Rodolfo A. Ugalde

A chromosomal cluster of genes encoding ADP-glucose synthetase, glycogen synthase and phosphoglucomutase in agrobacterium tumefaciens

A chromosomal region from Agrobacterium tumefaciens that complements exoC (pgm) mutations was cloned and sequenced. A cluster of three open reading frames (ORF1, ORF2 and ORF3) was identified. These genes are oriented in the same direction and are involved in the synthesis of glycogen and other polysaccharides. ORF1 encodes a 420-amino-acid (aa) protein with 55.9% homology to Escherichia coli GlgC (ADP-glucose synthetase, EC 2.7.7.27). ORF2 encodes a 480-aa protein with 42.2% homology to E. coli GlgA (glycogen synthase, EC 2.4.1.21). Based on Tn5 mutagenesis and protein homology, ORF3 was identified as the structural gene encoding phosphoglucomutase (Pgm, EC 2.7.5.1). ORF3 encodes a 542-aa protein with 52.6% homology to rabbit Pgm. There is no significant homology (less than 20%) to the Xanthomonas campestris XanA protein, which displays phosphomannomutase (Pmm) and Pgm activities [Koplin et al., J. Bacteriol 174 (1992) 191-199]. An A. tumefaciens pgm::Tn5 mutant retains Pmm activity.

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble β-(1–3),β-(1–6) glucansA preliminary account of this work was presented at the 5th International Symposium on the Molecular Genetics of Plant-Microbe Interactions, Interlaken, Switzerland, September 1990. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg2+. Gel chromatography of soluble glucans resolved a cyclic β-(1–3) glucan with a degree of polymerization of eleven from a family of β-(1–3),β-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce β-(1–3),β-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize β-(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of β-(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.

Lipid-bound sugars in Rhizobium meliloti

Abstract Incubation of an enzyme preparation of Rhizobium meliloti with labeled uridine diphosphate glucose led to the formation of radioactive substances soluble in organic solvents. These substances are probably polyprenyl diphosphate saccharides. They behaved like these on treatment with ammonia or with hot phenol and were decomposed by heating for 10 min at pH 2 yielding a mono- and a disaccharide. The monosaccharide was identified as galactose by paper chromatography. The disaccharide gave glucose and galactose by acid hydrolysis. Following reduction with borohydride it yielded glucose and galactitol. After treatment with periodate followed by paper chromatography only galactose was detectable. The disaccharide was hydrolyzed by β- but not by α-glucosidase. Therefore the disaccharide is glucosyl β1-3-galactose.

Identification of toxigenic Vibrio cholerae from the Argentine outbreak by PCR for ctx A1 and ctx A2-B.

A polymerase chain reaction (PCR) to detect a region of the A1 cholera toxin gene was applied to the identification of 43 Vibrio cholerae strains isolated from the recent outbreak in Argentina. A good correlation was observed between the GM1‐enzyme‐linked immunosorbent assay (GM1‐ELISA) to detect the B subunit of the enterotoxin and PCR. However, a V. cholerae non‐01 strain that was negative by the ELISA test, was positive by the PCR assay for the Al region. A second PCR test to detect the A2‐B coding region was developed to solve this case. We propose that routine detection of toxigenic V. cholerae by PCR should include analysis of A2‐B coding region or the whole cholera toxin operon.