loti chromosome (Fig. 1, bottom). The numbering of the genes is fixed in the RhizoBase (genome database for Rhizobia, http://bacteria.kazusa.or.jp/rhizobase/). The first enzyme, pyridoxine 4-oxidase, is encoded by the mll6785 gene (Yuan et al., 2004); the second, pyridoxal 4-dehydrogenase, by mlr6807 (Yokochi et al., 2006); the third, 4-pyridoxolactonase, by mlr6805 (Funami et al., 2005); the fourth, 4-pyridoxic acid dehydrogenase, by mlr6792 (Ge et al., 2008); the fifth, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic
acid (FHMPC) dehydrogenase, by mlr6793 (Yokochi et al., 2009); the sixth, 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid (HMPC) decarboxylase, by mlr6791 (Mukherjee et al., 2007); the seventh, 3-hydroxy-2-methylpyridine-5-carboxylic acid (HMPC) oxygenase, by mlr6788 (Yuan learn more et al., 2006; McCulloch buy PI3K Inhibitor Library et al., 2009); and the eighth, AAMS amidohydrolase, by mlr6787 (Mukherjee et al., 2008; Yuan et al., 2008). Pyridoxamine is converted into pyridoxal by pyridoxamine-pyruvate aminotransferase
encoded by mlr6806 (Yoshikane et al., 2006). Thus, the genes form a cluster, from mll6785 to mlr6807, including several genes of unknown function. The expression of genes involved in bacterial catabolic pathways is often regulated by one or several transcriptional regulators (Tropel & Van der Meer, 2004). The GntR family proteins are well known transcription factors and comprise more than 8500 members in the Pfam database (Hoskisson & Rigali, 2009). They are distributed throughout the bacterial world. The GntR regulators are subdivided into the AraR, DevA, FadR, HutC, MocR, PlmA, and YtrA subfamilies based on the sequence FER similarity of their C-terminal effector-binding oligomerization domains. The FadR subfamily is the most representative GntR subfamily and can be divided into FadR and VanR subgroups based on the number of α-helices (10 and 9, respectively) in their secondary structures (Rigali et al., 2002). In the cluster of genes involved in the degradation of pyridoxine (Fig. 1b) there is one gene (mll6786) that encodes a probable transcriptional regulator protein. The primary structure and deduced secondary structure suggested that mll6786
encodes a regulator protein that belongs to the VanR subgroup. As far as we know, no study has been done on the regulation mechanism for the degradation pathway for pyridoxine. Here, we identified the protein PyrR encoded by mll6786 as a transcriptional repressor protein. The recombinant repressor protein was over-expressed and characterized as the first step of elucidation of the regulatory mechanism for the pyridoxine-degradation pathway in M. loti cells. Escherichia coli strains BL21(DE3) and JM109 were purchased from Novagen (San Diego, CA) and Takara (Tokyo, Japan), respectively. Escherichia coli S17-1 was obtained from the National Bioresource Project (Mishima, Japan). Mesorhizobium loti MAFF303099 was obtained from the MAFF GenBank (Tsukuba, Japan).