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...00) or YMB (g l21: K2HPO4, 0.5; MgSO4.7H2O, 0.2; NaCl, 0.1; mannitol, 10; yeast extract, 0.4) (Tombolini et al., 1995) at 30 uC. Escherichia coli strains were grown at 37 uC in Luria– Bertani medium (=-=Miller, 1972-=-). Antibiotics were used at the following concentrations: 100 mg ampicillin ml21, 20 mg kanamycin ml21, 25 mg chloramphenicol ml21, 50 mg rifampicin ml21, 50 mg spectinomycin ml21 and 50 mg streptomyc...

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...029. The insert was confirmed by DNA sequence analysis. A unique BamHI site was identified within the glgA2 coding region 759 bp downstream from the translation start codon, and the VSpSm interposon (=-=Prentki & Krisch, 1984-=-) was ligated into this site to make pKS031. The insert fragment construct was then subcloned into pK19mobsac using EcoRI to make pMS001. Gene replacement in Rm5000 was carried out by first introducin...

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...ated onM. truncatula andM. sativa. METHODS Bacterial strains, plasmids and growth conditions. Strains and plasmids used in this work are shown in Table 1. S. meliloti strains were grown in TY medium (=-=Beringer, 1974-=-), MOPS (Mendrygal & Gonzalez, 2000) or YMB (g l21: K2HPO4, 0.5; MgSO4.7H2O, 0.2; NaCl, 0.1; mannitol, 10; yeast extract, 0.4) (Tombolini et al., 1995) at 30 uC. Escherichia coli strains were grown at...

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... M. loti and R. leguminosarum bv. phaseoli, glgA mutations were reported to have no effect on symbiosis (Lodwig et al., 2005). There are two predicted glycogen-synthase-encoding genes in S. meliloti (=-=Galibert et al., 2001-=-). The glgA1 gene is located within the glgPBCApgm operon on the chromosome, while the glgA2 gene is located on megaplasmid pSymB (Finan et al., 2001; Galibert et al., 2001). Under growth-limiting con...

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.... sativa were used as hosts to test the nodulation of the S. meliloti strains. Germinated seedlings were transferred to growth pouches (MEGA International). Each pouch contained 5 ml Fahraeus medium (=-=Fahraeus, 1957-=-) with six seedlings. Pouches were put in a growth chamber under the following conditions: 18 h day/6 h night cycle (22 uC light/18 uC dark), and 60% relative humidity. After 4 days in the growth pouc...

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...ce EPS are unable to invade and thus form ineffective nodules (Leigh & Walker, 1994). S. meliloti has at least two types of EPS: succinoglycan (EPSI) (Leigh & Walker, 1994) and galactoglucan (EPSII) (=-=Glazebrook & Walker, 1989-=-). A correlation between PHB metabolism and EPSI synthesis has been demonstrated (Aneja et al., 2004). However, mechanisms of regulation have not been characterized, and it is not known whether EPSI s...

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...into the cells of the developing nodule (Udvardi & Day, 1997). Once released from the infection threads, the bacterial cells differentiate into bacteroids, able to convert atmospheric N2 to ammonium (=-=Schultze & Kondorosi, 1998-=-). Nitrogen fixation within legume nodules results from a complex metabolic exchange between the bacterium and its plant host. Rhizobia are strict aerobes; therefore dicarboxylates provided by plants ...

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...equires the action of additional signal molecules, such as the exopolysaccharides (EPSs) produced by S. meliloti. The infecting bacteria grow and divide as they progress through the infection thread (=-=Gage et al., 1996-=-), which is then degraded and the bacteria are taken into the cells of the developing nodule (Udvardi & Day, 1997). Once released from the infection threads, the bacterial cells differentiate into bac...

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... mg rifampicin ml21, 50 mg spectinomycin ml21 and 50 mg streptomycin ml21. M9 minimal medium with various carbon sources, each at a final concentration of 15 mM, was prepared as described previously (=-=Charles & Finan, 1991-=-; Charles et al., 1997). Sucrose was added to the medium at 5% (w/v) when required. Media were solidified by the addition of 1.5% (w/v) agar. Liquid cultures were shaken at 220 r.p.m. Genetics and mol...

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...(Fraysse et al., 2003) and plays a signalling role during nodulation (Niehaus & Becker, 1998), and mutants of S. meliloti unable to produce EPS are unable to invade and thus form ineffective nodules (=-=Leigh & Walker, 1994-=-). S. meliloti has at least two types of EPS: succinoglycan (EPSI) (Leigh & Walker, 1994) and galactoglucan (EPSII) (Glazebrook & Walker, 1989). A correlation between PHB metabolism and EPSI synthesis...

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...creased infection and is important for N2 fixation. This may be partially due to the reduction of EPS in mutants containing an interrupted phbC gene. EPS is essential for the early infection process (=-=Fraysse et al., 2003-=-) and plays a signalling role during nodulation (Niehaus & Becker, 1998), and mutants of S. meliloti unable to produce EPS are unable to invade and thus form ineffective nodules (Leigh & Walker, 1994)...

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...dM. sativa. METHODS Bacterial strains, plasmids and growth conditions. Strains and plasmids used in this work are shown in Table 1. S. meliloti strains were grown in TY medium (Beringer, 1974), MOPS (=-=Mendrygal & Gonzalez, 2000-=-) or YMB (g l21: K2HPO4, 0.5; MgSO4.7H2O, 0.2; NaCl, 0.1; mannitol, 10; yeast extract, 0.4) (Tombolini et al., 1995) at 30 uC. Escherichia coli strains were grown at 37 uC in Luria– Bertani medium (Mi...

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...that much of the carbon flow in the bacteroidmay be linear, with the substrate diverted to storage pools such as polyhydroxybutyrate (PHB) and glycogen, and some of it may be secreted as amino acids (=-=Lodwig & Poole, 2003-=-). As a result of this complex situation, many of the bacterial metabolic processes that occur in the free-living state are not the same as in the nodule. PHB and glycogen are major carbon storage com...

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...i. The infecting bacteria grow and divide as they progress through the infection thread (Gage et al., 1996), which is then degraded and the bacteria are taken into the cells of the developing nodule (=-=Udvardi & Day, 1997-=-). Once released from the infection threads, the bacterial cells differentiate into bacteroids, able to convert atmospheric N2 to ammonium (Schultze & Kondorosi, 1998). Nitrogen fixation within legume...

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...ally due to the reduction of EPS in mutants containing an interrupted phbC gene. EPS is essential for the early infection process (Fraysse et al., 2003) and plays a signalling role during nodulation (=-=Niehaus & Becker, 1998-=-), and mutants of S. meliloti unable to produce EPS are unable to invade and thus form ineffective nodules (Leigh & Walker, 1994). S. meliloti has at least two types of EPS: succinoglycan (EPSI) (Leig...

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...f PHB, and the accumulation of PHB requires nitrogen fixation (Cermola et al., 2000). However, bacteroids of indeterminate nodules (e.g. alfalfa nodules) do not accumulate PHB (Aneja & Charles, 1999; =-=Hirsch et al., 1983-=-). In the symbiosis between S. meliloti and M. sativa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & Charles, 1999; Aneja e...

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... in Table 1. S. meliloti strains were grown in TY medium (Beringer, 1974), MOPS (Mendrygal & Gonzalez, 2000) or YMB (g l21: K2HPO4, 0.5; MgSO4.7H2O, 0.2; NaCl, 0.1; mannitol, 10; yeast extract, 0.4) (=-=Tombolini et al., 1995-=-) at 30 uC. Escherichia coli strains were grown at 37 uC in Luria– Bertani medium (Miller, 1972). Antibiotics were used at the following concentrations: 100 mg ampicillin ml21, 20 mg kanamycin ml21, 2...

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...dition of 1.5% (w/v) agar. Liquid cultures were shaken at 220 r.p.m. Genetics and molecular biology techniques. Bacterial conjugations and wM12 transductions were carried out as described previously (=-=Charles & Finan, 1990-=-, 1991). DNA manipulations were performed using standard methods (Ausubel et al., 1997). Oligonucleotide primers were purchased from Sigma-Aldrich. DNA amplification by PCR using Pfu DNA polymerase (P...

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...meliloti and M. sativa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & Charles, 1999; Aneja et al., 2005; Cai et al., 2000; =-=Povolo et al., 1994-=-; Willis & Walker, 1998), but are less competitive than the wild-type (Aneja et al., 2005; Willis & Walker, 1998). Recently, it has been shown that S. meliloti phbC mutants are not only unable to prod...

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...ccumulate high levels of PHB, and the accumulation of PHB requires nitrogen fixation (Cermola et al., 2000). However, bacteroids of indeterminate nodules (e.g. alfalfa nodules) do not accumulate PHB (=-=Aneja & Charles, 1999-=-; Hirsch et al., 1983). In the symbiosis between S. meliloti and M. sativa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & C...

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... Zevenhuizen, 1981). This suggests that glycogenmetabolism may fulfil a similar role to PHB, and the carbon flux between the two compounds is relatively plastic and appears subject to common control (=-=Povolo & Casella, 2000-=-; Encarnacion et al., 2002; Dunn et al., 2002). In S. meliloti, it has been reported that under oxygen-limiting conditions free-living cells can use intracellular glycogen to generate ATP while mainta...

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...biosis between S. meliloti and M. sativa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & Charles, 1999; Aneja et al., 2005; =-=Cai et al., 2000-=-; Povolo et al., 1994; Willis & Walker, 1998), but are less competitive than the wild-type (Aneja et al., 2005; Willis & Walker, 1998). Recently, it has been shown that S. meliloti phbC mutants are no...

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... are major carbon storage compounds in rhizobia. The roles of PHB and glycogen in rhizobia–legume symbiosis are not fully understood (Lodwig & Poole, 2003; Mandon et al., 1998; Marroqui et al., 2001; =-=Trainer & Charles, 2006-=-). For instance, bacteroids of determinate nodules (e.g. bean nodules) often accumulate high levels of PHB, and the accumulation of PHB requires nitrogen fixation (Cermola et al., 2000). However, bact...

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...LB plates containing appropriate antibiotics. For assays in the growth chamber, plants were grown in sterile pots (464 inches, 9 plantlets per pot) containing Jensen’s N-free plant nutrient solution (=-=Jensen, 1942-=-) treated vermiculite (Sta-Green, St Louis, USA), as described previously (Aneja & Charles, 1999). The plants were inoculated 5 days after germination, with the appropriate S. meliloti strains. Each s...

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...by anthrone, according to the procedure of Chun & Yin (1998). Cell number was determined by serial dilution and plating on TY medium. PHB assays were performed using the spectrophotometric technique (=-=Law & Slepecky, 1961-=-) as modified by Peoples & Sinskey (1989). Cells were harvested from S. meliloti cultures grown to saturation in YMB medium (50 ml). Following a saline wash and resuspension of cells in 50 ml saline, ...

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...hesis of the EPS succinoglycan (EPSI) (Aneja et al., 2004). It has been hypothesized that PHB accumulated by the bacteria prior to infection serves to fuel infection and/or bacteroid differentiation (=-=Lodwig et al., 2005-=-). Glycogen is synthesized in bacteria through the action of glycogen synthase on ADP-glucose (Ugalde et al., 2003), resulting in the formation of a-1,4-linked glucan. A common genetic organization of...

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...s suggests that glycogenmetabolism may fulfil a similar role to PHB, and the carbon flux between the two compounds is relatively plastic and appears subject to common control (Povolo & Casella, 2000; =-=Encarnacion et al., 2002-=-; Dunn et al., 2002). In S. meliloti, it has been reported that under oxygen-limiting conditions free-living cells can use intracellular glycogen to generate ATP while maintaining their PHB content (P...

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...odule. PHB and glycogen are major carbon storage compounds in rhizobia. The roles of PHB and glycogen in rhizobia–legume symbiosis are not fully understood (Lodwig & Poole, 2003; Mandon et al., 1998; =-=Marroqui et al., 2001-=-; Trainer & Charles, 2006). For instance, bacteroids of determinate nodules (e.g. bean nodules) often accumulate high levels of PHB, and the accumulation of PHB requires nitrogen fixation (Cermola et ...

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... mg spectinomycin ml21 and 50 mg streptomycin ml21. M9 minimal medium with various carbon sources, each at a final concentration of 15 mM, was prepared as described previously (Charles & Finan, 1991; =-=Charles et al., 1997-=-). Sucrose was added to the medium at 5% (w/v) when required. Media were solidified by the addition of 1.5% (w/v) agar. Liquid cultures were shaken at 220 r.p.m. Genetics and molecular biology techniq...

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...acteria prior to infection serves to fuel infection and/or bacteroid differentiation (Lodwig et al., 2005). Glycogen is synthesized in bacteria through the action of glycogen synthase on ADP-glucose (=-=Ugalde et al., 2003-=-), resulting in the formation of a-1,4-linked glucan. A common genetic organization of the glycogen metabolism genes is found in the closely related Agrobacterium tumefaciens, Rhizobium tropici, Rhizo...

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...a, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & Charles, 1999; Aneja et al., 2005; Cai et al., 2000; Povolo et al., 1994; =-=Willis & Walker, 1998-=-), but are less competitive than the wild-type (Aneja et al., 2005; Willis & Walker, 1998). Recently, it has been shown that S. meliloti phbC mutants are not only unable to produce PHB but are also de...

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...et al., 2005; Willis & Walker, 1998). Recently, it has been shown that S. meliloti phbC mutants are not only unable to produce PHB but are also deficient in synthesis of the EPS succinoglycan (EPSI) (=-=Aneja et al., 2004-=-). It has been hypothesized that PHB accumulated by the bacteria prior to infection serves to fuel infection and/or bacteroid differentiation (Lodwig et al., 2005). Glycogen is synthesized in bacteria...

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...ation with its host plants, including Medicago truncatula (barrel medic) and Medicago sativa (alfalfa). A successful symbiosis involves a complex series of interactions between the host and bacteria (=-=Brewin, 1991-=-; Long, 1989, 2001; van Rhijn & Vanderleyden, 1995). Initially, plants release flavonoids that attract S. meliloti cells from the surrounding environment to the plant’s roots, and induce the productio...

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...tabolism may fulfil a similar role to PHB, and the carbon flux between the two compounds is relatively plastic and appears subject to common control (Povolo & Casella, 2000; Encarnacion et al., 2002; =-=Dunn et al., 2002-=-). In S. meliloti, it has been reported that under oxygen-limiting conditions free-living cells can use intracellular glycogen to generate ATP while maintaining their PHB content (Povolo & Casella, 20...

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...ile the glgA2 gene is located on megaplasmid pSymB (Finan et al., 2001; Galibert et al., 2001). Under growth-limiting conditions, free-living rhizobial cells produce glycogen simultaneously with PHB (=-=Tsien & Schmidt, 1977-=-; Zevenhuizen, 1981). This suggests that glycogenmetabolism may fulfil a similar role to PHB, and the carbon flux between the two compounds is relatively plastic and appears subject to common control ...

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...., 1983). In the symbiosis between S. meliloti and M. sativa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild-type (Aneja & Charles, 1999; =-=Aneja et al., 2005-=-; Cai et al., 2000; Povolo et al., 1994; Willis & Walker, 1998), but are less competitive than the wild-type (Aneja et al., 2005; Willis & Walker, 1998). Recently, it has been shown that S. meliloti p...

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...t al., 2001; Trainer & Charles, 2006). For instance, bacteroids of determinate nodules (e.g. bean nodules) often accumulate high levels of PHB, and the accumulation of PHB requires nitrogen fixation (=-=Cermola et al., 2000-=-). However, bacteroids of indeterminate nodules (e.g. alfalfa nodules) do not accumulate PHB (Aneja & Charles, 1999; Hirsch et al., 1983). In the symbiosis between S. meliloti and M. sativa, it has be...

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...ired EPSs and initiate adherence, signalling, and infection of the nodule. Previous ultrastructural studies have indicated that PHB granules are present in S. meliloti cells inside infection threads (=-=Paau et al., 1978-=-), but they disappear when cells are (a) (d) (e) Starch Starch Starch Starch Starch 200 mm 200 mm 200 mm 200 mm 200 mm (b) (c) Fig. 4. Light micrographs of longitudinal sections of M. truncatula nodul...