A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interaction with soybean and other legumes

RESEARCH ARTICLE Summary.Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5´- monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution. [Int Microbiol 2007; 10(3):169-176] Key words: Sinorhizobium fredii HH103 · Macrotyloma axillare ·pyrF · symbiotic defects · soybean · legumes Introduction In the symbiotic relationship that is established between rhizobia and their host legumes, a complex set of molecular interactions takes place during the development and maintenance of nitrogen-fixing nodules on the plant roots. Inside the nodules, rhizobia provide the plant with an assimilable source of nitrogen. In return, the legume supplies compounds that can be used by the bacteria as sources of carbon and energy [14]. Analyses of the symbiotic properties of auxotrophic rhizobial mutants are one way to identify some of the metabolic functions that are necessary to establish a successful symbiotic relationship. Symbiotically-defective auxotrophic mutants have been isolated from several fast-growing strains of rhizobia, and their effects in the nodulation process are highly variable [reviewed in 14]. Auxotrophic mutants for leucine, histidine, ornithine, aspartic acid, or cysteine induce ineffective nitrogen-fixing nodules. However, the nodules induced by Sinorhizobium meliloti mutants affected in glutamine biosynthesis are nitrogen-fixing, which indicates that this amino acid is not essential for symbiotic nitrogen fixation in alfalfa plants. Furthermore, S. meliloti auxotrophic mutants for glycine are more effective in nitrogen fixation than their parental prototrophic strain. INTERNATIONAL MICROBIOLOGY (2007) 10:169-176 DOI: 10.2436/20.1501.01.24 ISSN: 1139-6709 www.im.microbios.org *Corresponding author: A.M. Buendía-Clavería Departamento de Microbiología, Facultad de Biología Universidad de Sevilla Av. Reina Mercedes, 6 41012 Sevilla, Spain Tel. +34-954557121. Fax +34-954557830 E-mail: [email protected] §These two authors contributed equally to this work. Juan C. Crespo-Rivas,1§ Isabel Margaret,1§ Francisco Pérez-Montaño,1 Francisco J. López-Baena,1José M. Vinardell,1Francisco J. Ollero,1 Javier Moreno,2José E. Ruiz-Sainz,1Ana M. Buendía-Clavería1* 1Department of Microbiology, Faculty of Biology, University of Sevilla, Sevilla, Spain. 2Department of Cellular Biology, Faculty of Biology, University of Sevilla, Sevilla, Spain Received 9 April 2007 · Accepted 26 July 2007 A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes 170 INT. MICROBIOL. Vol. 10, 2007 Purine auxotrophs (pur mutants) of most rhizobia species are unable to effectively nodulate their host plants [7,12,14]. While adenine auxotrophs of Rhizobium leguminosarum bv. viciae are unable to nodulate pea plants, those of S. meliloti form nodules on alfalfa that fail to fix nitrogen [14]. Apurine auxotrophic mutant of Rhizobium sp. NGR234 elicited roothair curling and nodule-meristem initiation on Macroptilium atropurpureum, but infection threads (inward-facing tubular structures through which rhizobia penetrate the root) were not observed [14]. A purL mutant of S. fredii HH103 induces pseudonodules on soybean roots but it is still able to induce a few nitrogen-fixing nodules on Glycyrrhiza uralensis [7]. In contrast, a purF mutant of R. leguminosarum bv. trifolii L1 has been described to retain the capacity to induce fully developed nitrogen-fixing nodules on its legume host [24]. A pyrimidine auxotrophic R. etli mutant, unable to synthesize uracil, induces small ineffective pseudonodules on Phaseolus vulgaris (bean). Similarly, uracil auxotrophic derivatives of S. meliloti 2001 and S. fredii HH303 only induce pseudonodules on alfalfa and soybean, respectively [14]. S. meliloti Rmd201 pyrimidine auxotrophic mutants affected in carbamoyl phospate synthetase (car) and in pyrimidine biosynthesis (pyr) induce white ineffective nodules on alfalfa plants. The nodules induced by pyr mutants are more advanced than those induced by car mutants [22]. In some cases, rhizobia require a particular intermediate of a biosynthetic pathway (but not the end products) to establish successful symbiotic interactions. Studies with tryptophan auxotrophic mutants of S. meliloti suggested that anthranilate synthesis, rather than tryptophan, is required for bacteroid development in alfalfa nodules [14]. Information about the importance of the different metabolic pathways for the Sinorhizobium fredii symbiotic capacity is scarce. Interestingly, S. fredii strains show a broad host-range of nodulation, a characteristic that facilitates studies of the impact of any particular mutation on the bacterial symbiotic capacity with legumes in which determined or undetermined nodules are formed [8]. In this work, we characterized genetically and symbiotically a uracil auxotrophic mutant of S. fredii HH103 affected in the pyrF gene. Material and methods Strains, media, and genetic manipulations. Bacterial strains and plasmids used in this work are listed in Table 1. Sinorhizobium strains were grown in TY [2], yeast mannitol (YM) [23], B– [20], or MMB [1] minimal medium at 28ºC. Auxotrophic strains were plated on MMB solidified with 1.5% (w/v) agarose. Escherichia coli was cultured in Luria-Bertani (LB) medium [16] at 37ºC. When required, the media were supplemented with the appropriate antibiotics, as described by Lamrabet et al. [10], or with uracil at 12 µg/ml. Plasmids were transferred by conjugation as described by Simon [17]. Tn5-Mob mutagenesis of S. fredii strain SVQ269 was carried out using the suicide plasmid pSUP5011 [17]. Sinorhizobium plasmid profiles were obtained using the Ekhardt method, as described by Ollero et al. [13]. Those substances required by the auxotrophic mutant SVQ292 to grow in minimal media were identified as described by Beringer et al. [3]. DNA was manipulated according to the general protocols of Sambrook et al. [16]. The primer pdad2 (5′AGATTTAGCCCAGTCG 3′), located inside the IS50R of transposon Tn5-Mob, was used as a primer to sequence the rhizobial DNA adjacent to the right site of the inserted transposon in mutant strain SVQ292. The UWGCG program was used for basic DNA sequence analysis and assembly. The NCBI ORF-Finder [http://www.ncbi.nlm.nih.gov/gorf/ gorf.html] and BLAST [http://www.ncbi.nlm.nih.gov/BLAST/] programs CRESPO-RIVAS ET AL Table 1. Bacterial strains and plasmids used in this work Strains Derivation and relevant properties Source or reference Sinorhizobium fredii SVQ269 HH103 Rifr[6] SVQ292 SVQ269 pyrF::Tn5-Mob This work Escherichia coli HB101 Restriction-minus, recA background, Strr[4] Plasmids pBluescript Cloning vector, AprStratagene® pSUP202 pBR325 containing the Mob region subcloned in Sau3A, Apr, Cmr, Tcr [18] pSUP5011 Suicide plasmid carrying the Tn5-Mob transposon [19] pMUS390 pSUP202 derivative plasmid harboring about 0.5 kb of SVQ292 DNA adjacent to right of Tn5-Mob transposon This work pMUS344 Cosmid pLAFR1 carrying the pyrF gene of S. fredii HH103 This work 171 INT. MICROBIOL. Vol.10, 2007 were used for open reading frame (ORF) identification and homology searches, respectively. The ClustalW and Box-shade programs were used to align peptide sequences. Nucleotide and/or amino acid sequence data are to be found at the EMBL database as accession no. AF311321. In vivo isolation of SVQ292 mutation. The mutated gene in SVQ292 was cloned following the method described by Madinabeitia et al. [11] and was used to construct plasmid pMUS390. This plasmid harbors about 0.5 kb of the SVQ292 DNA adjacent to the IS50R of the inserted transposon. Plant assays. Nodulation assays on Glycine max (L.) Merr. cv. Williams, Cajanus cajan (L.) Millsp., Desmodium canadense (L.) DC, Macroptilium atropurpureum (Moc.& Sessé ex DC) Urb, Indigofera tinctoria (L.), and Macrotyloma axillare (E. Mey) Verdc, were carried out as described by Buendía-Clavería et al. [6]. Germinated seeds were transferred to Leonard jars containing sterilized vermiculite supplemented with Fåhraeus nutrient solution [23]. Each Leonard jar contained two plants. Each plant was inoculated with about 108bacteria and then grown for 40 days with a 16 h-photoperiod at 25ºC in the light and 18ºC in the dark [8]. Nitrogen fixation was determined by acetylene reduction assay [5]. Plant tops were dried at 80ºC for 48 h and weighted. Bacteria were isolated from surface-sterilized nodules as previously reported [10]. When required, the Fåhraeus plant nutritive solution was supplemented with uracil at 12 µg/ml. Lipo-chitin oligosaccharide (LCO) detection. Bacteria were grown in 5 ml of B–medium in the presence of the inducer naringenin (3.6 µM) and N-acetyl[14C] glucosamine. The 14C-labeled LCO were analyzed by reverse thin-layer chromatography, as described by Spaink et al.[20]. Lipopolysaccharide SDS/PAGE analysis. To investigate lipopolysaccharide (LPS) profiles, bacterial cultures were grown on solid TY medium. Bacterial cells were washed in 0.9% NaCl and pelleted by centrifugation. Treatments applied to bacterial pellet, electrophoresis of crude bacteria extracts, and the silver-staining procedures were done as described by Buendía-Clavería et al.[7]. Microscopic studies. Nodules and pseudonodules were fixed in 4% (v/v) glutaraldehyde prepared in 0.1 M cacodylate buffer, pH 7.2, for 3 h at 4°C and post-fixed in 1% OsO4(v/v) for 2 h at 4°C. Samples were dehydrated in an acetone series and embedded in Epon (epoxy embedding medium). Toluidine blue-stained semi-thin sections (0.5 µm thick) were viewed in a Leitz (Aristoplan) light microscope. Results Isolation and characterization of a S. fredii HH103 Tn 5 -Mob auxotrophic mutant.S. fredii SVQ269 (= HH103 Rifr) was subjected to Tn5-Mob mutagenesis using plasmid pSUP5011, after which transconjugant colonies were selected on TY supplemented with rifampicin and kanamycin. In order to select auxotrophic mutants, transconjugants were replica-plated on MMB solidified with agarose. One of the colonies that failed to grow in MMB was selected for further investigation (auxotrophic mutant SVQ292). SVQ292 was able to grow in MMB if the minimal medium was supplemented with uracil. In order to identify and subclone the gene mutated in SVQ292, plasmid pMUS390 was obtained as described in Material and Methods. This plasmid harbors about 0.5 kb of the rhizobial DNA adjacent to the IS50R of the Tn5-Mob inserted in mutant SVQ292. An EcoRI/XhoI 0.9-kb fragment of pMUS390 was cloned into pBluescript and about 450 bp were sequenced by using primer pdad2. Computer analysis of this sequence indicated homology with the pyrF gene of S. meliloti, which encodes an orotidine-5´-monophosphate decarboxylase (EC 4.1.1.23). This enzyme catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine5´-monophosphate (OMP) to uridine-5´-monophosphate. S. FREDII PYRIMIDINE MUTANT Int. Microbiol. Fig. 1. Toluidine blue-stained semithin sections of pseudonodule induced by the pyrF mutant SVQ292 (A) and by the parental strain SVQ269 (B) on soybean Glycine max cv. Williams. The arrows mark nodule cells full of bacteroids. (Magnification, ca. 16×) 172 INT. MICROBIOL. Vol. 10, 2007 SVQ292 only induced pseudonodules on soybean cv. Williams that did not fix nitrogen. Microscopic analysis showed that plant cells of these pseudonodules were devoid of bacteria, while many cells of nodules induced by the parental strain SVQ269 were filled of rhizobia (Fig. 1A,B). This symbiotic defect was used to construct a genomic library of the wild-type strain HH103 (in cosmid pLAFR1) and then to isolate the wild-type pyrF gene. The genomic library was transferred “en masse” to mutant SVQ292, and the tetracycline-resistant transconjugants were used to inoculate soybean cv. Williams plants. Soybean roots developed many pseudonodules and a few nitrogen-fixing nodules. All bacteria re-isolated from the effective nodules were Tcr(presence of cosmid pLAFR1), Nmr, and Kmr(presence of Tn5Mob) and contained the same cosmid. This cosmid, named pMUS344, was isolated, introduced into E. coli HB101, and then re-transferred to the pyrF mutant SVQ292. Strain SVQ292 (pMUS344) was able to grow in MMB without uracil and formed normal nitrogen-fixing nodules on soybean. A 1.5-kb PstI fragment of cosmid pMUS344, which positively hybridized to the 0.9-kb EcoRI/XhoI fragment of plasmid pMUS390, was subcloned into pBluescript. The 1511 bp sequence appears in the EMBL nucleotide data library as accession number AF311321. By matching this sequence with that obtained by partial sequencing of pMUS390 using the IS50R primer pdad2, it was possible to locate the insertion of the transposon Tn5-Mob at nucleotide 955 of the 1511-bp sequence. The sequenced fragment comprised one complete and two partial ORFs, each with the same polarity and with a high probability of encoding proteins, as indicated by the Testcode algorithm. The left side of the sequenced fragment contained the 3´ fragment of a putative ORF that is 87.5% identical to smc00413, a putative S. meliloti gene of unknown function (GenBank accession no. AL591783). The putative protein encoded by this HH103 ORF was 66% identical to protein AGR_C_57 of Agrobacterium tumefaciens C58 (NP_353329) and contained the conserved domain GpmB, which is characteristic of fructose 2,6 biphosphatases (EC 3.1.3.46). The second ORF was 85% identical to the pyrF gene of S. meliloti. This ORF began at position 397 and extended for 702 bp, encoding a deduced polypeptide of 234 amino acids with a predicted molecular mass of 24.5 kDa. In mutant SVQ292, the Tn5-Mob transposon was inserted 558 nucleotides after the start codon of the pyrF gene. At position –79/–66 there is a putative GGN10GC motif (GGcaatctc tttGC), described as a promoter [21]. The percentages of identity between the S. fredii HH103 PyrF protein and those of different rhizobia and other gram-negative bacteria are shown in Table 2. In addition, alignment of the PyrF proteins of S. fredii HH103 and different rhizobial strains is shown in Fig. 2. The final 175 nucleotides (positions 1323–1511) corresponded to the 5´ region of a putative ORF that is 82% identical to the S. meliloti gene smc00411, which encodes a hypothetical protein of 95 amino acids. In the S. fredii sequence, the intergenic region between pyrF and sm00411 spans 221 CRESPO-RIVAS ET AL Table 2. Comparison of HH103 PyrF protein with PyrF proteins from different bacteria Bacterial strains Identity (%) Similarity (%) Length of the PyrF protein (amino acids) Amino-acid overlap Accession number Sinorhizobium meliloti 86 94 234 233 CAC41769 Sinorhizobium medicae 83 91 232 231 ABR62357 Mesorhizobium sp. BNC1 79 89 249 223 ZP_00194064 Rhizobium leguminosarum bv. viciae 79 89 235 228 CAK05821 Rhizobium etli 78 89 235 228 ABC89136 Agrobacterium tumefaciens 77 88 255 225 AAK86113 Mesorhizobium loti 74 87 237 225 BAB51336 Brucella suis 71 85 238 227 AAN31018 Brucella melitensis 70 85 259 227 AAL53180 Bartonella bacilliformis 68 84 235 225 Q44843 Bradyrhizobium japonicum 54 72 237 229 CAB91880 Vibrio cholerae 40 60 231 215 AAF95059 Pseudomonas aeruginosa 38 57 232 231 CAA46564 Escherichia coli 36 55 245 227 NP_415797 173 INT. MICROBIOL. Vol.10, 2007 nucleotides, instead of the 106 nucleotides present in the corresponding region of S. meliloti. Symbiotic properties of mutant SVQ292 on several legumes. S. fredii HH103 is considered a broad host-range nodulation strain. Thus, we studied whether the mutation in pyrF could affect the symbiotic capacity of HH103 with different legumes, as it did for soybean. Mutant SVQ292 formed pseudonodules with Glycine max cv. Williams, M. atropurpureum, I. tinctoria, and D. canadense, and ineffective nodules with C. cajan. Inoculation of M. axillare with mutant SVQ292 did not induce any macroscopic root response. Plant tests were carried out to determine whether the presence of uracil in the plant nutritive solution would compleS. FREDII PYRIMIDINE MUTANT Int. Microbiol. Fig. 2. Alignment of the PyrF proteins of different rhizobia. Sf, Sinorhizobium fredii; Sm, S. meliloti; Sme, S. medicae; Msp, Mesorhizobium sp.; Rlv, Rhizobium leguminosarum bv. viciae; Re, Rhizobium etli; At, Agrobacterium tumefaciens; Ml, Mesorhizobium loti. 174 INT. MICROBIOL. Vol. 10, 2007 CRESPO-RIVAS ET AL ment the symbiotic defects of mutant SVQ292. The presence of uracil did not enhance the symbiotic capacity of SVQ292 with any of the legumes tested. Since SVQ292 was symbiotically defective, we decided to investigate whether this mutant was altered either in symbiotic signals (such as LCO and LPS) or in its plasmid profile. The LCO and LPS profiles produced by SVQ292 were similar to those observed for the parental strain SVQ269, as demonstrated by thin-layer chromatography and SDS-PAGE, respectively (data not shown). However, plasmid agarose electrophoresis showed that, apparently, SVQ292 lacked a cryptic plasmid of about 85 MDa (Fig. 3). Discussion Purine and pyrimidine auxotrophic rhizobial mutants are usually unable to nodulate their host plants or to induce nitrogenfixing nodules [7,14]. Vineetha et al. [22] obtained different pyrimidine auxotrophs of S. meliloti that induce white ineffective nodules on alfalfa plants. These reports suggest that the expression of some purine and pyrimidine biosynthetic genes is essential for an effective symbiosis of rhizobia with host plants. We cloned and sequenced a 1.5-kb PstI DNA fragment that contained the pyrF gene of S. fredii HH103. This DNA fragment harbored three ORFs (5´ sm00413-pyrF-smc00411 3´) with the same organization as the corresponding ORFs of S. meliloti. The only difference detected was in the intergenic region between pyrF and smc00411. In S. meliloti, this region spans 106 nucleotides. Of these, only the six immediately after pyrF and the 54 preceding smc00411 are conserved in HH103. Moreover, between these two conserved fragments, HH103 harbors 161 additional nucleotides without homology to any known DNA sequence. In M. loti, a gene homologous to smc00411 is located 17 nucleotides after the end of pyrF. However, the mrl7505 gene (NC_002678), which is homologous to smc00413, is at a distance of 3000 kb from the other two genes. In this work, we found that strain SVQ292, a pyrF mutant of S. fredii HH103 that requires uracil to grow, induced Fix– pseudonodules on plants—including Glycine max cv. Williams and even Macrotyloma axillare—that were effectively nodulated by the parental strain. These results suggested that in S. fredii HH103 the pyrF gene, which encodes orotidine-5´-monophosphate decarboxylase, is essential for the establishment of an effective symbiosis between the bacterium and its hosts. The symbiotic impairment with M. axillare should occur at the very early stages of nodulation, since neither pseodunodules nor any other macroscopic root responses to the presence of SVQ292 were observed. Pseudonodules induced by SVQ292 on Williams soybean were devoid of bacteria (Fig. 1A). Similarly, pyrimidine auxotrophs of S. meliloti 2011 induced white ineffective nodules in alfalfa plants. The alfalfa nodules induced by S. meliloti pyrE/pyrF mutants are more developed than those induced by a pyrC mutant, and the latter are more advanced than those induced by car mutants [22]. In the pyrimidine biosynthetic pathway, enzymes encoded by car genes act earlier than Pyr enzymes. All these findings indicated that the pyrimidine biosynthetic pathway, or a particular intermediate, is essential for nodule morphogenesis. Also, the impaired nodule development caused by the auxotrophy varies according to the position at which the biosynthetic pathway is blocked. The symbiotic defect of SVQ292 was not reverted by the addition of uracil to the nutritive plant solution. Similar results were described for pyrimidine auxotrophic mutants of R. etli CFN42, S. fredii HH303, and S. meliloti Rmd2, in which the addition to the plant nutrient medium of uracil or any other intermediate in the biosynthetic pyrimidine pathway does not restore the symbiotic defects [14,22]. However, in different rhizobial purine auxotrophs, the addition of AICA-riboside (5-aminoimidazole-4-carboxamide 1-β-Dribofuranoside, an intermediate in the biosynthetic purine pathway) to the plant nutritive solution significantly enhances nodule development [7,12,14]. This is also the case Int. Microbiol. Fig. 3. Plasmid profiles of S. fredii SVQ269 and its pyrF derivative SVQ292. The indicated molecular masses of the plasmids are approximate. 175 INT. MICROBIOL. Vol.10, 2007 of other auxotrophic rhizobial mutants that induce ineffective nodules but are able to effectively nodulate their host plants if the auxotrophic requirement is added to the plant nutritive solution. For example, histidine auxotrophs of Bradyrhizobium japonicum and S. meliloti and a riboflavin-requiring mutant of R. leguminosarum induce nitrogen-fixing nodules on their host plants only when the nutritive plant solution is supplemented with the required growth factor [14]. SVQ292 lacked a cryptic plasmid of about 85 MDa (Fig. 3) but this was not the cause of the mutant’s symbiotic defect because SVQ292 harboring cosmid pMUS344, which contained the wild-type pyrF gene, induced nitrogen-fixing nodules on soybean. These results suggested that the cryptic plasmid cured in mutant SVQ292 is not essential for the establishment of an effective symbiosis between S. fredii HH103 and soybean. Nodulation factors (LCO) and rhizobial surface polysaccharides act as signal molecules in the nodulation process. Rhizobial mutants that do not produce LCO are totally unable to initiate nodule formation [10,20]. Many rhizobial mutants affected in the production of bacterial surface polysaccharides form ineffective pseudonodules that are devoid of bacteria [14]. Since a leucine auxotrophic mutant of S. meliloti is unable to produce LCO [15] and a purine auxotrophic mutant of S. fredii has an altered LPS profile [7], we investigated whether mutant SVQ292 produced normal LCO and LPS profiles. Indeed, the the LCO and LPS profiles produced by SVQ292 were similar to those produced by its wild-type parental strain HH103. Thus, the symbiotic impairment of SVQ292 was not apparently due to alterations in these two symbiotic signals. Although the possible symbiotic role of amino acids, purines, and pyrimidines is not clear, a recent report provided new insights indicating that purines and pyrimidines, or any intermediary of their biosynthetic pathways, play an important role in triggering nodule formation [9]. Photosynthetic Bradyrhizobium sp. strain ORS278 does not require LCO production for its symbiotic interaction with the legume Aeschynomene sensitiva. However, different mutants affected in the biosynthesis of purines and a strain ORS278 pyrF mutant only formed a few pseudonodules on this legume. Thus, purines and pyrimidines are essential for the formation of nodules, regardless of whether the mechanism of nodule organogenesis requires the presence of bacterial nodulation factors. Acknowledgements. This work was supported by the Spanish Ministry of Education and Science grants BIO2005-08691-C02-02 and AGL2006-13758-C05-03/AGR. We are grateful to Ahmed Moussaid, Rocío Gutiérrez, Ángeles Hidalgo, and Remedios García-Navarro for technical assistance. References 1. Bergensen FJ (1961) The growth of Rhizobium in synthetic media. Aust J Biol Sci 14:349-360 2. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188-198 3. Beringer JE, Ruiz-Sainz JE, Johnston AWB (1984) Methods for the genetic manipulation of Rhizobium. In: Grainger JM, Lynch JM (eds) Microbiological methods for environmental biotechnology. Academic Press, New York, pp 79-94 4. Boyer HW, Roulland-Dossoix D (1969) Complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459-472 5. Buendía-Clavería AM, Ruiz-Sainz JE, Cubo-Sánchez T, Pérez Silva J (1986) Studies of symbiotic plasmids in Rhizobium trifolii and fastgrowing bacteria that nodulate soybean. J Appl Bacteriol 17:155-160 6. Buendía-Clavería AM, Chamber M, Ruiz-Sainz JE (1989) A comparative study of the physiological characteristics, plasmid content and symbiotic properties of different Rhizobium fredii in European soils. Syst Appl Bacteriol 17:55-160 7. Buendía-Clavería AM, Moussaid A, Ollero FJ, et al. (2003) A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide. Microbiology 149:1807-1818 8. de Lyra MCCP, López-Baena FJ, Madinabeitia N, et al. (2006) Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases symbiotic capacity with soybean. Int Microbiol 9:125-133 9. Giraud E, Moulin L, Vallenet D, et al. (2007) Legumes symbioses: absence of nod genes in photosynthetic bradyrhizobial. Science 316:1307-1312 10. Lamrabet Y, Bellogín RA, Cubo T, et al. (1999) Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans. Mol PlantMicrobe Interact 12:207-217 11. Madinabeitia N, Bellogín RA, Buendía-Clavería A, et al. (2002) Sinorhizobium fredii HH103 has a truncated nolO gene due to a –1 frameshift mutation that is conserved among other geographically distant S. fredii strains. Mol Plant-Microbe Interact 15:150-159 12. Newman JD, Rosovitz MJ, Noel KD (1995) Requirement for rhizobial production of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) for infection of bean. Mol Plant-Microbe Interact 8:407-414 13. Ollero FJ, Espuny MR, Bellogín RA (1989) Mobilization of the symbiotic plasmid from a strain of Rhizobium sp. (Hedysarum coronarium). System Appl Microbiol 11:217-222 14. Randhawa GS, Hassani R (2002) Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in symbiosis. Indian J Exp Biol 40:755-764 15. Sanjuán-Pinilla JM, Muñoz S, Nogales J, Olivares J, Sanjuán J (2002) Involvement of the Sinorhizobium meliloti leuA gene in activation of nodulation genes by NodD1 and luteolin. Arch Microbiol 178:36-44. 16. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Lab Press, Cold Spring Harbor, NY 17. Simon R (1984) High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-Mob transposon. Mol Gen Genet 196:413-420 18. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784-791 S. FREDII PYRIMIDINE MUTANT 176 INT. MICROBIOL. Vol. 10, 2007 19. Simon R, Quandt J, Klipp W (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions, and induction of genes in gram-negative bacteria. Gene 80:161-169 20. Spaink HP, Aarts A, Stacey G, Bloemberg GV, Lugtenberg BJJ, Kennedy EP (1992) Detection and separation of Rhizobium and Bradyrhizobium Nod metabolites using thin-layer chromatography. Mol Plant-Microbe Interact 5:72-80 21. Strych U, Wohlfarth S, Winkler UK (1994) Orotidine-5´-monophosphate decarboxylase from Pseudomonas aeruginosa PAO1: cloning, overexpression, and enzyme characterization. Curr Microbiol 29: 353-359 22. Vineetha KE, Vij N, Prasad CK, Hassani R, Randhawa GS (2001) Ultrastructural studies on nodules induced by pyrimidine auxotrophs of Sinorhizobium meliloti. Indian J Exp Biol 39:371-377 23. Vincent JM (1970) The modified Fåhraeus slide technique. In: Vincent JM (ed) A manual for the practical study of root nodule bacteria. Appendix III. Blackwell Scientific Pub, Oxford, pp 144-145 24. Worland S, Guerreiro N, Yip L, Djordjevic MA, Djordjevic SP, Weinman J, Rolfe BG (1999) Rhizobium purine auxotrophs, perturbed in nodulation, have multiple changes in protein synthesis. Aust J Plant Physiol 26:511-519 CRESPO-RIVAS ET AL