IDENTIFICATION AND CHARACTERIZATION OF A BASIC CELL SURFACE-LOCATED PROTEIN FROM LACTOBACILLUS-FERMENTUM BR11

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an app arent molecular mass of 32 kDa, A clone encoding an immunoreactive 32- kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. f ermentum BR11, Sequence determination of the insert in the clone revea led a complete 795-bp opera reading frame (ORF) that defines a 28,625- Da polypeptide (BspA), N-terminal sequencing of the LiCl-extracted pol ypeptide from L. fermentum BR11 confirmed that it is the same as the c loned BspA, BspA was found to have a sequence similar to those of fami ly III of the bacterial solute-binding proteins, The sequences of two ORFs upstream of bspA, are consistent with bspA being located in an op eron encoding an ATP-binding cassette-type uptake system, Unusually, B spA contains no lipoprotein cleavage and attachment motif (LXXC), desp ite its origin in a gram-positive bacterium, Biotin labelling and tryp sin digestion of whole cells indicated that this polypeptide is expose d on the cell surface, The isoelectric point as predicted from the put ative mature sequence is 10.59, It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction w ith acidic groups on the cell surface. It, was shown that BspA could b e selectively removed from the surface by extraction with an acidic hu ller, thus supporting this hypothesis.