Ca2+/calmodulin (CaM)–dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca2+ signals induced by microbial symbionts. However, it is unclear how CCaMK is activated by these microbes. Here, we investigated in vivo activation of CCaMK in symbiotic signaling, focusing mainly on the significance of and epistatic relationships among functional domains of CCaMK. Loss-of-function mutations in EF-hand motifs revealed the critical importance of the third EF hand for CCaMK activation to promote infection of endosymbionts. However, a gain-of-function mutation (T265D) in the kinase domain compensated for these loss-of-function mutations in the EF hands. Mutation of the CaM binding domain abolished CaM binding and suppressed CCaMKT265D activity in rhizobial infection, but not in mycorrhization, indicating that the requirement for CaM binding to CCaMK differs between root nodule and arbuscular mycorrhizal symbioses. Homology modeling and mutagenesis studies showed that the hydrogen bond network including Thr265 has an important role in the regulation of CCaMK. Based on these genetic, biochemical, and structural studies, we propose an activation mechanism of CCaMK in which root nodule and arbuscular mycorrhizal symbioses are distinguished by differential regulation of CCaMK by CaM binding.

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wildtype and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.

AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding β-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and β-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain β-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family.

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Qipengyuania sediminis gen. nov., sp. nov., a member of the family Erythrobacteraceae isolated from subterrestrial sediment Oil and Gas Survey, China Geological Survey, Beijing 10029, PR China A Gram-reaction-negative, non-motile, facultatively aerobic bacterium, designated strain M1 T , was isolated from a subterrestrial sediment sample of Qiangtang Basin in Qinghai-Tibetan plateau, China. The strain formed rough yellow colonies on R2A plates. Cells were oval or short rod-shaped, catalase-positive and oxidase-negative. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate belonged to the family Erythrobacteraceae and showed 96.2-96.4 % 16S rRNA gene sequence similarities to its closest relatives. Chemotaxonomic analysis revealed ubiquinone-10 (Q10) as the dominant respiratory quinone of strain M1 T and C 17 : 1 v6c (44.2 %) and C 18 : 1 v7c (13.7 %) as the major fatty acids. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, sphingoglycolipid, three unidentified glycolipids, one unidentified phosphoglycolipid and one unidentified lipid. The DNA G+C content of strain M1 T was 73.7 mol%. On the basis of phenotypic, phylogenetic and genotypic data presented in this study, strain M1 T represents a novel species of a new genus in the family Erythrobacteraceae, for which the name Qipengyuania sediminis gen. nov., sp. nov. is proposed. The type strain of the type species is M1 T (5CGMCC 1.12928 T 5JCM 30182 T ).