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 apparent 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. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.
Turner
M S
MS
Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Brisbane, Australia.
Timms
P
P
Hafner
L M
LM
Giffard
P M
PM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000737:chemistry* / Q000235:genetics; D018021:Lithium Chloride; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000378:metabolism
D001426:Bacterial Proteins; C105995:BspA protein, bacteria; D008565:Membrane Proteins; D018021:Lithium Chloride