In all of the experiments, E. coli BL21(DE3) cells expressing either the authentic CGL1 or CGL2 proteins or the carbohydrate-binding defective variant CGL2(W72G), or control transformants were fed to C. elegans wild type N2. (A) CGL2 inhibits C. elegans development. C. elegans L1 larvae were seeded onto lawns of above bacteria (left panel) or fed with increasing concentrations of purified CGL2 together with equal amounts of empty vector-containing BL21(DE3) in liquid culture (right panel) and scored for the fraction developing to the L4 stage within 72 h and 96 h, respectively. Columns represent the average of 6 and 12 replicates, respectively. Error bars indicate standard deviations. The fraction of animals reaching L4 was significantly lower on CGL1- or CGL2-expressing bacteria (p<0.01) than on bacteria expressing CGL2(W72G) or containing empty vector. No significant difference was observed between latter two conditions (p>0.5). In the liquid assay, worm development decreases significantly at CGL2 concentrations higher than 150 µg/ml. (B) CGL2 inhibits C. elegans reproduction. C. elegans hermaphrodites were placed as L4 animals on plates seeded with above bacteria and scored for total progeny counts per hermaphrodite. The broods of eight to ten hermaphrodites were averaged per data point. The standard deviations are indicated. The differences between the L4 fractions on CGL2 and vector control and CGL2(W72G) were statistically significant (p>0.01). None of the progeny on wild type CGL2 developed to adulthood within 96 h post hatch. (C) CGL2 ultimately kills C. elegans. 10 L4 staged wild type C. elegans (N2) were seeded onto lawns of CGL2-, CGL2(W72G)-expressing and empty vector control-containing E. coli BL21(DE3) cells. Each day, the plates were checked for surviving animals which were then transferred to novel bacterial lawns of the same type. The data points represent the average of five replicates. Error bars indicate standard deviations. The survival rate of the worms was significantly impaired on CGL2-expressing bacteria compared to CGL2(W72G) and vector control (p<0.01), whereas there was no significant difference between latter two conditions (p>0.5). (D) CGL2 damages C. elegans intestine. C. elegans L4 larvae were fed with CGL2-expressing (panels i-vi) and control E. coli BL21(DE3) cells (panels i-iii) and examined after 24 h under the stereomicroscope (panels i,iv), by differential interference contrast (DIC) microscopy (panels ii, v) and by transmission electron microscopy (TEM) (panels iii,vi). The size bars in panels iii and vi are 200 nm.