2b). Comparisons with known lipopolysaccharide profiles from other gram-negative bacteria suggests that the LMW band corresponds to the rough lipopolysaccharide (lipid A plus core) and the HMW bands to the smooth lipopolysaccharide (complete lipopolysaccharide molecules with different number of attached O-antigen units) (Choudhury et al., 2005; Vilches et al., 2007). The mutant did not produce the smooth lipopolysaccharide bands and showed faint LMW lipopolysaccharide bands with
different electrophoretic mobility from the parental bands (Fig. 2b). The results indicated that BM07-59 was damaged in the production of normal lipopolysaccharide. These Androgen Receptor high throughput screening results were not unexpected, as the galU mutants in Pseudomonas aeruginosa and Aeromonas hydrophila produced truncated lipopolysaccharide core and lacked the O-antigen (Choudhury et al., 2005; Vilches et al., 2007). UDP-glucose formed through the GalU catalyzed reaction can serve as glucose donor for core and O-antigen polysaccharide biosynthesis in the production of lipopolysaccharide (Dean & Goldberg, 2002). To determine why the O-antigen is missing in BM07-59, we analyzed the composition of lipopolysaccharide from wild-type and mutant strains grown in M1 medium containing 70 mM fructose at 30 °C. Purified lipopolysaccharide from wild type and BM07-59 is predominantly
composed of a lipid, with 10.7% and 3.5% of the lipopolysaccharide Gefitinib solubility dmso composed of carbohydrate, respectively. The carbohydrate fraction of lipopolysaccharide from wild-type strain contained rhamnose, xylose, mannose, glucose, N-acetyl glucosamine and 3-deoxy-d-manno-oct-2-ulsonic acid (KDO) in a mole ratio of 31.8 : 1.7 : 0.3 : 50.2 : 14.9 : 1.1, respectively, whereas the carbohydrate fraction of lipopolysaccharide
from BM07-59 contained rhamnose, glucose, N-acetyl glucosamine and KDO in a mole ratio of 3.9 : 11.2 : 30.8 : 54.1, respectively. Thus, in comparison with the wild-type lipopolysaccharide, the lipopolysaccharide from BM07-59 contained a much smaller molar amount of rhamnose and glucose but a much larger (50-fold) molar amount of KDO was detected in the mutant lipopolysaccharide. oxyclozanide This significant sugar compositional difference of lipopolysaccharide between wild-type and mutant strains clearly reflects the fact that BM07-59 is unable to supply UDP-glucose for O-antigen and core lipopolysaccharide synthesis. To further confirm that galU gene is involved in lipopolysaccharide and exobiopolymer production, a complementary assay was performed. Plasmid pBBR-KT galU harboring galU gene from P. putida KT2440 was introduced into BM07-59 to recover GalU activity. As expected, the complement BM07-59 (KT GalU) restored the parental phenotype for colony morphology (Fig. 1a), autoagglutination phenotype (Fig. 2a), exobiopolymer production (Fig. 1b) and lipopolysaccharide synthesis (Fig. 2b). These results indicated that the expression of galU gene from P.