Construction of exoF::TnphoA fusion To generate plasmid-borne exoF::TnphoA fusions, plasmid pD82, a cosmid clone carrying the S. meliloti exoF gene and surrounding region of the genome [26], was introduced into the S. meliloti exoF::TnphoA fusion

strain Rm8369 [27]. This construct was subsequently www.selleckchem.com/products/abc294640.html transferred into E. coli strain MT607, by triparental conjugation using E. coli strain MT616 as the mobilizer. Transconjugants were selected on LB KmTc, and the nature of the fusion was confirmed by testing for inability to confer YMA mucoidy on the exoF::TnphoA mutant Rm7055. The resulting construct was named pD82 exoF::TnphoA. Biochemical assays Alkaline phosphatase activity of exoF::TnphoA fusions in S. meliloti strains was measured according to the method of Brinkmann and Beckwith [46]. Cells were grown to an OD600 of 0.7. 1 ml of culture was washed twice in 1 M Tris-HCl (pH 8.0), and resuspended in 1 ml 1 M Tris-HCl (pH 8.0). The OD600 of this cell suspension was then measured. Following

a 10 min equilibration period at 37°C, 50 μl of 4 mg/ml p-nitrophenyl phosphate (NPP) was added to start the reaction. check details The reaction was allowed to continue for 11 min at 37°C before being stopped by the addition of 50 μl of 1 M K2HPO4. The cells were pelleted and 50 μl of the supernatant was diluted in 450 μl of 1 M Tris-HCl (pH 8.0) and OD420 was measured. Units (U) of alkaline phosphatase activity were calculated using the formula: (1) Assuming a molar coefficient of 16,000 for p-nitrophenyl phosphate, 1 U is equal to 0.062 nmol of NPP hydrolyzed per min at a cell OD600 of 1. Therefore: (2) For PHB assays, 50 ml cultures were grown at 30°C to stationary phase in YMB. Cells were harvested and washed in 0.85% NaCl solution before resuspension in 50 ml 0.85%

NaCl. PHB was extracted from a 2 ml fraction of this suspension and the remaining 48 ml was used for cell dry weight determination by incubation of the pellet at 60°C until the pellet was dry and no further loss in mass was recorded. PHB content was measured by the method of Law and Slepecky [47] and expressed as a percentage of total cell dry weight. All glassware was washed in hot chloroform and ADP ribosylation factor rinsed in ethanol before use, to eliminate plasticizers. A standard curve was constructed by dissolving known quantities of PHB (Sigma) in hot chloroform to a final volume of 1 ml. The chloroform was allowed to evaporate before addition of 10 ml of H2SO4 and PHB was processed as described elsewhere [47]. Carbon starvation assay Saturated TY cultures were washed twice to remove traces of nutrients, and were subcultured 1:50 into carbon-free M9 medium. These cultures were incubated at 30°C, shaking at 180 rpm. Viable cell counts were monitored at weekly intervals by plating on TY agar. Samples at t = 0 were each given a relative value of 1, and all subsequent samples are compared to this starting value. Values recorded are the means from triplicate cultures.