Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes. The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners. We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation. Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated. This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation. Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN. The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions. The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression. In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids. A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis.Through the symbiotic nitrogen fixation process, bacteria belonging to the family Rhizobiaceae convert atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ), which can be effectively used by host legume plants. The establishment of a rhizobiumlegume symbiosis requires induction of new developmental programs in the partners. The symbiotic interaction begins with signal exchanges of flavonoids and Nod factors (lipochitooligosaccharides) between the two partners (6). In legume nodules, microaerobic environments trigger the rhizobial expression of nitrogen-fixing genes, such as nif and fix, via an oxygen-sensing system (13). However, the establishment of nitrogen-fixing symbiosis probably requires more complex steps triggered by reciprocal signal exchanges that lead to the organogenesis of nodules, differentiation of microsymbionts, and efficacy of nodulation (27). In addition to this symbiotic lifestyle, rhizobia survive in soils with many environment stresses, such as nutrient starvation.Lotus japonicus is a promising model legume for studying molecular interactions between symbiosis partners (20). Schauser et al. (40) first identified the plant regulatory gene nin, which is responsible for the nodule organogenesis program, in this legume. Recently, the receptor-like kinase genes have...

Inhibitors of ethylene synthesis or its physiological function enhanced nodulation in Lotus japonicus and Macroptilium atropurpureum. In contrast, the application of 1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene biosynthesis, reduced the nodule number in these legumes. These results suggest that an ethylene-mediated signaling pathway is involved in the nodulation process even in the determinate nodulators.

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.The symbiotic interactions between a legume and (brady) rhizobia result in a unique, nitrogen-fixing plant organ, the nodule. Recent studies have shown that the phytohormone ethylene inhibits nodule formation in some legumes (8,9,16,24,25). Application of 1-aminoocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, inhibits nodulation in Medicago truncatula (24).Rhizobitoxine [2-amino-4-(2-amino-3-hydropropoxy)-transbut-3-enoic acid] is an ethylene synthesis inhibitor that is produced by the legume symbiont Bradyrhizobium elkanii (15, 17-19, 22, 39). It is thought that production of this compound enhances nodulation of the host legume because of its inhibitory effect on ethylene synthesis. However, some reports have shown that there is not a significant difference in nodule number between plants inoculated with B. elkanii USDA61 and plants inoculated with rhizobitoxine-deficient mutants during nodulation of Glycine max, Glycine soja, Vigna unguiculata, and Macroptilium atropurpureum (26,39). Recently, Duodu et al. observed a significant difference in nodule number between plants inoculated with isogenic variants of USDA61 during nodulation of Vigna radiata (7). Although these findings do not seem to be consistent with the hypothesis that rhizobitoxine has a positive effect on nodulation, the inconsistency can be explained by differences in the ethylene sensitivity of nodulation among leguminous species; nodulation of G. max is generally not sensitive to ethylene (10, 31, 38), while nodulation of V. radiata is sensitive (7). The inconsistency could also result from differences in the abilities of the strains used in the experiments to produce rhizobitoxine; strain USDA61 is a weak producer of rhizobitoxine (39).In addition to G. max, the leguminous plant M. atropurpureum is a nodulating host for B. elkanii and Bradyrhizobium japonicum (12,15). Although the effect of ethylene on nodulation has been studied in many leguminous host plants so far, the effect of ethylene in M. atropurpureum is not known. B. elkanii was found to be more competitive than B. japonicum for nodulation of M. atropurpureum in a multistrain environment when a field soil was inoculated with a mixture of several strains isolated from the field soil (21). In general, B. elkanii accumulates rhizobitoxine in cultures and in nodules, while B. japonicum does not (5,15,18,19). These results led us to investigate the role of rhizobitoxine production on the nodulation and competitiveness of B. elkanii on M. atropurpureum by using a B. elkanii strain that produces...

Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the ␤-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fixation regulator gene, nifA. The histochemical GUS assay showed that there was exclusive expression of acdS in mature root nodules. Two homologous nifA genes, mll5857 and mll5837, were found in the symbiosis island of M. loti and were designated nifA1 and nifA2, respectively. Quantitative reverse transcription-PCR demonstrated that nifA2 disruption resulted in considerably diminished expression of acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption slightly enhanced expression of the acdS transcripts and suppressed nifH to some extent. These results indicate that the acdS gene and other symbiotic genes are positively regulated by the NifA2 protein, but not by the NifA1 protein, in M. loti. The mode of gene expression suggests that M. loti acdS participates in the establishment and/or maintenance of mature nodules by interfering with the production of ethylene, which induces negative regulation of nodulation.The formation of nitrogen-fixing root nodules is the result of a series of interactions between (Brady)rhizobium and its legume host plants (8). The host legumes have several mechanisms for regulating nodule formation (28, 36). The plant hormone ethylene is also known to have inhibitory effects on rhizobial infection and the formation of nodule primordia and to limit nodule number (21,24,31). Rhizobia often interfere with ethylene biosynthesis in the host plants by means of rhizobitoxine (25, 37, 38) or 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (19,33) and reduce host ethylene emission to overcome the negative regulation.ACC deaminase (EC 4.1.99.4) catalyzes the degradation of an ethylene precursor, ACC, into ammonium and ␣-ketobutyrate (13). The ACC deaminase structural gene (acdS) has been found in many rhizosphere bacteria (10, 13), including fast-and slow-growing rhizobia such as Rhizobium leguminosarum bv. viciae 128C53K (19), Bradyrhizobium japonicum USDA110 (16), Mesorhizobium loti MAFF303099 (15), and M. loti R7A (32). In the rhizobacterium Enterobacter cloacae UW4, promoter analysis of the acdS gene showed that expression of this gene requires both ACC and a leucine-responsive regulatory protein (LRP)-like protein and that anaerobic conditions enhance the expression (10). In B. japonicum USDA110 and R. leguminosarum bv. viciae 128C53K, the acdS genes are also probably regulated by an LRP-like protein and a 70 promoter (16,19).In M. loti MAFF303099, acdS was found in the symbiosis island (15). The enhancing effect of the acdS gene on nodulation of Lotus japonicus MG-20 Miyakojima roo...

Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. To examine how and when endogenous ethylene inhibits rhizobial infection and nodulation, we produced transgenic Lotus japonicus carrying the mutated melon ethylene receptor gene Cm-ERS1/H70A that confers ethylene insensitivity and fixes the transgene in the T(3) generation. The resultant transgenic plants showed reduced ethylene sensitivity because of 1-aminocyclopropane-1-carboxylate resistance and increased flowering duration, probably due to a dominant negative mechanism. When inoculated with Mesorhizobium loti, transgenic plants showed markedly higher numbers of infection threads and nodule primordia on their roots than did either wild-type or azygous plants during the early stage of cultivation period as well as during later stages, when the number of mature nodules had reached a steady state. In addition, transcripts of NIN, a gene governing infection thread formation, increased in the inoculated transgenic plants as compared with the wild-type plants. The infection responses of transgenic plants were similar to those of wild-type plants treated with ethylene inhibitors. These results imply that the endogenous ethylene in L. japonicus roots inhibits rhizobial infection at the primary nodulation, probably via NIN gene, and suggest that ethylene perception assists negative feedback regulation of secondary nodule initiation.

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Nitrogen-fixing nodules are formed as a result of a series of interactions between rhizobia and leguminous plants. Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process and nodulation competitiveness by inhibiting ethylene biosynthesis in host roots. In addition, the gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase was recently found in some rhizobia, such as Mesorhizobium loti, Bradyrhizobium japonicum and Rhizobium sp. ACC deaminase also facilitates symbiosis by decreasing ethylene levels in host roots. The cumulative evidence reveals general strategies by which rhizobia produce an inhibitor and an enzyme to decrease ethylene levels in host roots and thereby enhance nodulation. In this review, we compare these strategies and discuss how they function and have evolved in terms of genetics, biochemistry, and ecology. These rhizobial strategies might be utilized as tools in agriculture and biotechnology.

p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities.