- Research article
- Open Access
The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571
- Kyung-Bum Lee†1, 2,
- Philippe De Backer†3, 4,
- Toshihiro Aono1,
- Chi-Te Liu1,
- Shino Suzuki1,
- Tadahiro Suzuki1,
- Takakazu Kaneko5,
- Manabu Yamada5,
- Satoshi Tabata5,
- Doris M Kupfer6,
- Fares Z Najar6,
- Graham B Wiley6,
- Bruce Roe6,
- Tim T Binnewies7,
- David W Ussery7,
- Wim D'Haeze4,
- Jeroen Den Herder3, 4,
- Dirk Gevers3, 4, 8,
- Danny Vereecke3, 4,
- Marcelle Holsters3, 4 and
- Hiroshi Oyaizu1Email author
© Lee et al; licensee BioMed Central Ltd. 2008
- Received: 28 January 2008
- Accepted: 04 June 2008
- Published: 04 June 2008
Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem.
The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor.
The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.
- Biological Nitrogen Fixation
- Symbiosis Region
- Symbiotic Nitrogen Fixation
- Nonredundant Protein Database
- Strain ORS571
Biological nitrogen fixation is carried out by a limited number of prokaryotes that all possess a nitrogenase enzyme complex that reduces molecular dinitrogen to ammonia. Nitrogen-fixing bacteria can be divided in two major groups: free-living nitrogen fixers or diazotrophs that directly assimilate ammonia for growth and symbiotic nitrogen fixers that pass ammonia to a eukaryotic host and indirectly profit from nitrogen fixation by occupying a particular ecological niche or by supporting the population through better feeding. In the latter group, the symbiosis between leguminous crop plants and rhizobia is of great importance for agriculture. The term "rhizobia" is used for bacteria that induce the formation of new organs, nodules, on the roots of a specific legume host. Inside the nodule, rhizobia are internalized in plant cells where they differentiate into nitrogen-fixing bacteroids [for a recent review on legume nodulation, see ].
Nitrogen-fixing nodules typically occur on roots; however, some members of the subfamilies Papilionoideae (Aeschenomyne sp., Sesbania sp., and Discolobium pulchellum) and Mimosoideae (Neptunia oleracea) form stem-located, aerial nodules . These legumes grow in waterlogged soils of tropical regions and are characterized by dormant, stem-located adventitious root primordia that can develop into stem nodules upon inoculation with an appropriate microbial partner. Although stem and root nodulation are similar, in the latter the nodular vascular system is connected to that of the stem via the vascular bundles of the adventitious root primordium .
A particularly well-studied case of stem nodulation occurs in Sesbania rostrata Brem. upon inoculation with the microsymbiont Azorhizobium caulinodans . S. rostrata, a fast-growing annual shrub from the Sahel region of West-Africa, carries numerous adventitious root primordia that protrude through the stem cortex and epidermis, creating a circular fissure, where bacteria can invade and proliferate . The growth properties and the high rate of nitrogen fixation of stem-nodulated plants make S. rostrata well fit as green manure in rice cultivation and, possibly, as a pioneer plant for wetland improvement .
The bacterium, isolated from stem nodules  and originally designated Rhizobium sp. strain ORS571, was renamed Azorhizobium caulinodans inspired by the stem (cauli-)nodulating capacity and by the diazotrophic properties of the strain (azo-rhizobium). Its host range for effective nodulation is very narrow: although nodulation of several Sesbania sp. has been reported, nitrogen-fixing nodules are formed only on S. rostrata and S. punctata . A. caulinodans also induces Fix nodules on Phaseolus vulgaris and Leucaena leucocephala . Two features distinguish A. caulinodans from other rhizobia: its taxonomic position and its dual capacity for free-living and symbiotic nitrogen fixation. The latter is exceptional  and implies a regulatory mechanism to either assimilate the ammonia or donate it to the plant in the symbiotic interaction. The first taxonomic study of A. caulinodans strain ORS571  showed that it belongs to the Rhodopseudomonas palustris rRNA branch of purple bacteria, but that it is quite distinct from both Rhodopseudomonas and Bradyrhizobium spp. Based on numerical analysis of phenotypes, protein patterns, and DNA-DNA and DNA-rRNA hybridizations, A. caulinodans was considered as a separate genus with Xanthobacter as closest relative . Xanthobacter sp. are diazotrophic bacteria found in diverse soil habitats and in association with rice (Oryza sativa) roots [12, 13]. Comparison of 16S rRNA sequences indicated that X. flavus and A. caulinodans are strongly related .
Here, we present the genome sequence of the A. caulinodans strain ORS571 and discuss the annotation in function of the organism's biology with reference to comparative genomics. This information will stimulate the research on an organism that has real potential for novel applications in agriculture.
Overview of the functional categories of proteins present in the A. caulinodans genome according to the classification of Riley .
Amino acid biosynthesis
Biosynthesis of cofactors, prosthetic groups and carriers
Central intermediary metabolism
Fatty acid, phopholipid, and sterol metabolism
Purines, pyrimidines, nucleosides, and nucleotides
DNA replication, recombination and repair
Transport and binding proteins
Three rRNA clusters are ordered as 5S-23S-16S (located between the protein-coding genes AZC_0613-AZC_0614, AZC_4195-AZC_4196, and AZC_4435-AZC_4436) and all have an insertion of a tRNA-Ile and a tRNA-Ala between the 16S and 23S genes. A total of 53 tRNA genes representing 44 tRNA species for all 20 amino acids were assigned by sequence similarity and computer prediction with the tRNAscan-SE program . Most of the tRNA genes are dispersed on the genome and are probably transcribed as single units. Thirty out of 57 ribosomal protein genes occur in a cluster (AZC_2529-AZC_2559), whereas the others are scattered over the genome (Additional file 1).
Phylogeny and comparative genomics
For a broader view of the gene relationships, the occurrence and organization of the proteins encoded by these 45 genomes were evaluated (Methods). Each gene of a total data set of 146,315 was classified in one of four groups: orphans, genes without homologs in other bacteria of the data set; singletons, genes with one representative in the genome and homologs in other genomes; phage or integrase-related genes; and duplicated genes or paralogs with more than one paralog in the genome. The distribution of each of these categories differed in the surveyed genomes (Figure 5B). Paralog representation ranged from 5% for the Neorickettsia sennetsu strain Miyayama (genome size 0.86 Mb) to 44% for Rhizobium leguminosarum bv. vicae (strain 3841) (genome size 7.79 Mb), whereas A. caulinodans had 36% paralogs (genome size 5.37 Mb). The data confirmed the observation that the number of paralogs strongly correlates with the genome size in a linear regression .
Altogether, these analyses demonstrate that currently X. autotrophicus is the closest sequenced relative of A. caulinodans. However, a comparison of the genomes with the ARTEMIS comparison tool  revealed a very low degree of synteny (Additional file 3). Although short sequence stretches are conserved, extensive rearrangements have taken place. The occurrence of four prophages and numerous transposases in the A. caulinodans genome suggests a high genome plasticity. In A. caulinodans, 1412 proteins have no counterpart in X. autotrophicus of which 544 (38%) are catalogued as unknown or hypothetical (Additional file 4). In the remaining group of functionally classified proteins, 46% have GC and GC3 contents different from the genome averages, suggesting recent acquisition.
Functional protein classes and metabolic pathways
These analyses revealed the presence of many regulatory genes (8%) and several RNA polymerase σ factors, among which two household σ70 factors (AZC_3643 and AZC_4253), two σ54 factors (AZC_2924 and AZC_3925; see below), and five σ factors of the extracytoplasmic subclass (AZC_0389, AZC_1202, AZC_2427, AZC_2453, and AZC_3238), implying responsiveness to many environmental triggers. As A. caulinodans is a motile bacterium, a large gene cluster is present (AZC_0615-AZC_0666) for the formation of a type-III flagellum. A significant number of chemotaxis genes predicts the capacity to respond to a wide array of molecules (Additional file 5). While no complete quorum sensing system could be detected, the presence of no less than five LuxR-type response regulators suggests that A. caulinodans has the potential to listen in on acyl-homoserine lactone-mediated communication in its surroundings.
A variety of encoded proteins might offer protection against toxic compounds in the environment (Additional file 6). Examples are two cytochrome P450 monooxygenases and pathways to degrade or modify plant-derived molecules, such as protocatechuate, and xenobiotics, such as cyanate, 1,4-dichlorobenzene, octane, and gallate. Several multidrug efflux pumps, antibiotic-modifying enzymes, and heavy metal translocation systems probably confer resistance to deleterious compounds. The production of the siderophores enterobactin and aerobactin might guarantee iron acquisition from the surroundings.
The surface of bacteria is important for recognition, attachment, and colonization during the interaction with a host. Exopolysaccharides and lipopolysaccharides are involved in nodulation as protective compounds against defense molecules generated by the plant and as communication signals [28–30]. Other functions could relate to surface structures, important for interaction with the host (Additional file 7), e.g. putative adhesion proteins, antigens, and 29 genes that code for proteins with GGDEF/EAL domains. The latter typically play a role in the transition from a motile planktonic form to a sessile biofilm by controlling the formation and degradation of the secondary messenger cyclic di-GMP . Hormones also play an important role in plant-microbe interactions. Both a structural (AZC_0267) and a regulatory gene (AZC_0266) mediating degradation of the ethylene precursor 1-aminocyclopropane-1-carboxylate, are present in the genome.
Over 15% of the genes are dedicated to "transport and binding", of which more than 50% belong to the ATP-binding-cassette (ABC) transporter class. With 118 complete systems (consisting of a solute-binding protein, a permease, and an ABC component for the uptake systems, or an ABC component and a permease for the export systems), and numerous orphan subunits scattered over the genome, the transporter complement of A. caulinodans equals that of many other soil bacteria. These high-affinity transport systems are dedicated to the uptake of peptides, amino acids, sugars, polyamines, siderophores, nitrate/sulfonate/bicarbonate, or C4-dicarboxylate and many unknown substrates (Additional file 8). Accordingly, catabolic pathways are predicted for compounds, such as amino acids (including citrulline and ornithine), glucuronate, galactonate, galactarate, gluconate, quinate, L-idonate, creatinine, and 4-hydroxymandelate. Sugars, such as glucose, fructose, sucrose, ribose, xylose, xylulose, and lactose are not metabolized by A. caulinodans; instead, dicarboxylic acids are used as primary carbon source , as reflected by the presence of multiple C4-dicarboxylic acid transport systems. The occurrence of 16 putative alcohol dehydrogenase genes suggests that ethanol could be a major carbon source under flooded conditions. A. caulinodans is also capable of oxidizing hydrogen, an obligatory by-product of the nitrogenase, and the required hup, hyp, and hox A genes are located in a large gene cluster (AZC_0594-AZC_0613) . Encoded energy metabolism pathways include glycolysis, Entner-Doudoroff, and TCA cycle. The absence of a gene encoding phosphofructokinase indicates the lack of a functional Emden-Meyerhof pathway.
Nitrogen fixation and related functions
Nitrogen fixation-related genes
PTS IIA-like nitrogen-regulatory protein
Nitrogenase MoFe cofactor biosynthesis
Nitrogenase MoFe cofactor biosynthesis
Nitrogenase MoFe cofactor biosynthesis
Nitrogenase MoFe protein β-chain
Nitrogenase MoFe protein α-chain
Nitrogen regulatory protein
ntr B/ntr Y
Signal transduction histidine kinase
RNA polymerase σ54 factor
Translation regulator of nif A
Signal transduction histidine kinase
Signal transduction histidine kinase
Nitrogen assimilation-regulatory protein
Mobilization of Fe for Fe-S cluster synthesis and repair
Nitrogenase cofactor synthesis protein
Fe and S donor for MoFe cofactor biosynthesis
Nitrogenase MoFe cofactor biosynthesis
Electron-transferring flavoprotein oxidoreductase
Electron-transferring flavoprotein oxidoreductase
RNA polymerase σ54 factor
cyt N/fix N
Cytochrome c oxidase subunit 1
cyt O/fix O
Cytochrome c oxidase subunit 2
cyt Q/fix Q
Cytochrome c oxidase subunit 3
cyt P/fix P
Cytochrome c oxidase subunit 4
Assembly and stability of the FixNOQP complex
Assembly and stability of the FixNOQP complex
Transcriptional regulatory protein
The transcriptional activator NifA (AZC_1049) acts together with a σ54 factor RpoN (AZC_3925) to control the nif/fix gene expression . Nitrogen regulation of nifA expression is under control of the NtrBC (AZC_3086-AZC_3087) and NtrYX (AZC_3083-AZC_3084) two-component systems [35, 36] that respond to the intracellular and extracellular nitrogen status, respectively. The expression of these two loci depends on a hypothetical σ54 factor RpoF , which presumably corresponds to AZC_2924. Oxygen control of nif A expression is mediated by FixLJ (AZC_4654 and AZC_4655) , and the transcription factor FixK (AZC_4653) . The nif A gene is further controlled at the transcriptional level by a LysR-type regulator  and at the translational level by the nrf A gene product (AZC_3080) . FixK also activates transcription of the cyt NOQP operon (AZC_4523-AZC_4526), encoding the high-affinity terminal oxidase cytochrome cbb3 that is induced under microaerobiosis [41, 42]. Mutants in cyt NOQP still fix nitrogen under free-living conditions, suggesting the occurrence of another terminal oxidase [41, 43]. The survey of the genome excluded the presence of a second cytochrome cbb3 complex, but revealed two cytochrome bd complexes (AZC_1353-AZC_1354 and AZC_3759-AZC_3760).
A symbiosis region
The three nod loci are not adjacent and have a GC content lower than that of the surrounding sequences (Figure 3C). The shifts in GC content correspond to the location of repeated elements that are flanked by insertion sequences or tRNAs (Figures 1 and 6). The constitutively expressed nod D gene (AZC_3792) [45, 46] codes for a LysR-type regulator that activates transcription of the two other flavonoid-inducible nod loci. The inducible operon nod ABCSUIJZnoe CHOP (AZC_3818-AZC_3807) [47–49] encodes most of the enzymatic machinery for NF backbone synthesis, decoration, and secretion. The biochemical role of these proteins has been extensively described, except for the last four open reading frames noe CHOP that are involved in NF arabinosylation and are still under study. Based on similarity with proteins involved in arabinosylation of the cell wall in Mycobacterium tuberculosis, noe C (AZC_3810), noe H (AZC_3809), and noe O (AZC_3808) might encode the synthesis of a D-arabinose precursor [50–52]. The third locus encodes the inducible nol K gene responsible for GDP-fucose synthesis for NF decoration (AZC_3850) [53, 54].
The symbiosis region also contains two conjugation-related gene clusters with GC and GC3 contents comparable to the genome averages. The cluster AZC_3844-AZC_3826 – flanked by two transposases – consists of repA and genes encoding conjugal transfer, partition, and plasmid stabilization proteins (Additional file 1). In the cluster AZC_3856–3877, flanked by a transposase and an integrase, genes are found that are homologous to the trb BCDEJLFGI genes, a type-IV secretion system involved in conjugative transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens  (Figure 6).
The genome annotation indicates the presence of a few additional nodulation-related genes outside of the symbiosis region (Additional file 1). Two response regulators (AZC_1361 and AZC_2281) homologous to nod W genes of Bradyrhizobium japonicum and part of a two-component signal transduction system might be involved in the response to host-exuded flavonoids . A nod T-related gene (AZC_3288)  might act as the outer-membrane component in NF secretion together with the inner-membrane NodIJ proteins. None of these four potential nodulation genes has a different GC or GC3 content, in contrast to the nod genes of the symbiosis region.
Azorhizobium caulinodans is a member of the α-proteobacteria, a group with diverse genome architectures. Several plant-associated representatives, such as Agrobacterium and Sinorhizobium, have quite considerable genomes and large circular or linear plasmids. In contrast, A. caulinodans has a single circular chromosome of 5.37 Mb and no auxiliary replicons. The GC content and the coding density are in range with other rhizobia and soil bacteria. A. caulinodans is a motile, nitrogen-fixing, hydrogen-oxidizing, aerobic bacterium with a preference for organic acids as carbon source. This lifestyle is reflected in the metabolic pathways and in clusters for flagellum synthesis, motility, and chemotaxis. A high number of genes are dedicated to transport and regulation, indicating that a wide range of substrates can be taken up, but that the pathways are tightly regulated to limit the metabolic burden. Besides the well-described role of surface polysaccharides during plant-microbe interactions, the genome of A. caulinodans encodes functions that might be involved in biofilm formation, possibly facilitating the interaction with a host. Ongoing functional analysis will undoubtedly reveal new players in the ecology of the dual lifestyle of A. caulinodans [58, 59].
Genome analysis combined with phylogenetic studies has shed new light on bacterial evolution and taxonomy. Core functions can be identified that are highly conserved between related groups, but that may acquire individual characteristics through accessory genes . Analysis of a family of core proteins and 16S rDNA sequence comparison revealed that the closest relative of A. caulinodans is Xanthobacter autotrophicus. Xanthobacter sp. are free-living nitrogen fixers and the nif and fix genes can thus be considered part of the core functions of the Azorhizobium-Xanthobacter group. The major difference in the lifestyle of both organisms is that A. caulinodans has acquired the ability to establish a symbiosis with S. rostrata.
The nodulation capacities are related to the presence of a symbiosis region with distinct GC and GC3 contents and codon usage. The association with tRNA loci, which presumably act as targets for the integration of foreign DNA, and multiple transposons suggest a high plasticity of this region, as reflected in its composition. The symbiosis region contains three subclusters related to nodulation, nod ABCSUIJZnoe CHOP, nod D, and nol K that are flanked by sequences suggestive of independent horizontal acquisition. The repeated elements could be the relics of insertion elements that once played a role in the evolution of the A. caulinodans nodulation genes that have all the characteristics of archetypal accessory genes.
To study the evolution of nod genes, A. caulinodans forms an interesting case. The azorhizobial nod genes are only distantly related to their counterparts in other rhizobia. Phylogenetic comparisons demonstrated that the nod A and nod C genes from rhizobia that nodulate temperate legumes (e.g. S. meliloti, R. leguminosarum bv. viciae and bv. trifolii, and R. galegae) are grouped together and the genes from rhizobia that nodulate tropical legumes (e.g. B. japonicum, B. elkanii, R. loti, R. tropici, and R. etli) form a second cluster [61, 62]. However, the nod A, nod B, and nod C genes of A. caulinodans belong neither to the tropical nor the temperate clusters [62, 63]. Also, the genetic distance between the nod SU genes of A. caulinodans and their counterparts in other rhizobia is much greater than the mutual genetic distance between the nod SU genes of these rhizobia . The organization of the nod ABCSUIJ genes in A. caulinodans resembles the situation in B. japonicum, but the upstream and downstream regions are different [64, 65].
At present we do not know the origin of the symbiotic genes of A. caulinodans. The Rhizobiaceae, which have been historically considered a true family in phylogenetic terms, now seem a rather diverse group of bacteria, including Methylobacterium, Ralstonia, and Burkholderia that share variant, relatively recently acquired, symbiotic gene clusters. Possibly, the A. caulinodans nod genes are derived from unexplored rhizobia or even from obligate endophytes. Undoubtedly, the recent and ongoing explosion in meta-genomic projects will provide more insight into the origin of the nodulation functions.
Extension of symbiotic nitrogen fixation to non-legume cereal plants is a challenging long-standing goal. Especially, there is a growing interest in nitrogen-fixing organisms that could establish an endophytic, beneficial relation with important crops, such as rice and wheat (Triticum aestivum). Interestingly in this context, the occurrence of A. caulinodans has been reported in intercellular infection pockets located in the cortex of roots of Arabidopsis thaliana and wheat . In fields where S. rostrata and rice are grown as rotation crops, A. caulinodans seems to survive very well in the rhizosphere of the rice plants and in the soil . Moreover, the bacterium invades emerging lateral roots of rice, and rice seedlings inoculated with A. caulinodans have a high nitrogenase activity . Finally, A. caulinodans fixes nitrogen under relatively high oxygen tension as a free-living organism, invades the host via cracks, and establishes intercellular colonies. Altogether, these features might be advantageous for primary infection of nitrogen-starved root systems and highlight the potential of A. caulinodans as a candidate model organism. The genome sequence data provide new opportunities for exploring the regulatory aspects of Azorhizobium nitrogen fixation and the essential features that implement the ability for endosymbiosis.
The nucleotide sequence of the entire genome of A. caulinodans ORS571 was determined by the whole-genome shotgun strategy method. For shotgun cloning, DNA fragments of 2 to 3 kb were cloned into the Hin cII site of pUC118. For gap closing, the pCC1Fos vector (Epicentre, Madison, WI, USA) was used, and approximately 35-kb clones were prepared. The accumulated sequence files were assembled with the Phrap program . A total of 71,424 random sequence files corresponding to approximately 7.7 genome equivalents were assembled to generate draft sequences. Finishing was carried out by visual editing of the sequences, followed by gap closing, and additional sequencing to obtain sequence data with a Phred score of 20 or higher [70, 71]. The integrity of the final genome sequence was assessed by comparing the insert length of each fosmid clone with the computed distance between the end sequences of the clones. The end sequence data facilitated gap closure as well as accurate reconstruction of the entire genome. The final gaps in the sequences were filled by the primer walking method. A lower threshold of acceptability for the generation of consensus sequences was set at a Phred score of 20 for each base. The nucleotide sequence is available in the DDBJ/EMBL/GenBank databases under the accession number AP009384.
Structural and functional annotation
Coding regions were assigned by a combination of computer prediction and similarity search. Briefly, the protein-coding regions were predicted with the Glimmer 2.02 program  and all regions equal to or longer than 90 bp were translated into amino acid sequences that were subjected to similarity searches against the nonredundant protein database at NCBI with the BLASTP program . In parallel, the entire genomic sequence was compared with those in the nonredundant protein database with the BLASTX program  to identify genes that had escaped prediction and/or were smaller than 90 bp, especially in the predicted intergenic regions. For predicted genes without sequence similarity to known genes, only those equal to or longer than 150 bp were considered as candidates. Functions were assigned to the predicted genes based on sequence similarity of their deduced products to that of genes of known function. For genes that encode proteins of 100 amino acid residues or more, an E-value of 10-20 was considered significant, whereas a higher E-value was significant for genes encoding smaller proteins (E-value treshold of 10-10). Genes for structural RNAs were assigned by similarity search against the in-house structural RNA database that had been generated based on the GenBank data. tRNA-encoding regions were predicted by the tRNAscan-SE 1.21 program  in combination with the similarity search.
MetaCyc analysis [27, 74] detected 229 metabolic pathways, containing 1037 reaction steps. To assess the presence or absence of a metabolic pathway and to decrease the likelihood of being misled by the many enzymes that are shared among multiple pathways, the analysis was emphasized on the presence of enzymes that are unique to a pathway.
Construction of a phylogenetic tree
The Maximum-likelihood tree was based on 108 core proteins of 45 α-proteobacteria  whose sequence data and annotation files were available and downloaded from the NCBI Microbial Genome Resource database . The set of core genes was determined by an all-against-all BLAST at protein level. Best reciprocal hits were selected, taking into account a cut-off value defined as 20% similarity and an overlap of at least 150 amino acids. Only proteins present in all 45 genomes as single copy were considered as "core proteins" and used to construct the phylogenetic tree. The total alignment contained 32,327 amino acids. The tree was constructed with the Phyml program  and a WAG substitution model  and 100 bootstrap replicates were run. Unless indicated otherwise, bootstraps are 100 (Figure 5A).
This work was supported by grants from the Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan and the "Geconcerteerde Onderzoeksacties" (GOA 01GA0105) of the Ghent University.
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