Eight mutants completely abolished exobiopolymer production and O-antigen lipopolysaccharide synthesis and showed increased polyhydroxyalkanoates accumulation compared with the wild type. One mutant named BM07-59 was chosen for further study because it showed the greatest increase in polyhydroxyalkanoates level. Arbitrary PCR was used to determine the precise location of the transposon insertion (Wang et al., 2008). Sequencing of the region in BM07-59 flanked Cell Cycle inhibitor by the transposon revealed that the transposon was inserted into the gene that has high similarity to galU from Pseudomonas spp. The full galU
gene obtained from BM07 was found to have a sequence encoding a protein exhibiting a high sequence homology with UDP-glucose pyrophosphorylase (GalU). GalU catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate (PPi) from UTP and glucose 1-phosphate.
UDP-glucose not only functions as sugar nucleotide precursor for polysaccharide biosynthesis (Dean & Goldberg, 2002) but is also involved in the biosynthesis this website of several cell wall components (Sandlin et al., 1995). UDP-glucose is the substrate for the synthesis of UDP-glucuronic acid, and is also required for the interconversion of galactose and glucose by the Leloir pathway (Frey, 1996). A relevant role for GalU in virulence has also been recognized in several bacterial species, as this enzyme is required for the synthesis of UDP-glucose, which is the main glucosyl donor in lipopolysaccharide and unless capsule biosynthesis (Sandlin et al., 1995; Dean & Goldberg, 2002). The colony morphology of BM07-59 was distinct from that of the wild type. The mutant colony exhibited an alteration in slime production and appeared less glossy than the parent strain (Fig. 1a). Cultivation of BM07-59 in M1 minimal medium with 70 mM fructose at 10 °C did not lead to the production of exobiopolymer (Fig. 1b). After centrifugation, the supernatant from BM07-59 was clear, whereas the supernatant
from BM07 wild type was very turbid due to the presence of water-insoluble colloidal exobiopolymer particles in the supernatant (Zamil et al., 2008). When tested on LB medium containing 0.3% agar, the wild-type strain was able to swim, whereas BM07-59 had lost its motility (Fig. 1c). In LB or M1 medium with 70 mM fructose, the mutant exhibited the tendency to precipitate (autoagglutination) (Fig. 2a). Autoagglutination in unshaken liquid medium is a common phenotype displayed by rhizobia with lipopolysaccharide defects (Priefer, 1989). Therefore, BM07-59 was investigated for its lipopolysaccharide production. The lipopolysaccharide from the parental and mutant strain were extracted with proteinase K, resolved by SDS-PAGE, and silver stained.