Microbes Environ. Vol. 26, No. 2, 165–171, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10213 Identification of Mesorhizobium loti 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 Lotus 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 nitrogen fixation (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 rhizobia. Key words: Mesorhizobium loti, signature-tagged mutagenesis, symbiosis, competitiveness, root nodule Rhizobia are a group of soil bacteria 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).