A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.

Fucosyltransferases (FucT) from differentHelicobacter pylori strains display distinct Type I (Gal␤1,3GlcNAc) or Type II (Gal␤1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both ␣1,3 and ␣1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive ␣1,3 activity. Our domain swapping study ( Helicobacter pylori is associated with gastritis and peptic ulcer formation and is a risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma. One of the virulence factors of H. pylori is the lipopolysaccharide, which contains lipid A, core oligosaccharide and O-antigens. The O-antigens of H. pylori lipopolysaccharide contain fucosylated oligosaccharides, predominantly the Type II blood group antigens, Lewis X (Gal␤1,4(Fuc␣1,3)GlcNAc) and Lewis Y (Fuc␣1,2Gal␤1,4(Fuc␣1,3)GlcNAc) (1), but a small number of H. pylori strains also express the Type I blood group antigens, Lewis A (Gal␤1,3(Fuc␣1,4)GlcNAc), and Lewis B (Fuc␣1,2Gal␤1,3(Fuc␣1,4)-GlcNAc) (2).The role of Lewis antigens in H. pylori pathogenesis is still ambiguous. It has been suggested that Lewis antigens play a role in H. pylori adhesion to (3, 4) or internalization by (5) the gastric epithelial cells. Nevertheless, conflicting evidence argues that Lewis X and Lewis Y are not required for colonization of human gastric epithelium (6) or mouse stomach (7,8). Lewis antigens may also play an important role in the persistence of H. pylori infection by molecular mimicry, helping the bacteria to evade the host immune response (2, 9, 10). Environmental changes such as pH influence the expression of H. pylori O-antigens, particularly Lewis X and Lewis Y. This may aid in adaptation of the bacterium to its niche in the stomach (10).Fucosyltransferases (FucTs) 3 are enzymes responsible for the last steps in the synthesis of Lewis antigens in H. pylori (11,12). ␣1,2 and ␣1,3 or ␣1,3/4 FucTs have been identified and characterized in H. pylori (13-18). These FucTs catalyze the transfer of the L-fucose moiety from guanosine diphosphate ␤-L-fucose (GDP-Fuc) to the OH-2 of the galactose moiety and the OH-3 or the OH-3 and the OH-4 positions of the GlcNAc moiety in glycoconjugate acceptors, respectively. The H. pylori genome contains two homologous ␣1,3 or ␣1,3/4 FucT genes, futA and futB (19,20), but they do not always encode functional proteins. For instance, only the futA gene encodes an active FucT in H. pylori strains NCTC11639 and UA948 (13,17). Bacterial ␣1,3/4 FucTs are functionally equivalent to the mammalian ␣1,3/4 FucTs, which have been well characterized. Mammalian FucTs are Type II membrane proteins with a short N-terminal cytoplasmic tail, transmembrane domain, stem region, and C-terminal catalytic domain. H. pylori FucTs share weak homology with their mammalian counterparts in two small segments within the catalytic domain, called ␣1,3 FucT motifs (14, 21). H. pylori ␣1,3/4 FucTs lack the N...

The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.

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