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Applied and Environmental Microbiology, October 2007, p. 6650-6659, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01514-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis ^,

Shino Suzuki, Toshihiro Aono,^,* Kyung-Bum Lee, Tadahiro Suzuki, Chi-Te Liu, Hiroki Miwa, Seiji Wakao, Taichiro Iki, and Hiroshi Oyaizu

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 5 July 2007/ Accepted 17 August 2007

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The molecular and physiological mechanisms behind the maturation and maintenance of N₂-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N₂-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N₂ fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C₄ metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

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Symbiosis between rhizobia and legumes results in the formation of nitrogen-fixing nodules. The symbiotic interaction begins with the induction of bacterial nod genes by flavonoids secreted from the plant roots (11). The nod genes encode proteins that synthesize nodulation factor (Nod factor), which initiates many of the developmental changes seen in the host plant early in the nodulation process (11, 26, 55). After the initial exchange and bacterial attachment at the surface, cortical cells begin dividing to form the nodule primordia. Bacteria penetrate the developing nodule primordia via host-derived infection threads (11, 26, 55). Upon release from the infection threads, bacteria invade the plant cell cytoplasm, where they differentiate into bacteroids and provide ammonium to the host plant by reducing atmospheric dinitrogen in exchange for carbon and amino acid compounds (18, 54, 58).

It is deduced that multiple stages exist in the establishment of complete nitrogen-fixing symbiosis and that signal exchange between rhizobia and legumes might occur at each stage. The finding that the bacterial Nod factor switches on the nodulation program in the plant and the characterization of the plant genes of the Nod factor receptor or perception complexes have revealed a remarkable early event in the process of nodule development (23, 46, 47, 60, 75). However, the molecular and physiological mechanisms behind the maturation and maintenance of nodules still remain unclear. The transcriptional analysis of this process has been well-described in several legume-rhizobium symbiosis systems, and the results have revealed that drastic transcriptional changes occur during nodule development (2, 5, 7, 76). However, to our knowledge, large-scale in vivo studies targeting the nodule maturation process have not been previously reported. In this study, in order to elucidate the nodule development process, we aimed to identify rhizobial factors required for the maturation and maintenance of nodules by performing a large-scale in vivo screening.

Azorhizobium caulinodans ORS571 is a microsymbiont of the water-tolerant tropical legume Sesbania rostrata (20-22). N₂-fixing nodules are formed by A. caulinodans on the stems and roots of S. rostrata. Stem nodules occur at the site of adventitious root primordia located on the stems via crack entry. Root nodules are formed at the curled root hair under well-aerated conditions or at the base of lateral roots under hydroponic conditions (31, 32). During crack entry invasion, bacteria proliferate in the epidermal fissures at the lateral root base or at the adventitious root primordia on the stems (32). Cortical infection pockets are formed by Nod factor-dependent local cell death induction and subsequent colonization of bacteria (16). From the infection pockets, infection threads guide bacteria towards the cells in nodule primordia for symbiotic uptake (17).

A. caulinodans ORS571 shows N₂-fixing ability in the free-living state, but most of the other rhizobia do not (22). This characteristic is convenient for the purpose of distinguishing the defect in nitrogenase activation from the defect in establishing symbiosis. Also, the characteristics of the Azorhizobium-Sesbania system that forms nodules on the stems allow for the easy recognition of different nodule phenotypes. Additionally, lateral root base invasion is useful for analyzing the molecular process downstream of epidermal primordial formation (32). On the basis of the abovementioned advantages provided by the Azorhizobium-Sesbania symbiotic system, we selected this system for a large-scale in vivo screening. In this study, over 10,000 Tn5 transposon-mutagenized A. caulinodans strains were inoculated on the stems of S. rostrata, and over 100 mutants inducing various ineffective stem nodules were obtained. This study allowed us to identify some novel rhizobial factors required for stem nodule maturation and maintenance.

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Bacterial media and growth.
A. caulinodans ORS571 (20) and its derivatives were grown at 37°C in TY medium (9) or synthetic nitrogen-deficient medium (L2 medium) with appropriate antibiotics. L2 medium is a modified LO medium (22) that contains lower vitamin concentrations (0.2 mg ml^–1 biotin, 0.4 mg ml^–1 nicotinic acid, and 0.4 mg ml^–1 pantothenic acid). Escherichia coli strains were grown in Luria broth (LB) medium.

Construction of insertional A. caulinodans ORS571 mutants by conjugation.
A spontaneous mutant resistant to nalidixic acid (Nx) was obtained by subculturing A. caulinodans ORS571 in TY medium supplemented with 5 to 20 µg ml^–1 Nx and designated ORS571-NX. Transposon Tn5 mutagenesis of A. caulinodans ORS571-NX was carried out by transconjugation, using E. coli strain S17-1 pir carrying pFAJ1819 (79) as a donor. Transconjugants were selected on TY plates supplemented with 25 µg ml^–1 Nx and 50 µg ml^–1 kanamycin (Km). From 10 mutagenesis experiments, 10,080 clones were randomly picked and stored at –80°C as glycerol stocks.

Plant growth and bacterial inoculation.
S. rostrata seeds were treated with concentrated sulfuric acid for 1 hour to induce rapid and uniform germination, rinsed with sterile water, and soaked in sterile water on trays. The trays were placed for 3 days at 37°C under dark conditions suitable for germination. The germinated seedlings were transferred into 50-ml plastic tubes, which had a hole at the bottom, and filled with a commercial horticulture soil (Kureha Chemical, Japan). The soil contained 0.4 g N/kg. As S. rostrata can form stem nodules even under 6.5 mM NO₃^– conditions (3), stem nodule formation was not inhibited by the use of this soil. The tubes containing the seedlings were placed on trays filled with tap water and grown for 2 weeks prior to bacterial inoculation at 35°C under a 24-h light regimen at an intensity of 50,000 lx.

In vivo screening.
Individual Tn5 mutants were grown overnight in 500 µl of TY medium supplemented with Nx (25 µg ml^–1) and Km (50 µg ml^–1) in 96-well plates. The cultures were inoculated on the stems of S. rostrata, and plants were grown. Stem nodules were observed 12 days after inoculation, and the mutants that induced stem nodules, which were smaller than those induced by strain ORS571-NX or phenotypes defective in size and color on cross sections of stem nodules, were selected. Three rounds of screenings were carried out for selected strains. The numbers of replicates in the first-, second-, and third-round screenings were one, three, and five, respectively. At the third screening round, three of five plants, for each mutant, were chosen, and the size and N₂ fixation ability of stem nodules were measured to determine the phenotypes.

Stem nodule size.
The diameters of the stem nodules and stems were measured using a digital micrometer caliper. The mean diameter of all stem nodules on the second stem internode of each plant was defined as the nodule size of each plant. The diameter of the stem in the middle of the second stem internode of each plant was measured, and the ratio of stem nodule to stem diameter was defined as the ratio of nodule/stem.

N₂ fixation activity of stem nodules.
Ten stem nodules were peeled off from the stem, and their acetylene reduction activity (ARA) was measured. The stem nodules were placed in 20-ml glass vials sealed with butyl rubber septa, and the air in the vials was replaced with 10% CH₄ in air, following by incubation at 37°C for 2 h. After incubation, 100-µl gas samples from the vials were sampled, and the ethylene concentration was assayed using a gas chromatograph (model GC-17A; Shimazu, Japan) equipped with a fused silica column (Rt-U PLOT; RESTEK, Bellefonte, PA).

N₂ fixation activity of bacterial culture.
Cultures grown in TY medium were centrifuged, washed with 50 mM potassium phosphate buffer (pH 6.8), and suspended in L2 medium to an optical density at 600 nm (OD₆₀₀) of 0.1. Cultures were incubated for 24 h at 37°C. Following incubation, 1-ml aliquots of culture were transferred into 20 ml glass vials sealed with butyl rubber septa. The air in the vials was replaced with N₂ gas containing 3% O₂ and 10% CH₄, and the vials were incubated at 37°C for 16 h. After incubation, 100-µl gas samples from the vials were sampled, and the ethylene concentration was assayed as described above. The OD₆₀₀ of each sample was measured before and after incubation.

Bacterial growth rate.
Cultures grown overnight in TY medium were centrifuged and washed with 50 mM potassium phosphate buffer (pH 6.8) and suspended in TY medium supplemented with or without 100 mM LiCl, 100 mM NaCl, and 15% sucrose, providing an initial OD₆₀₀ of 0.01. Culture aliquots of 200 µl were transferred into 96-well plates and incubated for 24 h at 37°C. After incubation, the OD₆₀₀ of each culture was measured.

Determination of the Tn5-containing regions.
For most mutants, Tn5-containing regions were determined by an inverse PCR method. Genomic DNA isolated from each mutant was digested with Sau3AI or MstI and self-ligated. The region bordering Tn5 was amplified by PCR using the self-ligated genomic DNA as a template and primers specific for the sequences of gusA on pFAJ1819. The sequences of primers were 5'-ATAAGGGACTCCTCCTTAGC-3' and 5'-GCCTGTGGGCATTCAGTC-3' for Sau3AI-digested DNA and 5'-ATAAGGGACTCCTCCTTAGC-3' and 5'-GAATTGATCAGCGTTGGTG-3' for MstI-digested DNA. The amplified fragments were sequenced directly. For some mutants, genomic DNA was digested with XhoI and ligated into the XhoI site of pBluescript SK(+) (Stratagene, La Jolla, CA). E. coli DH5 was transformed with the ligation product and selected on LB agar containing Km (25 µg ml^–1). The plasmid carrying a part of the transposon was purified and sequenced.

Nucleotide sequence accession number.
The genomic sequence of A. caulinodans was deposited in DDBJ/GenBank/EMBL under accession number AP009384.

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Construction of the Tn5 mutant library and determination of free-living and symbiotic characteristics.
To carry out a large-scale screening, 10,080 Tn5 mutants of A. caulinodans were prepared by transconjugation. Details of construction of the mutant library are described in Materials and Methods. In the first round of screening, all mutants were inoculated individually onto the stems of S. rostrata, and about 10% of the mutants, which formed nodules having defective phenotypes in size and interior color, were selected. The selected mutants were inoculated again, in the second and third screenings. Finally, 108 mutants were obtained. As the aim of this study was to determine the genes that are required for the maturation and maintenance of stem nodules, but not for infection and interaction at early stages, we did not select the mutants that were unable to induce stem nodules. That is, mutants that did not even induce bumps on the stems were excluded.

At the third round of screening, the diameters of stem nodules, the diameter ratios of stem nodules to stems, and the ARA of stem nodules were measured, in addition to observing the color of stem nodules on cross sections. In this study, the diameter ratios of stem nodules to stems were measured because the nodule size depended on the stem size under our experimental conditions (Fig. S1 in the supplemental material). Table 1 shows the phenotypes of the stem nodules induced by the mutants compared with those of the wild-type strain. The obtained mutants were classified into seven types on the basis of the phenotypes of the stem nodules (Table 2). Figure 1 shows examples of stem nodules included in each type.

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TABLE 1. Phenotypes and Tn5-inserted regions of mutants

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TABLE 2. Classification of mutants based on the size and N2-fixing ability of stem nodules

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FIG. 1. Typical appearances of stem nodules formed by mutants categorized by type.

To determine whether the abnormality in stem nodule formation by mutants was due to a problem of general metabolism and/or growth, the growth rate of each mutant was compared to that of the wild-type strain in TY medium (Table 1). The growth rates of most mutants were more than 80% that of the wild-type. Thirteen mutants grew more slowly than the wild type (below 80% of the wild type growth rate), and these mutants were defined to be defective in growth. It is thought that tolerance to salt and hyperosmotic stress is important for effective nodule formation (53). Therefore, the growth rate of each mutant was compared to the wild-type strain in TY medium supplemented with 100 mM LiCl, 150 mM NaCl, or 15% sucrose (Table 1). In this study, when the growth rates of mutants in each media were less than 80% of the wild-type growth rate, they were defined as growth-defective mutants. Forty-four, 17, and 20 mutants were defective in growth in LiCl, NaCl, and sucrose-containing media, respectively. All 13 mutants that were defective in growth under normal conditions were, unsurprisingly, defective in growth under stress conditions. Therefore, these mutants, except for the 13 growth-defective mutants, were defined as mutants defective in growth under stress conditions.

To determine whether the defectiveness in stem nodule formation was due to a problem of bacterial N₂-fixing ability in itself, the ARA for mutant cultures in the free-living state was measured (Table 1). The ARA of 42 mutants was less than 80% of the wild-type ATA and defined as defective in ARA in the free-living state. On the basis of these free-living phenotypes of mutants, the mutants were categorized into six types (A to F), as shown in Fig. 2.

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FIG. 2. Venn diagram summarizing the phenotype in the free-living state. When the growth rates of mutants in TY medium were more than 80% that of the wild type, they were defined as normal in growth. When the growth rates of mutants in TY medium containing 100 mM LiCl, 100 mM NaCl, or 15% sucrose were more than 80% that of the wild type, they were defined as tolerant to stresses. When the ARA were more than 80% that of the wild type, they were defined as normal in N2 fixation. Based on this diagram, mutants were classified into six types (types A to F). The number of mutants for each type is shown.

Determination of Tn5-containing regions.
For 96 of the 108 mutants, the Tn5-containing genomic region was cloned, and the respective insertion sites were determined (Table S1 in the supplemental material). For the remaining mutants, the Tn5-containing region could not be cloned. For 79 of the 96 mutants with determined insertion sites, Tn5 was inserted within or upstream of different open reading frames. Annotations of Tn5-containing regions and the locus tags on the entire genome of A. caulinodans ORS571 are listed in Table 1. The genomic sequence of A. caulinodans was completed (unpublished).

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The well-known genes, which are associated with symbiotic systems, such as the nod genes (nodD, nodI, nodJ, nodU, and nodZ) (28, 30, 73), nif genes (nifA, nifB, nifD, nifE, nifS, and nifW) (52), fix genes (fixB and fixC) (4, 41), ntrC (57, 62), ndvC (10), dctA (24), dctB (78), rpoN (74), and hfq (40) were identified in this screening (Table 1). The genes relating to bacterial surface polysaccharides, such as putative lipopolysaccharide (LPS) biosynthesis genes (rfaD, rfaE, and rfaF) (35, 71), putative exopolysaccharide biosynthesis genes (expE5) (8, 29), and putative K antigen biosynthesis genes (rkpA) (43) were also identified. These polysaccharides are important in symbiosis, either as structural components or as signaling molecules (58, 72). These results indicate that our screening methods were appropriate.

In addition to the already known nodule-related genes described above, genes that had not been reported to be involved in nodule formation and genes encoding proteins of unknown function, with or without homology to other rhizobial genes, were also isolated.

Type 1 mutants.
Type 1 mutants formed very small nodules called bumps, and the nodule-like structure did not demonstrate any N₂ fixation activity. Only one mutant, Ao88-F08, could not grow normally in TY medium, and four mutants (Ao44-H06, Ao62-F02, Ao24-F03, and Ao75-H02) were sensitive to ionic, salt, or hyperosmotic stress. These stress-sensitive mutants may lack the ability to survive on the surface of or in the stem of plants. Four mutants (Ao1-A11, Ao23-C06, Ao24-F03, and Ao88-F08) lacked N₂-fixing ability in the free-living state.

In the Ao38-C12 mutant, the cheW gene was disrupted. CheW is an adaptor protein of CheA that has a central role in bacterial chemotaxis mechanisms (42). Chemotaxis and motility have important roles for rhizobia in offering a competitive advantage during the early events of infection (1, 6, 13, 81). However, chemotaxis and motility are not required for the nodulation process after infection, and the expression of the related genes is downregulated during nodulation (2, 15, 51, 66, 67, 80). Since, in this study, bacterial cultures were inoculated directly on the root primordia on the stems, it is unlikely that chemotaxis and motility play a role in movement towards sites of infection. Rather, they may have roles in crack entry and colonization within the stem.

In the Ao23-C06 mutant, the transposon was inserted 26 bp upstream of nrfA, a homologue of E. coli's hfq. Hfq promotes efficient translation of rpoS mRNA in E. coli (50) and Salmonella enterica serovar Typhimurium (12) and alters the stability of several other mRNAs (34, 77). In A. caulinodans, NrfA regulates NifA expression, and nrfA mutants lack N₂-fixing ability (40), and in this study, Ao23-C06 did not show any N₂-fixing ability. Hfq is also involved in the virulence of pathogenic bacteria, for example, Brucella abortus (65), Vibrio cholerae (19), Listeria monocytogenes (14), Legionella pneumophila (49), and Salmonella enterica serovar Typhimurium (70). In S. enterica serovar Typhimurium, Hfq is a key regulator of multiple aspects of virulence, including the regulation of motility and outer membrane protein expression, in addition to invasion and intracellular growth (70). Ao23-C06 formed only bump nodules in this study, suggesting NrfA may also be involved in infection, and it is possible that lack of motility gave rise to the defective nodules.

Type 2 and type 4 mutants.
Type 2 and 4 mutants formed white or beige nodules, with little or undetectable N₂-fixing ability. Many mutants with disruption in the nif and fix genes and other genes related to N₂ fixation activities were categorized as type 2 and 4 mutants. Although all mutants showed no or little N₂-fixing abilities under symbiotic conditions, more than half of them showed high N₂-fixing abilities in the free-living state. These strains may lack the ability to adapt to nodules in the developing environment, which is caused by a deficiency in metabolism or tolerance to stress and/or the ability for signaling/response.

Several mutants showed disruption in the genes involved in carbon and amino acid metabolism. In Ao32-F09, A53-E04, and Ao62-G07 strains, the zwf gene encoding glucose-6-phosphate dehydrogenase, the thrA gene encoding homoserine dehydrogenase, and the betB gene encoding betaine aldehyde dehydrogenase, respectively, were disrupted. Recently, transcriptome and proteome analysis have revealed that rhizobia alter their metabolism in such a way that numerous genes, which are normally silent during free-living growth, are expressed and proteins are produced that appear to carry out unique bacteroid functions. At the same time, many genes that are expressed in free-living growth are repressed (5, 51). The zwf, thrA, and betB mutants were grown normally and showed high N₂ fixation in the free-living state in this study, suggesting that these genes are not indispensable for growth and N₂ fixation by A. caulinodans in the free-living state but are essential in sustaining high levels of N₂ fixation under symbiotic conditions.

In the Ao56-C08 mutant, the gene encoding a helix-turn-helix motif protein was disrupted. This mutant showed N₂ fixation in the free-living state but induced nodules showing no N₂ fixation. The homologous gene in Sinorhizobium medicae WSM419, phrR, is a low-pH-inducible gene and is not required for nodule formation (63). It should be investigated why the phrR gene is required for nodulation in A, caulinodans yet is not required in S. medicae. It will be of interest to understand what sorts of genes are regulated by PhrR and whether the gene encodes a transcriptional regulator.

In Ao29-A09 and Ao59-H05 mutants, the kup gene encoding a K⁺ uptake system was disrupted. A Rhizobium tropici kup mutant is sensitive to salt stress and forms nodules possessing low but detectable N₂ fixation activity (53). However, in this study, A. caulinodans kup mutants were not sensitive to salt-induced or hyperosmotic stress and formed nodules showing no N₂ fixation. In E. coli, Kup has an important role in K⁺ uptake under low pH conditions (68). It is possible that Kup, in A. caulinodans, is also functional at low pH.

In the Ao34-D03 mutant, the ntpA gene encoding a Na⁺/phosphate symporter was disrupted. Although ntpA genes are found in rhizobial genomes, characterization of the genes has not been reported. Some other phosphate transporters are known to be involved in nodulation. In Sinorhizobium meliloti, three phosphate transport systems, PhoCDET, open reading frame A (OrfA)-Pit, and PstSCAB, have been characterized, and the expression of only a single functional Pi transport system, be it OrfA-Pit, PstSCAB, or PhoCDET, is necessary and sufficient for symbiotic N₂ fixation (82). However, the ntpA mutant showed no N₂ fixation in free-living and symbiotic states, suggesting that the functions of NptA may not be complemented by the other phosphate transporters in A. caulinodans.

The composition of LPS is important for the establishment of mature nodules (48). Bacterial LPS typically consists of a lipid A, core oligosaccharide (OS), and O-antigen. The core OS is divided into two regions, inner core and outer core. The outer core region provides an attachment site for the O-antigen. The inner core contains residues of 3-deoxy-D-manno-2-octulosonic acid (Kdo) and heptose, and a Kdo connects the inner core to lipid A (35, 61). Three LPS mutants were isolated in this screening. Ao77-C09 and Ao80-F04 were categorized as type 4 mutants, while Ao13-C11 was a categorized as type 5 mutant. The rfaD, rfaE, and rfaF genes were disrupted in Ao77-C09, Ao80-F04, and Ao13-C11, respectively. The rfaD gene encoding ADP-L-glycero-D-manno-heptose-6-epimerase and the rfaE gene encoding ADP-heptose synthase are involved in the inner core OS biosynthesis, whereas the rfaF gene encoding ADP-heptose-LPS heptosyltransferase II is necessary for inner core OS assembly (35, 45, 69). Based on this information, the LPSs attached to the outer membrane in Ao77-C09 and Ao80-F04 may be only a unit of lipid A-Kdo, while LPS attached to the outer membrane in Ao13-C11 may be a unit of lipid A-Kdo with one heptose. These three mutants may lack the O-antigen in LPS. The A. caulinodans oac2 mutant produces the modified O-antigen, having lower rhamnose content in LPS, and induces multilobed, ineffective nodules lacking functional central tissues taking a long time to develop (27). The three mutants isolated in the present study formed small nodules, but the shapes of these nodules were normal, unlike those formed by the oac2 mutant. Furthermore, Ao13-C11 formed N₂-fixing nodules. These observations suggest that mutants lacking O-antigen of LPS formed more developed nodules compared with mutants having a modified O-antigen.

Type 3 and type 5 mutants.
Type 3 and Type 5 mutants induced nodules, which demonstrated N₂ fixation activities, but the sizes of the nodules were smaller than those observed in the wild type. Four mutants (Ao1-C11, Ao8-C05, Ao2-C07, and Ao13-C11) showed low N₂ fixation activities in the free-living state. In these mutants, the insufficient N₂ fixation activity may have resulted in the small nodules. In the other 15 mutants, small nodules were perhaps caused by factors other than N₂ fixation.

In the Ao83-B10 mutant, the typA gene encoding a GTP-binding protein was disrupted. TypA and its homologues are global regulators involved in growth at low temperatures (33), flagellum-mediated cell motility (25), and resistance to certain antimicrobial peptides (59). In S. meliloti, the typA gene is required for housekeeping functions, the improvement of survival under some stress conditions, and symbiosis with certain Medicago truncatura lines (44). In E. coli, BipA, the homologue of TypA, is a translational factor required specifically for the expression of the transcriptional modulator Fis (56). Important transcriptional regulators in rhizobia, such as NifA and NtrC, also have a helix-turn-helix motif categorized for the Fis family. It will be noteworthy to investigate what Fis-type transcriptional factors are targeted by TypA in A. caulinodans.

Type 6 and type 7 mutants.
Type 6 and type 7 mutants produced almost the same size of nodules as those of the wild type. Although all of the mutants showed high N₂ fixation activities in the free-living state, type 6 mutants formed nodules that showed little or no N₂-fixing activity, while type 7 mutants formed nodules with significantly lower activity of N₂ fixation than that of the wild type.

In the Ao10-B03 mutant, the gene encoding a protein having an autotransporter domain was disrupted. The functions of the autotransporter passenger domains are diverse; they act as adhesin, protease, elastase, toxin, and so on (36). The putative protein disrupted in Ao10-B03 has a domain of filamentous hemagglutinin (FHA) containing a beta-solenoid motif (38, 39) which functions as an adhesin in Bordetella pertussis (64). FHA plays important roles also in immunomodulation, suggesting that FHA may be related to host specificity and/or specific disease characteristics (37). If FHA can function as a recognition molecule in the eukaryote-prokaryote interaction, the molecule may possibly play an important role in legume-rhizobium symbiosis.

View of the future study of legume-rhizobium symbiosis.
The list of mutants that showed aberrations in nodule development at various stages shows the complexity of nodule development and maturation. The genes and their products are considered to have important roles in the metabolic cycle of nodules or act as molecular signals to establish Azorhizobium-Sesbania symbiosis. Although a number of genes were previously reported to be involved in nodule development in other legume-rhizobium symbiosis systems, the biological functions of these genes are not always identical in the different symbiotic systems. Those results may indicate that the symbiosis-related genes of rhizobia evolved to adapt the functions of the genes to symbiotic conditions in a manner specific for each rhizobial strain. This may be one of the reasons behind the complexity of the mechanisms of nodule development. It remains an important task to elucidate which mutants obtained in this screening are conditioned to be function specific to the Azorhizobium-Sesbania symbiotic system and which ones can be extrapolated to general legume-rhizobium symbiosis.

 
R. Funamoto, H. Toyazaki, and N. Akiba are thanked for their technical assistance.

This work was supported by grants from the Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan.

 
* Corresponding author. Mailing address: Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81 3 5841 2407. Fax: 81 3 5841 2408. E-mail: uaono{at}mail.ecc.u-tokyo.ac.jp

Published ahead of print on 24 August 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to the work.

Present address: National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.

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Applied and Environmental Microbiology, October 2007, p. 6650-6659, Vol. 73, No. 20
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