Search

Aims This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). Methods and results The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. Conclusions C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi. [ABSTRACT FROM AUTHOR]

Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and [NAD.sup.+]) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and [NAD.sup.+] were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPHdependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophUus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fdl and Fd2 from this bacterium) containing [4Fel4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.

The assimilation of acetate generated in the anoxic rice field during the degradation of organic matter by microorganisms is studied using isotopes of carbon. The bacteria were found to utilize acetate under methanogenic conditions where carbon dioxide is the predominant electron acceptor on the polymerase chain reaction analysis of RNA isolated from the bacterial cells.