• Abstract and Figures
• Public Full-text

JOURNAL OF BACTERIOLOGY, Sept. 2004, p. 6042–6049 Vol. 186, No. 18 0021-9193/04/$08.00ϩ0 DOI: 10.1128/JB.186.18.6042–6049.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved. Sinorhizobium meliloti ExoR and ExoS Proteins Regulate both Succinoglycan and Flagellum Production Shi-Yi Yao,1 Li Luo,2 Katherine J. Har,1† Anke Becker,3 Silvia Ru¨berg,3 Guan-Qiao Yu,2 Jia-Bi Zhu,2 and Hai-Ping Cheng1* Biological Sciences Department, Lehman College, The City University of New York, Bronx, New York1; National Laboratory of Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institute of Biological Sciences, Chinese Academia of Science, Shanghai, People’s Republic of China2; and Institut fu¨r Genomforschung, Universita¨t Bielefeld, Bielefeld, Germany3 Received 1 August 2003/Accepted 22 June 2004 The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa)byS. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinogly- can and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagel- lum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes. Sinorhizobium meliloti and its legume host, alfalfa (Medicago (12). The exoR gene encodes a protein of 268 amino acids that sativa), establish an effective nitrogen-fixing symbiosis through shares no significant homology with any other protein in cur- a series of signal exchanges that starts with the exchange of rently available databases (23). The exoS gene encodes the Nod (nodulation) factors and flavonoids, which results in the membrane-bound sensor of the ExoS/ChvI two-component formation of curled alfalfa root hairs that are colonized by S. regulatory system, and the exoS96 mutation might have re- meliloti cells (13, 20, 32). The colonized curled root hairs de- sulted in the formation of a constitutively active version of the velop infection threads within the root hairs, which allow S. sensor (10). Two close homologs of the exoS gene, Agrobacte- meliloti cells to invade the developing root nodules (14, 16, 31). rium tumefaciens chvG (8) and Brucella abortus bvrS (29), are A successful invasion of nodules by S. meliloti will result in the essential to the pathogenicity of their respective hosts (8, 29). formation of pink nitrogen-fixing nodules. The pink color is The A. tumefaciens chvG gene is involved in regulating acid- due to the presence of leghemoglobin. Nodules that are not inducible genes (18), and B. abortus bvrS is involved in regu- occupied by S. meliloti and/or not capable of fixing nitrogen are lating membrane protein expression (15). Both the exoR95 and most often white due to the lack of leghemoglobin (20). exoS96 mutations resulted in the upregulation of the 22 succi- The formation of infection threads inside root hairs requires noglycan biosynthesis genes, with the exoYFQ operon as the the presence of an S. meliloti exopolysaccharide, succinoglycan primary target of the regulation (23). The exoY gene encodes a (9), in addition to the Nod factor (33). Succinoglycan is a galactose transferase that carries out the first step of succino- polymer that consists of different numbers of a repeating unit glycan biosynthesis, and the exoY210::Tn5 mutation completely consisting of one galactose and seven glucoses with three mod- blocks succinoglycan biosynthesis (17, 25). ification groups: acetyl, pyruvyl, and succinyl (17, 24). All three The exoR95 mutation resulted in a reduction of nodulation modifications must be present in order for the S. meliloti suc- efficiency so that some of the plants inoculated with the exoR95 cinoglycan to be active in eliciting infection thread formation mutants were tall and green with pink nodules, while the others (9). Surprisingly, overproduction of succinoglycan appears to were short and yellow with white nodules (12). The exoR95 reduce efficiency of nodulation (12). mutant also appeared to have a reduced efficiency in colonizing Two S. meliloti mutants, exoR95::Tn5 and exoS96::Tn5, were curled root hairs (9). The exoS96 mutation did not change the isolated based on their ability to overproduce succinoglycan nodulation efficiency significantly, even though it appeared to cause a reduction in the efficiency in colonizing curled root hairs (9). * Corresponding author. Mailing address: Biological Sciences De- The exoR95 and exoS96 mutants have been linked to the partment, Lehman College, The City University of New York, 250 Bedford Park Blvd., West, Bronx, NY 10468. Phone: (718) 960-7190. reduction of cellular motility, based on the findings that the Fax: (718) 960-8236. E-mail: [email protected]. two mutants formed smaller colonies on swarming plates (35). † Present address: Greenwich High School, Greenwich, CT 06830. Two S. meliloti mutations have been isolated that increase 6042 VOL. 186, 2004 REGULATION OF SUCCINOGLYCAN AND FLAGELLUM BIOSYNTHESIS 6043 TABLE 1. S. meliloti strains used in this study and LB supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LB/MC) was used for all S. meliloti strains (17). TY medium was used for growth of S. meliloti Source or Strain Relevant characteristic(s) strains for microarray analysis. Agar (1.5%) was added to make solid medium. reference Antibiotics were used at the indicated concentrations: chloramphenicol (Cm), 10 ␮ ␮ ␮ Rm1021 Wild type 17 g/ml; gentamicin (Gm), 15 g/ml; kanamycin, 25 g/ml; neomycin (Neo), 200 ␮ ␮ Rm7210 exoY210::Tn5 17 g/ml; streptomycin (Str), 500 g/ml. Rm7095 exoR95::Tn5 12 Nodulation efficiency. To characterize the functions of ExoR and ExoS/ChvI Rm7096 exoS96::Tn5 12 proteins, exoR95 and exoS96 mutants and other strains were analyzed for their Rm8395 exoR395::Tn5-233 G. C. Walker ability to establish nitrogen-fixing symbiosis with alfalfa by determining their Rm8396 exoS396::Tn5-233 G. C. Walker nodulation efficiency, which was measured by the color and the number of RmHC1 exoY210::Tn5 exoR395::Tn5-233 This work nodules on roots of alfalfa plants inoculated with these strains. The symbiotic RmHC2 exoY210::Tn5 exoR395::Tn5-233 This work ability of the bacterial strains can also be measured by their ability to colonize curled alfalfa root hairs (root hair colonization efficiency), to invade alfalfa roots through root hairs (root hair invasion efficiency), and to invade root nodules (nodule invasion efficiency). motility and the sizes of colonies on swarming plates and re- Nodulation efficiency assays were carried out as previously described (17). Alfalfa (M. sativa cv. Iroquois) seeds were surface sterilized and germinated in duce succinoglycan production (35). These findings raised the the dark. The bacterial cells (0.1 ml) were plated on the surface of solid plant possibility that the regulation of succinoglycan production and growth medium, Jensen’s agar (22), inside a square petri dish. For each bacterial cellular motility could be coupled. The coupling of the regu- strain, 0.1 ml of cell suspension was placed on top of solid plant growth medium, lation of bacterial exopolysaccharide production and the reg- Jensen’s agar. Ten 2-day-old alfalfa seedlings were spread out evenly across the ulation of cell motility has been found in Ralstonia solanacea- plate. A stack of 10 plates was wrapped with aluminum foil and placed in a plant growth chamber on one side of the petri dishes so that the shoots of the seedlings rum (5), Vibrio cholerae (1, 34), and Salmonella enterica (7), were pointing up. To determine the effect of the size of the inoculum on nodu- which presumably increases the ability of the cells to interact lation efficiency, plants were inoculated with decreasing amounts of inoculum. with their prospective host cells. One milliliter of cell suspensions at three different cell concentrations, 107,105, 3 S. meliloti cellular motility is supported by two to eight peri- and 10 cells/ml, was used to inoculate a set of 10 plants in one square petri dish. Ten plants were used for each strain at each cell concentration to minimize the trichous semirigid flagella which allow the bacterium to move influence of the individual alfalfa plant on the results. The alfalfa plants were effectively in viscous liquid (6). These flagella rotate in one checked for plant height, the numbers of nodules, and the percentage of nodules direction at different speeds, which allows the bacterial cells to that were pink each week. The growth of the plants was documented using a alter direction (27). The S. meliloti cellular motility requires Kodak digital camera. the function of the chemotaxis, flagellum, and motility genes, The nodule invasion efficiency was measured as the percentage of the nodules that were pink. The formation of pink nodules is the result of bacterial coloni- which are tightly regulated in a hierarchical order from class I zation of the plant cells inside the nodule, which elicits the production of leghe- to class III (30). Class I genes comprise the visN and visR genes moglobin (11). (30). Class II genes, which comprise flagellar assembly and Root hair invasion efficiency assays were carried out as previously described motor genes, are controlled by the class I genes (30).

Load more

  8

Copy Link