The Roles of the Malic Enzymes of Rhizobium (Sinorhizobium) Meliloti in Symbiotic Nitrogen Fixation

Abstract

The genome of 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 contains two genes for malic enzymes. One uses NAD⁺ as a cofactor (𝘥𝘮𝘦) and one utilizes NADP⁺ (𝘵𝘮𝘦). The two enzymes have been purified and the genes cloned and sequenced. Loss of TME enzyme function gives no detectable phenotype in either 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture or in bacteroids. Loss of DME function gives no detectable phenotype in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture but does result in bacteroids that are unable to fix nitrogen (Fix⁻). Expression of 𝘵𝘮𝘦 is reduced in bacteroids whereas 𝘥𝘮𝘦 expression remains unchanged. In order to overexpress 𝘵𝘮𝘦 in bacteroids a fusion gene was constructed with the 𝘥𝘮𝘦 promoter driving expression of the 𝘵𝘮𝘦 structural gene (𝘥𝘵𝘮𝘦). The 𝘥𝘵𝘮𝘦 gene was expressed and functional in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, but alfalfa plants inoculated with strains expressing only the 𝘥𝘵𝘮𝘦 gene were Fix⁻. In addition the NAD⁺-dependent malic enzyme gene from 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘣𝘰𝘷𝘪𝘴 (𝘮𝘢𝘦𝘌) was similarly cloned downstream of the 𝘥𝘮𝘦 promoter. The fusion gene 𝘥𝘮𝘢𝘦𝘌 was expressed in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, surprisingly plants inoculated with strains expressing only the 𝘥𝘮𝘢𝘦𝘌 gene showed a Fix⁻ phenotype. A truncated 𝘥𝘮𝘦 gene was constructed which contained only the N-terminal, malic enzyme domain of the protein (𝘥𝘮𝘦Δ𝘗𝘴𝘵). The truncated enzyme was expressed and active in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture and gave a Fix⁺ phenotype when inoculated onto alfalfa plants.