Simultaneous Display of Multiple Foreign Peptides in the FliD Capping and FliC Filament Proteins of the Escherichia coli Flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

THE ZOOSPORE OF CHLOROKYBUS ATMOPHYTICUS,A CHAROPHYTE WITH SARCINOID GROWTH HABIT

Chlorokybus atmophyticus has a sarcinoid growth habit and produces scale-covered zoospores. Flagella are laterally inserted and attached internally to a multilayered structure characteristic of the Charophyceae. There are two kinds of pyrenoid in each cell, a feature previously observed in only one scaly green flagellate. C. atmophyticus demonstrates that the sarcinoid growth habit arose independently at least twice in the green algae and cannot be used to define taxonomic groups unless combined with other criteria. It is further concluded that C. atmophyticus should be classified in a separate family Chlorokybaceae and a separate order Chloroky bales.     

Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.     

New labdane resin acids from Pinus elliottii

Slash pine needles and cortex oleoresin have been found to contain a new major diterpene constituent, imbricataloic acid. The closely related imbricatoloic acid, previously reported only in Araucaria imbricata, was found to be present in small amounts in slash pine needle extract. Spectral data are given for an unidentified diterpene alcohol isolated from the cortex oleoresin.