Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11

Abstract

Extraction ofCell surface polypeptides of gram-positive bacteria may be divided into a number of classes on the basis of the way in which they are anchored to the cell envelope. In two such classes, the LPXTG-containing proteins and the lipoproteins, attachment is covalent. The former are anchored to cell wall peptidoglycan via the threonine residue in the C-terminally located LPXTG motif (20), while the latter are anchored to cytoplasmic membrane lipids via the cysteine residue in the N-terminally located LXXC motif (29). Many lipoproteins of gram-positive bacteria are homologs of periplasmic solutebinding proteins of gram-negative bacteria, and it is currently thought that lipid anchoring of these proteins is necessary in gram-positive bacteria because there is no outer membrane to prevent their loss to the environment (29).A number of other surface-located proteins are anchored via charge interactions. Examples include S-layer proteins which form regular arrays covering the cell surface and the protein monomers that constitute polymeric structures such as fimbriae and flagella. A characteristic of S-layer proteins from lactobacilli is that they can be extracted from the cell surface by charge-occupying agents such as concentrated salt or guanidine hydrochloride (17, 18).The cell surfaces of lactobacilli are poorly understood in comparison to those of the related streptococci. Only S-layer proteins and surface-located enzymes have been extensively characterized (2,4,11,12,33). We are currently investigating the surface of the guinea pig female genital tract isolate Lactobacillus fermentum BR11 (26). Here we report the sequence of BspA, a surface-located polypeptide of this organism. BspA is selectively removed by extraction with 5 M LiCl and is clearly homologous to family III of the bacterial solute-binding proteins (30). Unlike solute-binding proteins from gram-positive bacteria, BspA does not contain a lipoprotein consensus sequence. It has a highly alkaline isoelectric point, and it appears likely that it is anchored by electrostatic interaction with negatively charged groups on the cell surface. MATERIALS AND METHODSStrains and plasmids. L. fermentum BR11 is a guinea pig vaginal isolate that has been previously described (26). It was grown in liquid or solid MRS medium (Oxoid) for 18 to 24 h at 37°C in the presence of 5% CO 2 . Escherichia coli JM109 (36) and recombinant derivatives were grown at 37°C in Luria-Bertani medium (27) supplemented with ampicillin (100 g/ml), isopropyl-␤-D-thiogalactopyranoside (IPTG) (0.25 mM), and 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) (40 l of a 20-mg/ml solution) as appropriate. Plasmid pUC18 has been described previously (36), while plasmids pMFT1, pMFT2, and pMFT3 are pUC18 derivatives described more fully in Results.L. fermentum BR11 extractions. Cell surface extractions were made from 35-ml stationary-phase cultures. The cells were harvested by centrifugation (10,000 ϫ g, 5 min), washed once with an equal volume of 0.15 M NaCl, resuspended in 500 l of ...

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