title: Characterization of a Mesorhizobium loti alpha-type carbonic anhydrase and its role in symbiotic nitrogen fixation.
abstract: Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.
authors.affiliation: Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.
authors.forename: Chrysanthi
authors.initials: C
authors.lastname: Kalloniati
authors.forename: Daniela
authors.initials: D
authors.lastname: Tsikou
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Lampiri
authors.forename: Mariangela N
authors.initials: MN
authors.lastname: Fotelli
authors.forename: Heinz
authors.initials: H
authors.lastname: Rennenberg
authors.forename: Iordanis
authors.initials: I
authors.lastname: Chatzipavlidis
authors.forename: Costas
authors.initials: C
authors.lastname: Fasseas
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Katinakis
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Flemetakis
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology*; D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000378:metabolism; D018533:Biomass; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017353:Gene Deletion; D015870:Gene Expression; D000070116:Lotus / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009586:Nitrogen Fixation; D016415:Sequence Alignment; D013559:Symbiosis
chemical_list: D001639:Bicarbonates; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases