) catalyzes the transfer of a methyl group fromN 5 -methyl-5,6,7,8-tetrahydrofolate tol -homocysteine duringl -methionine synthesis and uses methylcobalamin as an intermediate methyl donor. Numerous bacteria and plants alternatively employ a cobalamin-independent methionine synthase isozyme, MetE, that catalyzes the same methyl transfer reaction as MetH but usesN 5 -methyl-5,6,7,8-tetrahydrofolate directly as the methyl donor. The cobalamin auxotrophy ofSynechococcus sp. strain PCC 7002 was complemented by using themetE gene from the closely related cyanobacteriumSynechococcus sp. strain PCC 73109, which possesses genes for both methionine synthases. This result suggests that methionine biosynthesis is probably the sole use of cobalamin inSynechococcus sp. strain PCC 7002. Furthermore, a cobalamin-repressible gene expression system was developed inSynechococcus sp. strain PCC 7002 that was used to validate the presence of a cobalamin riboswitch in the promoter region ofmetE fromSynechococcus sp. strain PCC 73109. This riboswitch acts as a cobalamin-dependent transcriptional attenuator formetE in that organism.IMPORTANCE Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph because, like eukaryotic marine algae, it uses a cobalamin-dependent methionine synthase (MetH) for the final step ofl -methionine biosynthesis but cannot synthesize cobalaminde novo . Heterologous expression ofmetE , encoding cobalamin-independent methionine synthase, fromSynechococcus sp. strain PCC 73109, relieved this auxotrophy and enabled the construction of a truly autotrophicSynechococcus sp. strain PCC 7002 more suitable for large-scale industrial applications. Characterization of a cobalamin riboswitch expands the genetic toolbox forSynechococcus sp. strain PCC 7002 by providing a cobalamin-repressible expression system.