Microbes Environ. Vol. 26, No. 2, 165–171, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10213

Identification of Genes Relevant to Symbiosis by Using Signature-Tagged Mutants

NAOGANCHAOLU BORJIGIN1, KEISUKE FURUKAWA1, YOSHIKAZU SHIMODA2, SATOSHI TABATA2, SHUSEI SATO2, SHIMA EDA1, KIWAMU MINAMISAWA1, and HISAYUKI MITSUI1* 1Graduate School of Life Sciences, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan; and 2Kazusa DNA Research Institute, 2–6–7 Kazusa-kamatari, Kisarazu 292–0818, Japan

(Received December 30, 2010—Accepted March 2, 2011—Published online March 29, 2011)

Signature-tagged mutagenesis was applied to Mesorhizobium loti, a nitrogen-fixing root-nodule symbiont of the leguminous plant japonicus. We arranged 1,887 non-redundant mutant strains of M. loti into 75 sets, each consisting of 24 to 26 strains with a different tag in each strain. These sets were each inoculated en masse onto L. japonicus plants. Comparative analysis of total DNA extracted from inoculants and resulting nodules based on quantitative PCR led to the selection of 69 strains as being reduced in relative abundance during nodulation. Plant assays conducted with individual strains confirmed that 3 were defective in nodulation (Nod−) and that 10 were Nod+ but defective in (Fix−); in each case, the symbiosis deficiency could be attributed to the transposon insertion carried by that strain. Although the remaining 56 strains were Fix+, 33 of them showed significantly reduced competitiveness during nodulation. Among the mutants we identified are known genes that are diverse in predicted function as well as some genes of unknown function, which demonstrates the validity of this screening procedure for functional genomics in . Key words: Mesorhizobium loti, signature-tagged mutagenesis, symbiosis, competitiveness,

Rhizobia are a group of soil that establish a capacity and metabolic versatility. A number of genes have nitrogen-fixing symbiosis with leguminous plants. been assigned to functions required for symbiosis, such as Mesorhizobium loti is an α-proteobacterial species of the the biosynthesis of Nod factors, cell-surface polysaccharides, group and a symbiotic partner of Lotus japonicus; this and nitrogenase machinery, and to functions required for symbiotic pair is a well-documented model system for diverse metabolic pathways. Nevertheless, many of the genes molecular genetic studies (20, 31). Some rhizobia, including predicted from genome sequence data still need to be exam- M. loti, elicit root-hair curling and nodule organogenesis on ined for their functions, including their potential roles in roots of host plants at an early step during symbiosis. symbiosis. Functional genomic approaches are needed to Such plant responses are triggered specifically by signaling achieve breakthroughs in rhizobia research in this post- compounds, called Nod factors, produced by compatible genomics era. rhizobia. Rhizobia colonize curled root hairs and invade Signature-tagged mutagenesis (STM) is a technique for developing nodules via infection threads, which are formed identifying mutants that fail to grow or survive under a by invagination of the root-hair cell membrane. The rhizobia particular selection condition. It employs a set of trans- are then released into the host nodule cells (33, 42). posons, each equipped with a tag signature consisting of a These intracellular bacteria (bacteroids) reduce dinitrogen unique DNA sequence. When a set of mutants having inser- into ammonia by use of a carbon and energy source origi- tions with these tagged transposons is passed en masse nating from photosynthates. Host plants are supplied with through a selection condition, individual mutants can be the fixed nitrogen and thus are able to grow under distinguished from others by their unique tags, thus making nitrogen-poor conditions. In a free-living state, rhizobia can it possible to monitor changes in relative abundance during metabolize a wide range of carbon and nitrogen sources. the selection procedure. STM was originally developed with This metabolic versatility allows rhizobia to adapt to the Salmonella enterica serovar Typhimurium; in the original nutritional complexity of the rhizosphere, which is affected experiments, the transposon mutants were detected by by plant root exudates (14, 15, 25, 36). hybridization (12). STM has since been applied to a number Whole genome sequences have been determined for of pathogenic or symbiotic bacteria (for a review, see several rhizobial species; their genomes range in size from reference 27). An STM library was constructed for the 5.4 Mb (Azorhizobium caulinodans), 6.7 Mb (Sinorhizobium symbiotic bacterium S. meliloti by using 412 transposons meliloti) and 7.6 Mb (M. loti) to 8.5 Mb (Bradyrhizobium sp. with unique tags that can be detected by hybridization to BTAi1) and 9.1 Mb (Bradyrhizobium japonicum) (8, 10, microarrays (34). A pilot screening of this library showed 16, 17, 22). These large sizes can be explained by the need that a number of genes relevant to symbiosis or competition to accommodate the many genes responsible for symbiotic could be identified (35). A modified STM technique was developed to use real-time PCR to detect each transposon * Corresponding author. E-mail: [email protected]; mutant, with the advantage that a PCR-based procedure can Tel: +81–22–217–5685; Fax: +81–22–217–5685. decrease the occurrence of false positives resulting from 166 BORJIGIN et al. cross-hybridization between different tags (13). By using a template DNA, 15 pmol of each primer, 0.75 μL of dimethylsul- library arranged in sets containing 37 differently tagged foxide, and 7.25 μL of SYBR Premix ExTaq (Takara Bio, Otsu, mutants each, the authors successfully selected Burkholderia Japan), using an iCycler iQ Real-Time Detection system (Bio-Rad cenocepacia mutants that were attenuated for survival Laboratories, Hercules, CA, USA). The cycling conditions con- sisted of an initial 1 min at 94°C; 35 cycles at 94°C for 30 s, 60°C during lung infection in rats (13). We considered whether for 30 s, and 72°C for 15 s; and a final 10 min at 72°C. The reaction this technique could be effectively applied to the systematic mixture was then measured to determine the melting temperature of mutagenesis of M. loti. double-stranded DNA. Strains that showed a larger cycle number In this study, we used an STM library constructed by by more than 0.5 with the nodule DNA than with the inoculant random mutagenesis with tagged transposons (41); the tags DNA (measured at the time when fluorescence indicated exponen- used were developed by Hunt and associates (13). The tial amplification) were classified as mutants reduced in relative abundance during nodulation. genomes of 7,892 mutants from the library were mapped for We expected the DNA extracted from nodules to be contami- transposon insertions (41). Here, we report the application of nated with plant DNA. To test whether this possible contamination the PCR-based STM strategy in combination with a plant had any detectable effect on the PCR amplification from nodule nodulation assay to screening for M. loti mutants altered in DNA, we performed a real-time PCR experiment in which template symbiosis or competitive interactions. DNA was mixed with DNA extracted from nodules occupied by ML001 alone to see whether the amplification was attenuated quan- titatively according to the proportion of template DNA. The results Materials and Methods indicated that any contamination by plant DNA had no detectable effect on the assay of bacterial DNA from nodules (data not shown). Mesorhizobium loti strains and culture conditions The signature-tagged strains to be screened in this study Acetylene reduction assay were selected from a mutant library described previously (41). Whole roots from an L. japonicus plant at 42 to 45 days post- Additional signature-tagged strains were provided by the National inoculation were cut from the plant and transferred into a 18-mL BioResource Project (L. japonicus and G. max). Strain ML001, bottle, and a rubber stopper was inserted. The bottle was injected which we used as a control, is a streptomycin-resistant derivative of with 1.8 mL of acetylene gas and incubated at 25°C. At 0, 1, and 2 h the wild-type strain MAFF303099 (18). All the M. loti strains were after the injection in the case of the mutants, or at 0, 10, and 20 min grown aerobically at 30°C in tryptone-yeast (TY) medium, which in the case of ML001, approximately 0.7 mL of gas was removed contained (per liter) tryptone (5 g), yeast extract (3 g), CaCl2·2H2O from the bottle, and 0.5 mL was analyzed for the amount of (0.83 g), and agar (15 g, if needed) (pH 7.2). For the auxotrophy ethylene by using a gas chromatograph (GC-18A, Shimadzu, assay, we used M9 medium containing (per liter) glucose (2 g), Kyoto, Japan) equipped with a Porapak N column (mesh size, 80– CoCl2·6H2O (10 μg) and agar (15 g) (39); adenine, inosine, uridine, 100; length, 2 m; Waters, Milford, MA, USA) and a flame ioniza- serine, or methionine was added at a final concentration of 0.2 tion detector. The flow rate of nitrogen carrier gas was 60 mL min−1, mM. When necessary for strain selection, antibiotics were added at and the injector and column temperatures were 100°C and 40°C, the following concentrations: streptomycin, 100 μg mL−1; spectino- respectively. Under these conditions, ethylene typically eluted after mycin, 100 μg mL−1; and neomycin, 100 μg mL−1. 2.75 min. Plant nodulation assays Genetic techniques and DNA manipulations Seeds of L. japonicus B-129 Gifu were scarified with sandpaper, Plasmids were conjugatively transferred into M. loti strains by surface-sterilized in 1% (w/v) sodium hypochlorite solution, triparental mating using E. coli strain MT607 containing the helper immersed overnight in sterilized water, and germinated for 4 days plasmid pRK600 (6). Recombinant DNA techniques were on a 1% (w/v) agar plate. Ten seedlings were transferred onto performed according to standard protocols (39). sterilized vermiculite soaked with B&D medium (2) in an assembly of 300-mL plastic jars (Iwaki Scitech, Tokyo, Japan) that followed Generation of a reference strain for assay of competitive nodulation the design devised by Leonard (23). Mesorhizobium loti strains We amplified a 1.9-kb DNA fragment containing a translational were grown individually in liquid media and then combined if fusion of aphII and gusA from pCAM120 (44) by performing PCR required for the experiment. The cells were collected, washed with with primers 5'-CCGCTCGAGGATCGTTTCGCATGATTGAAC- sterilized water and resuspended in sterilized water to an optical 3' and 5'-ACGCGTCGACTCATTGTTTGCCTCCCTGCTGCG-3'. density at 660 nm of 0.05. The suspension (1 mL) was added to the (The underlined sequences denote XhoI and SalI sites, respectively, planted jar, which was then placed in a growth chamber (Koito, which were added for the convenience of cloning.) The amplified Tokyo, Japan) with a regime of 16 h illumination at 25°C and 8 h fragment was cloned into the SalI site of pK18mob (40), in the same darkness at 20°C. The photosynthetically active radiation on the orientation as lacZα, to produce pFK7. We also amplified a 370-bp rack in the growth chamber was 80 μmol m−2 s−1. DNA fragment, which is located 22 bp upstream of the translational start site of mlr5905 (nifH), by PCR from DNA of cosmid no. PCR-based screening of M. loti mutants 232, derived from the ordered genomic library of M. loti (11). Total DNA of M. loti cells grown before plant inoculation was Primers used for the PCR were 5'-CGCGAATTCCAATGATGG prepared as described previously (1), after the cultures in each set TAACTTCCATG-3' and 5'-CGCGGATCCATAGCGGTTACCGCG were combined. To extract total DNA of M. loti cells in nodules, ACTTC-3'; the underlined sequences denote EcoRI and BamHI we collected 131 to 323 nodules (0.26 to 0.77 g total wet weight) sites, respectively. We cloned this PCR product into pFK7 and from 20 plants in two jars at 2 months after inoculation. We crushed transferred the resulting plasmid into ML001 by conjugation. A the nodules in a homogenizer at room temperature, and extracted resulting neomycin-resistant transconjugant was designated strain DNA by using a DNeasy Plant Maxi kit (Qiagen, Hilden, Germany) BN3 and used as a reference for plant assays. according to the manufacturer’s instructions. Real-time PCR was conducted using either inoculant or nodule DNA as a template and Determination of nodule occupancy in dually inoculated plants each of 26 primer pairs consisting of a unique forward primer and We sampled 96 nodules larger than 1 mm in diameter from 10 to a common reverse primer that were previously described; a forward 12 plants that were grown for 2 months after inoculation with an primer to detect the 27th tag (no. 39) was not used (41). The reac- equal mixture of a test strain and BN3. Nodules produced after tion was performed in a 15-μL solution containing 50 ng of inoculation with an equal mixture of ML001 and BN3 at the Screening of Mesorhizobium loti Mutants by STM 167 same time were used as the control. The nodules were cut into a cosmid carrying a wild-type DNA region that covers the halves with a razor blade and immersed in a solution containing 5- transposon insertion site in that strain, confirming that the bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt −1 −1 insertions are each attributable to the symbiotic deficiency. (X-Gluc) (50 mg L ), sodium lauryl sulfate (2 g L ), methanol Ten of the 13 strains elicited small white nodules (Nod+), (20%, v/v), and sodium phosphate (20 mM, pH 7.0) on a 96-well − microtiter plate. After overnight incubation at 30°C, the nodules and the other three elicited no appreciable nodules (Nod ) + − were checked by eye for the presence of blue. The difference in the (Table 1). The symbiosis deficiency of one of the Nod Fix number of non-blue nodules between a test strain and ML001 was strains (31T03g03) was leaky: a large pink nodule was evaluated by chi-square test. formed on some of the inoculated plants together with a number of small white nodules. We have not yet tested Results whether the leakiness in 31T03g03 is specific to this strain or a general characteristic of mutants in mll4296. Screening of signature-tagged mutants through a plant assay The involvement of some M. loti genes in symbiosis We screened 1,887 non-redundant mutant strains from the or nitrogen fixation has been documented. mlr5909 (nifN) defined-mutant library (41) in 75 sets, each consisting of 24 appears to be necessary for the synthesis of the iron- to 26 differently tagged strains (supplementary Table S1). molybdenum cofactor of nitrogenase (16, 38). mll3875 The component strains were grown individually, combined shows 95% identity in deduced amino acid sequence to for each set, and inoculated onto L. japonicus plants. By serA, which is required for serine bio- assessing the abundance of each strain in the inoculum and in synthesis as well as symbiotic nitrogen fixation in the chick- the nodules with quantitative real-time PCR, we were able to pea host plant (5). Strain 02T02g01 was confirmed to be a detect the difference in proportion of each strain between the serine auxotroph (data not shown). mlr5265 encodes a homo- inoculant culture and the nodules. log of UDP-glucose dehydrogenase, which is 45% identical On the basis of this screen, we selected 69 strains as ones to S. meliloti RkpK (SMc02641). This enzyme catalyzes the that were reduced in relative abundance during nodulation oxidation of UDP-glucose to UDP-glucuronic acid, and its compared with the initial inoculant. We then inoculated the mutation affects the production of surface polysaccharides strains singly onto L. japonicus plants to examine whether containing glucuronic acid, such as extracellular polysaccha- any of them were deficient in symbiotic nitrogen fixation rides, capsular polysaccharides and lipopolysaccharides (Fix− phenotype). (19). This defect results in the symbiotic phenotype impaired in infection thread elongation as well as nodule Mutants with phenotypes of ineffective symbiosis formation for Rhizobium leguminosarum (21). mlr0384 Thirteen out of the 69 strains caused poor growth of plants (pdhAβ) and mlr0385 (pdhB) encode homologs of the E1 and compared with the wild-type strain ML001 on nitrogen- E2 components, respectively, of the pyruvate dehydrogenase free medium, indicating that they are Fix− (supplementary complex. The gene encoding a homolog of E3, mlr0388, Fig. S1). Each of the strains was complemented to Fix+ by is located downstream of those genes, raising the possibility

Table 1. Mesorhizobium loti mutants with phenotypes of ineffective symbiosis Acetylene reduction a Mutated gene c Deduced gene product Strain activityb Cosmid Nod−Fix− strains 03T01d01 n.d.d mll7962 320.1 PurM; 5'-phosphoribosyl-5-aminoimidazole synthetase 18T02f09 n.d. mlr5498 217 PheA; prephenate dehydratase 34T01b05 n.d. mlr5265 206.1 RkpK; UDP-glucose dehydrogenase Nod+Fix− strains 02T02g01 <0.5 mll3875 157 SerA; 3-phosphoglycerate dehydrogenase 20T02a09 <0.5 mlr5909 232 NifN; nitrogenase FeMo-co biosynthesis protein 02T03c03 1.4±1.5 (0.6%) mll7833 316 PurF; amidophosphoribosyltransferase 02T01b04 3.1±0.5 (1.4%) mll5838 230 DNA-binding protein 29T03b01 2.5±1.8 (1.1%) mlr0384 14.1 PdhAβ; pyruvate dehydrogenase E1 beta subunit 29T02e09 5.9±1.7 (2.6%) mlr0385 14.1 PdhB; pyruvate dehydrogenase E2 subunit 34T04f07 3.2±2.1 (1.4%) mlr8350 335.1 GuaB; inosine 5'-monophosphate dehydrogenase 29T03d06 43.0±16.6 (19%) mll0920 39 Protein of unknown function 34T01d10 68.8±17.5 (30%) mll1587 68.1 MetF; 5,10-methylenetetrahydrofolate reductase 31T03g03 n.d. mll4296 171 Lpd; E3 subunit of both 2-oxoglutarate and pyruvate dehydrogenases a The location of the transposon insertion for each strain can be retrieved from RhizoGenes2 (http://bacteria.kazusa.or.jp/rhizo_legacy/ Mesorhizobium/genes2/). All the strains can be obtained from the National BioResource Project (L. japonicus and G. m ax) at the University of Miyazaki. b Values are the mean±SD in nanomoles of ethylene evolved per hour per gram (fresh weight) of nodules from at least three measurements. The value for strain ML001 (wild type) was 225.7±15.6; percentage of that for ML001 is indicated in parentheses. c Numbers are IDs of cosmid clones that each complemented the mutants to Fix+. Those clones were selected from the ordered genomic library of M. loti MAFF303099, and information about the DNA fragment carried by each clone can be retrieved from the Home Page of lotus-loti (http://miya.bio.sci.osaka-u.ac.jp/cosmid-lb.html). d Not determined 168 BORJIGIN et al. that its expression is also affected by insertion mutations. mll1587 might slow a broad range of reactions in cellular Expression of the pdhAB genes in S. meliloti is enhanced metabolism such as the biosynthesis of amino acids, purine, in the symbiotic state, indicating a key role for carbon meta- and pantothenate. Strain 34T01d10 was confirmed to be a bolism in bacteroids (3). methionine auxotroph (data not shown). The three genes mll7962 (purM), mll7833 (purF), and We measured the acetylene reduction activity of nodules mlr8350 (guaB) are predicted to be involved in purine elicited by the Nod+Fix− strains, with the exception of the biosynthesis. The gene located downstream of mll7962, leaky one. Whereas the growth and nodulation of the inocu- mll7961 (purN), is predicted to be the same and could be lated plants were similar among the Nod+Fix− strains, the under the polar effect of the insertion mutation. In fact, none acetylene reduction activity roughly divided the plants into of the mutant grew in minimal medium (data not shown). two classes (Table 1). One class included the mlr5909, Nevertheless, they showed differences in both growth and mll3875, mll7833, mlr0384, mll5838, mlr8350, and mlr0385 symbiosis. Supplementation with adenine or inosine allowed mutants, whose activity (per fresh weight of nodules) ranged the mutant in mll7833 or mlr8350 to grow in minimal from undetectable to 2.6% of that of ML001. The markedly medium but not the mutant in mll7962 (data not shown). For reduced levels of activity account directly for the poor symbiosis, the former mutants elicited numerous ineffective growth of host plants on nitrogen-free medium. The other nodules, whereas the latter mutant elicited no discernible class included the mll0920 and mll1587 mutants, whose nodules; the growth defect appears to parallel the severity of activity (per fresh weight of nodules) corresponded to 19% symbiotic deficiency. and 30%, respectively, of that of ML001. The activities mll1587 (metF) encodes a homolog of methylenetetra- per plant, however, were approximately 5% and 10%, hydrofolate reductase, which is involved in the flux of one- respectively, of that of ML001, as the nodule weight per carbon units in the cell (26). Therefore, the mutation in plant of the Fix− strains was less than 30% of that of ML001

Table 2. Mesorhizobium loti mutant strains reduced for competitiveness

Relative nodule a Mutated gene Deduced gene product Strain occupancy (%)b 01T02b08 mlr7576 NodQ; bifunctional sulfate adenylyltransferase/adenylylsulfate kinase 16 02T03a06 mll5232 Sarcosine oxidase beta subunit 28 03T01c10 mlr2565 RfaD; ADP-L-glycero-D-mannoheptose-6-epimerase 19 06T02c07 mlr0378 Eno; enolase 49 06T02d05 mlr3523 Putative cell division protein FtsX 12 06T02h06 msl0067 PurS; phosphoribosylformylglycinamidine synthase 28 09T01h01_2 mlr1231 Corrinoid methyltransferase 2 10T01c04 mll7468 Protein of unknown function 18 10T01g06 mll5681 PyrD; dihydroorotate dehydrogenase 54 10T01h09 mll5942 Protein of unknown function 4 11T01d06 mll6127 Outer membrane protein of unknown function 28 11T01e05 mll5345 Dut; deoxyuridinetriphosphatase 2 11T02g07 mlr5848 NodZ; nodulation protein 0 17T01b09 mll8451 Protein similar to streptomycin phosphotransferase 25 20T02e04 mll0053 Protein of unknown function 4 22T01h04_2 mlr7269 Putative phosphoglycerate dehydrogenase 11 22T02a11 mll1244 Protein of unknown function 25 25T02a07 mlr3298 PolA; DNA polymerase I 18 25T02c02 mll9385 Putative oxidoreductase 18 27T01g08 mlr5272 ExoA; glycosyltransferase 20 27T02d11 mll3253 Protein of unknown function 39 27T02f02 msl7460 Protein of unknown function 35 29T02c09 mll2796 Protein of unknown function 7 36T02c04 mll1088 Protein of unknown function 16 36T02c05 mll1455 HflK; membrane-bound protease 29 38T01b08 mll2811 Protein of unknown function 35 38T01c09 mlr2436 Putative oxidoreductase 12 38T01e04 mlr0937 PhbC (PhaC); poly-β-hydroxybutyrate synthase 8 40T02g09 mlr7575 NodP; sulfate adenylyltransferase 9 41T01b07 mlr0237 Protein of unknown function 20 41T01c08 mlr7549 Nucleotide sugar epimerase 32 41T01g10 mll9145 Oligopeptide ABC transporter ATP-binding protein 34 41T02a03 mll3238 Putative penicillin-binding protein 27 a Information about each strain is the same as that described in the footnote to Table1. b The proportion of non-blue nodules relative to that seen in ML001 when each strain was inoculated with an equal amount of BN3. The value presented is that obtained from an assay representative of three. ML001 is set to 100 (%). All the strains produced significantly smaller numbers of non-blue nodules than ML001 (P<0.0001) (see text). Screening of Mesorhizobium loti Mutants by STM 169

Table 3. Competitiveness of Mesorhizobium loti strains that contain and strain 10T01g06 (mutant in mll5681) was a pyrimidine mutations allelic to those found in the mutants selected by auxotroph (data not shown). the STM procedure Next, we conducted the same type of mixed-inoculation a Relative nodule Strain Mutated gene b assays for 6 additional strains that contain mutations allelic occupancy (%) to those found in 6 of the strains with reduced competitive- 09T01a05 mll5232 52 ness (Table 3). All of them were shown to produce signifi- 09T05d03 mlr1231 3 cantly fewer non-blue nodules than ML001 (P<0.0001) con- 01T06e01 mll5942 16 sistently in two independent assays. This suggests that the 29T01f09 mlr3298 61 mutations in the identified genes are the causes of reduced 36T02e02 mlr0937 15 competitiveness; thus, these genes make a real contribution 23T04c04 mlr0237 37 a to nodulation efficiency despite being nonessential to symbi- Information about each strain is the same as that described in the foot- otic nitrogen fixation. note to Table 1. b The proportion of non-blue nodules relative to that seen in ML001 as described in the footnote to Table 2. The value presented is that Discussion obtained from an assay representative of two. All the strains produced significantly smaller numbers of non-blue nodules than ML001 The nodulation performed by symbiotic pairs of rhizobia P< ( 0.0001) (see text). and legumes falls into two morphological classes, deter- minate and indeterminate. These classes differ widely in the pattern of meristem growth as well as in the physiological (data not shown). These noticeable levels of activity suggest and genetic characteristics of the bacteroids that differentiate another cause of the Fix− phenotype than the reduced activity within host cells (7, 9). Mesorhizobium loti and S. meliloti per se. are symbiotic partners of the model legumes L. japonicus and Medicago truncatula, respectively; these pairs represent Mutants showing reduced competitiveness during nodulation determinate and indeterminate classes, respectively. One of The remaining 56 strains supported plant growth indistin- the differences in symbiosis between M. loti and S. meliloti is guishable from ML001 when inoculated individually. The that the latter bacteroid undergoes terminal differentiation phenotypes prompted us to test whether these strains were through endoreduplication, causing it to lose reproductive altered in competitiveness during nodulation. Each strain capacity, whereas the former maintains reproductive capac- was inoculated with an equal amount of reference strain ity during symbiosis (28). Therefore, it is significant that BN3, which was engineered to contain gusA under the con- large-scale mutant libraries of mapped transposon insertion trol of the putative mlr5905 (nifH) promoter in a genetically mutants have been constructed to enable STM screening of wild-type background (see Materials and Methods). All nod- both M. loti and S. meliloti (34, 41). Complementary func- ules from plants inoculated with BN3 alone turned blue when tional genomics in these two species should provide a treated with the chromogenic substrate X-Gluc. In contrast, complete picture of the symbiosis between rhizobia and all nodules elicited by ML001 alone developed no color after legumes. the same treatment. When BN3 and ML001 were inoculated Our work used a PCR-based STM procedure to screen M. in an equal mixture, blue nodules represented 37%±14% loti mutants. An advantage of this method is that it does not (mean ± standard deviation; n=13) of the nodules examined, require microarrays; thus, the use of our tagged transposons indicating that BN3 nodulates substantially under mixed- can be easily extended to other rhizobia. The validity of our inoculation conditions. For each of the 56 mutant/BN3 strain screening method was demonstrated by the fact that we mixtures, we determined the number of non-blue nodules successfully selected a number of M. loti strains that were after treatment with X-Gluc. A total of 33 strains produced Fix− or reduced in competitiveness during nodulation, though significantly fewer non-blue nodules than did ML001 not all of the strains with such phenotypes could be recov- (P<0.0001) consistently in three independent assays (Table ered. The confirmation of nodulation phenotypes in this 2). This indicates a reduction in competitiveness during study was based on repeated assays, indicating that the nodulation, though it has not been examined whether each results obtained from the screen are reproducible. In addi- insertion is the cause of the reduced competitiveness. The tion, the 13 genes identified from the Fix− strains were all genes with transposon insertions vary in their predicted shown by complementation tests to be necessary for effective function (Table 2), and some of them have been documented symbiosis. Also, the 6 genes for which additional mutant in M. loti as influencing the efficiency of nodulation. The alleles were tested were shown to make a real contribution M. loti mutant in nodZ (mlr5848), whose product adds the to competitiveness. They include genes previously described fucosyl residue to the reducing terminal residue of Nod fac- to be involved in symbiosis (Tables 2 and 3), confirming tor, was described to be severely impaired in competition on again the validity of our procedure. More notably, the genes L. japonicus (37). The nodPQ genes (mlr7575 and mlr7576) identified for the first time in this study will reveal new are required for the biosynthesis of 3'-phosphoadenosine-5'- classes of function for symbiosis or competition in rhizobia. phosphosulfate, which is used for the sulfation of cell- Purine auxotrophs of some rhizobia are defective in surface polysaccharides, but not of Nod factor, in M. loti. host invasion (29), but this is not the case with M. loti. 5- The mutant in nodPQ elicited effective nodules with reduced Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a nodulation kinetics (43). In addition, we confirmed that purine biosynthesis intermediate, was suggested to be a key strain 06T02h06 (mutant in msl0067) was a purine auxotroph compound promoting host invasion and nodule formation in 170 BORJIGIN et al.

M. loti (32). Our work showed, however, that mutants of lation efficiency, mlr0937 is notable for encoding poly-β- three genes possibly required for AICAR biosynthesis each hydroxybutyrate (PHB) synthase. The R. etli mutant displayed distinctive phenotypes: the mll7962 (purM) mutant defective in PHB biosynthesis formed nodules that prolonged was Nod−Fix−, the mll7833 (purF) mutant was Nod+Fix−, the capacity to fix nitrogen, increasing the total nitrogen and the msl0067 (purS) mutant was Fix+ but reduced in content and dry weight of host plants compared to the wild competitiveness. In addition, the mlr8350 (guaB) mutant, type (4). This has been explained by the possibility that which would be required for the synthesis of xanthosine PHB synthesis could compete with nitrogen fixation for the monophosphate in purine metabolism, was Nod+Fix−. These reductant. The result provides a clue as to the novel function results appear to challenge the previously suggested role of of PHB in promoting nodulation, in addition to its relevance purine-biosynthetic pathways in M. loti symbiosis, although to bacteroid metabolism. we cannot rule out the possibility that suppressor mutations alleviated the symbiotic deficiencies in those strains. Acknowledgements The mutant in mll5838 was Nod+Fix−. The wild-type We thank Kazuhiko Saeki for providing cosmid clones from the mll5838 gene encodes a putative DNA-binding protein that M. loti genomic library, and the National BioResource Project (L. is well conserved in Mesorhizobium, Sinorhizobium, and japonicus and G. max ) at the University of Miyazaki for providing Rhizobium species (S. meliloti SMb20817, 62% amino acid signature-tagged M. loti strains and L. japonicus seeds. This work was supported in part by Grants-in-Aid for Scientific Research on identify to mll5838; R. etli RHE_PE00447, 60% identity) but Priority Area Comparative Genomics and for Scientific Research not yet characterized in any species. Notably, mll5838 is (B) (no. 22380048), Special Coordination Funds for Promoting located 319 bp downstream of mll5840 (dctA), coding for a Science and Technology, PROBRAIN, and a grant from the C4-dicarboxylate permease, and 547 bp upstream of mll5837 Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics (nifA2), coding for a transcriptional regulator, within the for Agricultural Innovation, PMI-0002). symbiosis island of the M. loti genome; both of these genes are essential for symbiotic nitrogen fixation (30, 45). References Pyruvate dehydrogenase is a key enzyme for carbon metab- olism in both free-living and symbiotic states: it catalyzes 1. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. the oxidative decarboxylation of pyruvate into acetyl-CoA, Seidman, J.A. Smith, and K. Struhl. 1995. Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons, Hoboken, USA. which is fed into the TCA cycle and the pathway of fatty 2. Broughton, W.J., and M.J. Dilworth. 1971. Control of leghaemo- acid biosynthesis. Enhanced synthesis of this enzyme in S. globin synthesis in snake beans. Biochem. J. 125:1075–1080. meliloti is thought to fulfill the necessary production of 3. Cabanes, D., P. Boistard, and J. Batut. 2000. Symbiotic induction acetyl-CoA in bacteroids, where C4-dicaboxylate is supplied of pyruvate dehydrogenase genes from Sinorhizobium meliloti. Mol. Plant-Microbe Interact. 13:483–493. as the sole carbon source by host plants (3). Sinorhizobium 4. Cevallos, M.A., S. Encarnación, A. Leija, Y. Mora, and J. Mora. meliloti mutants in a single set of genes for pyruvate dehy- 1996. Genetic and physiological characterization of a Rhizobium drogenase has not been characterized to date. In contrast, two etli mutant strain unable to synthesize poly-β-hydroxybutyrate. J. paralogous genes, mlr0383-mlr0384-mlr0385 and mll3629- Bacteriol. 178:1646–1654. mll3628-mll3627, encode the E1α, E1β, and E2 components 5. Das, S.K., U.S. Gautam, P.K. Chakrabartty, and A. Singh. 2006. − Characterization of a symbiotically defective serine auxotroph of of the pyruvate dehydrogenase complex in M. loti. The Fix Mesorhizobium ciceri. FEMS Microbiol. Lett. 263:244–251. phenotype of the mlr0384 and mlr0385 mutants indicates 6. Finan, T.M., B. Kunkel, G.F. De Vos, and E.R. Signer. 1986. their importance in symbiosis compared with the other paral- Second symbiotic megaplasmid in Rhizobium meliloti carrying ogs. Moreover, M. loti possesses three paralogs, mlr0388, exopolysaccharide and thiamine synthesis genes. J. Bacteriol. 167: 66–72. mll4296 and mll4470, encoding the E3 (dihydrolipoamide 7. Franssen, H.J., I. Vijn, W.C. Yang, and T. Bisseling. 1992. dehydrogenase) component of both the pyruvate and 2- Developmental aspects of the Rhizobium-legume symbiosis. Plant oxoglutarate dehydrogenases. Our work showed that the Mol. Biol. 19:89–107. mutant in mll4296 was Fix−. 8. Galibert, F., T.M. Finan, S.R. Long, et al. 2001. The composite genome of the legume symbiont Sinorhizobium meliloti. Science It is remarkable that nodules elicited by the mutants in 293:668–672. mll0920 and mll1587 (metF) retained nitrogenase activity 9. Gibson, K.E., H. Kobayashi, and G.C. Walker. 2008. Molecular at considerable levels whereas the host plants exhibited a determinants of a symbiotic chronic infection. Annu. Rev. Genet. symptom of nitrogen starvation and the nodules were like 42:413–441. − 10. Giraud, E., L. Moulin, D. Vallenet, et al. 2007. Legume symbioses: those of other Fix mutants. This phenotype partly resembles Absence of nod genes in photosynthetic bradyrhizobia. Science that of the double aap bra mutants of R. leguminosarum bv. 316:1307–1312. viciae, which are defective for the transport of a broad range 11. Hattori, Y., H. Omori, M. Hanyu, N. Kaseda, E. Mishima, T. of amino acids. It has since been explained that ammonium Kaneko, S. Tabata, and K. Saeki. 2002. Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic assimilation in the host plant is compromised because the gene disruption and complementation analysis. Plant Cell Physiol. loss of transport inhibits the amino-acid cycling between the 43:1542–1557. bacteroid and the host plant (24). The mutant in mll1587 was 12. Hensel, M., J.E. Shea, C. Gleeson, M.D. Jones, E. Dalton, and confirmed to be a methionine auxotroph and this gene D.W. Holden. 1995. Simultaneous identification of bacterial viru- lence genes by negative selection. Science 269:400–403. potentially controls the flux of one-carbon units in the cell. It 13. Hunt, T.A., C. Kooi, P.A. Sokol, and M.A. Valvano. 2004. needs to be addressed whether the one-carbon metabolism on Identification of Burkholderia cenocepacia genes required for the rhizobial side specifically contributes to the mechanism bacterial survival in vivo. Infect. Immun. 72:4010–4022. by which the host plant can acquire fixed nitrogen. Among the genes suggested to be involved in nodu- Screening of Mesorhizobium loti Mutants by STM 171

14. Ito, N., M. Itakura, S. Eda, et al. 2006. Global gene expression 29. Noel, K.D., R.J. Diebold, J.R. Cava, and B.A. Brink. 1988. in Bradyrhizobium japonicum cultured with vanillin, vanillate, 4- Rhizobial purine and pyrimidine auxotrophs: Nutrient supplementa- hydroxybenzoate and protocatechuate. Microbes Environ. 21:240– tion, genetic analysis, and the symbiotic requirement for de novo 250. purine biosynthesis. Arch. Microbiol. 149:499–506. 15. Kahn, M.L., T.R. McDermott, and M.K. Udvardi. 1998. Carbon 30. Nukui, N., K. Minamisawa, S. Ayabe, and T. Aoki. 2006. and nitrogen metabolism in rhizobia, p. 461–485. In H.P. Spaink, A. Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase Kondorosi, and P.J.J. Hooykaas (ed.), The Rhizobiaceae: Molecular gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Biology of Model Plant-Associated Bacteria. Kluwer Academic Mesorhizobium loti MAFF303099. Appl. Environ. Microbiol. Publishers, Dordrecht, The Netherlands. 72:4964–4969. 16. Kaneko, T., Y. Nakamura, S. Sato, et al. 2000. Complete genome 31. Okabe, S., M. Oshiki, Y. Kamagata, et al. 2010. A great leap forward structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium in microbial ecology. Microbes Environ. 25:230–240. loti. DNA Res. 7:331–338. 32. Okazaki, S., Y. Hattori, and K. Saeki. 2007. The Mesorhizobium 17. Kaneko, T., Y. Nakamura, S. Sato, et al. 2002. Complete genomic loti purB gene is involved in infection thread formation and nodule sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium development in Lotus jaopnicus. J. Bacteriol. 189:8347–8352. japonicum USDA110. DNA Res. 9:189–197. 33. Oldroyd, G.E.D., and J.A. Downie. 2008. Coordinating nodule 18. Kawaharada, Y., S. Eda, K. Minamisawa, and H. Mitsui. 2007. A morphogenesis with rhizobial infection in legumes. Annu. Rev. Plant Mesorhizobium loti mutant with reduced glucan content shows Biol. 59:519–546. defective invasion of its host plant Lotus japonicus. Microbiology 34. Pobigaylo, N., D. Wetter, S. Szymczak, U. Schiller, S. Kurtz, F. 153:3983–3993. Meyer, T.W. Nattkemper, and A. Becker. 2006. Construction of a 19. Kereszt, A., E. Kiss, B.L. Reuhs, R.W. Carlson, Á. Kondorosi, and P. large signature-tagged mini-Tn5 transposon library and its application Putnoky. 1998. Novel rkp gene clusters of Sinorhizibum meliloti to mutagenesis of Sinorhizobium meliloti. Appl. Environ. Microbiol. involved in capsular polysaccharide production and invasion of the 72:4329–4337. symbiotic nodule: The rkpK gene encodes a UDP-glucose dehydroge- 35. Pobigaylo, N., S. Szymczak, T.W. Nattkemper, and A. Becker. 2008. nase. J. Bacteriol. 180:5426–5431. Identification of genes relevant to symbiosis and competitiveness 20. Kouchi, H., H. Imaizumi-Anraku, M. Hayashi, T. Hakoyama, T. in Sinorhizobium meliloti using signature-tagged mutants. Mol. Nakagawa, Y. Umehara, N. Suganuma, and M. Kawaguchi. 2010. Plant-Microbe Interact. 21:219–231. How many peas in a pod? Legume genes responsible for mutualistic 36. Prell, J., and P. Poole. 2006. Metabolic changes of rhizobia in symbioses underground. Plant Cell Physiol. 51:1381–1397. legume nodules. Trends Microbiol. 14:161–168. 21. Laus, M.C., T.J. Logman, A.A.N. van Brussel, R.W. Carlson, P. 37. Rodpothong, P., J.T. Sullivan, K. Songsrirote, D. Sumpton, K.W.J.-T. Azadi, M.-Y. Gao, and J.W. Kijne. 2004. Involvement of exo5 in Cheung, J. Thomas-Oates, S. Radutoiu, J. Stougaard, and C.W. production of surface polysaccharides in Rhizobium leguminosarum Ronson. 2009. Nodulation gene mutants of Mesorhizobium loti R7A– and its role in nodulation of Vicia sativa subsp. nigra. J. Bacteriol. nodZ and nolL mutants have host-specific phenotypes on Lotus spp. 186:6617–6625. Mol. Plant-Microbe Interact. 22:1546–1554. 22. Lee, K.B., P. De Backer, T. Aono, et al. 2008. The genome of the 38. Roll, J.T., V.K. Shah, D.R. Dean, and G.P. Roberts. 1995. versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC Characteristics of NIFNE in Azotobacter vinelandii strains. J. Biol. Genomics 9:271. Chem. 270:4432–4437. 23. Leonard, L.T. 1943. A simple assembly for use in the testing of 39. Sambrook, J., and D.W. Russell. 2001. Molecular Cloning: A cultures of rhizobia. J. Bacteriol. 45:523–525. Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, 24. Lodwig, E.M., A.H.F. Hosie, A. Bourdès, K. Findlay, D. Allaway, R. New York, USA. Karunakaran, J.A. Downie, and P.S. Poole. 2003. Amino-acid cycling 40. Schäfer, A., A. Tauch, W. Jäger, J. Kalinowski, G. Thierbach, and A. drives nitrogen fixation in the legume-Rhizobium symbiosis. Nature Pühler. 1994. Small mobilizable multi-purpose cloning vectors 422:722–726. derived from the Escherichia coli plasmids pK18 and pK19: Selection 25. Masuda, S., S. Eda, C. Sugawara, H. Mitsui, and K. Minamisawa. of defined deletions in the chromosome of Corynebacterium 2010. The cbbL gene is required for thiosulfate-dependent autotrophic glutamicum. Gene 145:69–73. growth of Bradyrhizobium japonicum. Microbes Environ. 25:220– 41. Shimoda, Y., H. Mitsui, H. Kamimatsuse, et al. 2008. Construction of 223. signature-tagged mutant library in Mesorhizobium loti as a powerful 26. Matthews, R.G. 1996. One-carbon metabolism, p. 600–611. In F.C. tool for functional genomincs. DNA Res. 15:297–308. Neidhardt, R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. 42. Spaink, H.P. 2000. Root nodulation and infection factors produced Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. by rhizobial bacteria. Annu. Rev. Microbiol. 54:257–288. Umbarger (ed.), Escherichia coli and Salmonella Cellular and 43. Townsend II, G.E., L.S. Forsberg, and D.H. Keating. 2006. Molecular Biology, 2nd ed., ASM Press, Washington DC, USA. Mesorhizobium loti produces nodPQ-dependent sulfated cell surface 27. Mazurkiewicz, P., C.M. Tang, C. Boone, and D.W. Holden. 2006. polysaccharides. J. Bacteriol. 188:8560–8572. Signature-tagged mutagenesis: Barcoding mutants for genome-wide 44. Wilson, K.J., A. Sessitsch, J.C. Corbo, K.E. Giller, A.D.L. screens. Nat. Rev. Genet. 7:929–939. Akkermans, and R.A. Jefferson. 1995. β-Glucuronidase (GUS) 28. Mergaert, P., T. Uchiumi, B. Alunni, et al. 2006. Eukaryotic control transposons for ecological and genetic studies of rhizobia and other on bacterial cell cycle and differentiation in the Rhizobium-legume Gram-negative bacteria. Microbiology 141:1691–1705. symbiosis. Proc. Natl. Acad. Sci. USA 103:5230–5235. 45. Yurgel, S.N., and M.L. Kahn. 2004. Dicarboxylate transport by rhizobia. FEMS Microbiol. Rev. 28:489–501.