Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus

Abstract

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

FIG. 1.

Analysis of proteins found in the culture supernatant of L. fermentum BR11 grown in MRS broth. Growth of L. fermentum BR11 was monitored over 24 h by optical density measurements at 600 nm. At various time points, indicated by a number in a circle, aliquots were taken and centrifuged, and the supernatant was filtered and precipitated with 5% trichloroacetic acid. The equivalent of 225 μl of culture supernatant was analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue G-250. The arrow indicates Sep.

FIG. 2.

(A) Amino acid identity between different regions of Sep from L. fermentum BR11 and APF1 from L. johnsonii ATCC 11506. SS indicates a secretion signal, LysM indicates a LysM domain, Q-rich indicates regions rich in glutamine amino acids, and the hatched boxes indicate the highly homologous carboxy-terminal regions. The arrows indicate cleavage sites of the secretion signals. Note: APF1 does not contain a LysM domain. (B) Multiple alignment of Sep, Efae0789 from E. faecium, SP0107 from S. pneumoniae, and APF1 from L. johnsonii ATCC 11506. Shaded residues indicate identity with the consensus sequence determined using the AlignX program in Vector NTI Suite 6.0. The secretion signals of Sep and APF1 are underlined. The amino-terminal LysM domains found in Sep, Efae0789, and SP0107, but not in APF1, are indicated by a line above the sequence (note: there is one LysM domain for these proteins which extends over the alignment gap). The core residues found in the YG motif in the amino-terminal LysM domains and near the carboxy termini are indicated by asterisks. (C) Alignment of the amino-terminal LysM domain of Sep with the consensus LysM domain pfam01476 (see text) and the core amino acids found in YG domains. Letters in boldface type indicate the consensus sequence amino acids for the LysM and YG core domains. H_y stands for a hydrophobic amino acid. Lines indicate amino acid matches.

FIG. 3.

Expression and subcellular location of a His₆-Sep fusion protein in L. fermentum BR11, L. rhamnosus GG, and L. lactis MG1363. (A) Arrangement of the constructs which were introduced into L. fermentum, L. rhamnosus, or L. lactis. The sep terminator (T sep) and DNA encoding the Sep secretion signal (ssSep), BspA secretion signal (ssBspA), and His₆ (grey box) are indicated. The DNA region which is the site of single-crossover homologous recombination into either the sep or bspA loci of L. fermentum BR11 is spotted and is marked with a cross below. (B) Western blot detection of fusion proteins in cell extracts and in the supernatant using an anti-His₅ antibody. The strains and constructs are indicated at the bottom of each blot. Sizes of molecular mass markers are indicated in kilodaltons on the left. The lanes containing cell extracts prepared by boiling in 2× SDS-loading dye (SDS), by sonication (son), and with 5 M LiCl (LiCl) and the precipitated supernatant fractions (SN) are indicated. The amount of cells or medium loaded in each lane is the equivalent to 500 μl (SDS), 50 μl (son), 160 μl (LiCl), and 675 μl (SN) of culture.

FIG. 4.

Expression and secretion of a human E-cadherin fusion protein by L. fermentum BR11 and L. rhamnosus GG. (A) Arrangement of the constructs which were introduced into L. fermentum or L. rhamnosus. The bspA terminator (T bspA) and DNA encoding the Sep secretion signal (ssSep), BspA secretion signal (ssBspA), and His₆ (grey box) are indicated. The DNA region which is the site of single-crossover homologous recombination into either the sep or bspA loci of L. fermentum BR11 is spotted and is marked with a cross below. (B) Western blots of fusion proteins in cell extracts and in the supernatant using an anti-His₅ antibody, except for the rightmost blot for which an anti-E-cadherin antibody was used. The strains and constructs are indicated at the bottom of each blot. The sizes of the molecular mass markers are indicated on the left. The lanes containing cell extracts prepared by boiling in 2× SDS-loading dye (SDS), by sonication (son), and with 5 M LiCl (LiCl) and the precipitated supernatant fractions (SN) are indicated. The amounts of cells or medium loaded in each lane are the equivalent to 500 μl (SDS), 50 μl (son), 160 μl (LiCl), and 675 μl (SN) of culture. For the two rightmost blots, the equivalent of 1.2 ml of culture supernatant from L. fermentum (wild-type) or L. fermentum containing BspA-6xHis-Ecad (BspA-6xHis-Ecad) was loaded in each lane.