Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein

The amino acid and carbohydrate content of chloroplastic glutamine synthetase from tobacco leaves has been analysed. The enzyme subunit contanins 5% carbohydrate, mainly represented by glucosamine, galactosamine, glucose, galactose and mannose residues. The enzyme subunit displayed a single band of molecular mass 44000 Da after sodium dodecyl sulphate (SDS) electrophoresis. However, when isoelectrofocussing electrophoresis was performed, four subunits were evident differing by their charge. Furthermore, the four different subunits stained positively when tested with periodic acid Shiff reagent, showing that sugars and amino sugars were present within all the subunits.     

Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain-swapping’strategy - replacement of the cell wall anchor of InIA by the membrane anchor of ActA - we show that the reduced ability to adhere and enter cells of strains expressing InIA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.