These data are shown in Table 2 and represent the average from three samples. Rm11430 demonstrates significantly increased PHB accumulation relative to Rm1021 suggesting that, while synthesis of PHB is not impaired, the lesion in phaZ inhibits degradation of PHB. The PHB accumulation CP-690550 clinical trial phenotype of Rm11430 is complemented by pMA157, demonstrating a clear relationship between the presence of phaZ and PHB accumulation. Table 2 PHB accumulation during free-living growth in Yeast-Mannitol Medium Strain Relevant Characteristics PHB Accumulation % cell dry mass Rm1021

wild-type 18.9 Rm11105 phaC::Tn5 0.240 Rm11430 phaZΩSmSp 28.6 Rm11430 pMA157 phaZΩSmSp pRK7813 phaZ 7.39 Effect on expression of succinoglycan synthesis genes The product of the exoF gene is involved in the transfer of the first sugar, galactose, to the lipid carrier, upon which the subunits of succinoglycan are assembled [25]. pD82exoF::TnphoA was constructed by homologous recombination between exoF carried on pD82 [26] and the chromosomal exoF::TnphoA fusion of strain Rm8369 [27]. The resultant plasmid was used to measure the transcriptional activity of exoF in different S. meliloti PHB mutant backgrounds when grown under different culture conditions. A Student’s t-test was used to analyze the data and determine statistical significance

of the observed differences. The results RG7112 nmr presented in Table 3 represent the mean of three independent samples. When analyzed using a two-tailed Student’s t-test, the 1.1-fold increase in exoF expression Mannose-binding protein-associated serine protease exhibited by

YMB-grown Rm11430 is statistically significant. Furthermore, the non-mucoid mutants Rm11105 and Rm11107 exhibit a reduction in exoF expression. This is consistent with the observation that colonies formed by Rm11430 appear larger and more mucoid on YMA than Rm11105 or Rm11107 (Table 1). Table 3 exoF::phoA Alkaline Phosphatase Assay Strain Relevant Characteristics Activity (U) Std Error Rm1021 wild-type 14.1 0.331 Rm11105 phaC::Tn5 9.68 a 0.264 Rm11347 phaBO 6.23 a 0.223 Rm11107 bdhA::Tn5 16.1 0.714 Rm11430 phaZ OSmSp 15.7 a 0.296 a These differences are statistically significant from the value recorded for Rm1021, when analysed using a two-tailed Student’s t-test Symbiotic phenotype of Rm11430 and bacteroid PHB accumulation Unlike bacteroids of determinate nodules, bacteroids of S. meliloti do not accumulate PHB during symbiosis (reviewed in [4]). Interestingly, a mutant of R. leguminosarum unable to cycle amino acids between the bacteroid and plants, showed apparent accumulation of PHB in the bacteroid within pea indeterminate nodules [11]. This suggests that the pathway for PHB metabolism can function within bacteroids of indeterminate nodules; however accumulation of PHB only occurs under extreme circumstances for example, when carbon is in excess and bacteroid metabolism is limited by the availability of a key nutrient. To confirm that S.