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Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.

ABSTRACT: BACKGROUND: Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-beta-N-acetylglucosaminidase (Mbg), as the anchor protein. RESULTS: Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. CONCLUSION: Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system.

SUBMITTER: Shao X

PROVIDER: S-EPMC2754439 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.

Shao Xiaohu X Jiang Mengtian M Yu Ziniu Z Cai Hao H Li Lin L

Microbial cell factories 20090916

<h4>Background</h4>Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-beta-N-acetylglucosaminidase (Mbg), as ...[more]

PMID: 19754974

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Project description:BACKGROUND: From fundamental studies to industrial processes, synthesis of heterologous protein by micro-organisms is widely employed. The secretion of soluble heterologous proteins in the extracellular medium facilitates their recovery, while their attachment to the cell surface permits the use of the recombinant host cells as protein or peptide supports. One of the key points to carry out heterologous expression is to choose the appropriate host. We propose to enlarge the panel of heterologous secretion hosts by using Streptococcus thermophilus LMD-9. This lactic acid bacterium has a generally recognised as safe status, is widely used in the manufacture of yogurts, fermented milks and cheeses, and is easy to transform by natural competence. This study demonstrates the feasibility of secretion of a heterologous protein anchored to the cell surface by S. thermophilus. For this, we used the cell envelope proteinase (CEP) PrtH of Lactobacillus helveticus CNRZ32 CIRM-BIA 103. RESULTS: Using S. thermophilus LMD-9 as the background host, three recombinant strains were constructed: i) a negative control corresponding to S. thermophilus PrtS- mutant where the prtS gene encoding its CEP was partially deleted; ii) a PrtH+ mutant expressing the L. helveticus PrtH pro-protein with its own motif (S-layer type) of cell-wall attachment and iii) a PrtH+WANS mutant expressing PrtH pro-protein with the LPXTG anchoring motif from PrtS. The PrtH+ and PrtH+WANS genes expression levels were measured by RT-qPCR in the corresponding mutants and compared to that of prtS gene in the strain LMD-9. The expression levels of both fused prtH CEPs genes, regardless of the anchoring motif, reached up-to more than 76% of the wild-type prtS expression level. CEPs were sought and identified on the cell surface of LMD-9 wild-type strain, PrtH+ and PrtH+WANS mutants using shaving technique followed by peptide identification with tandem mass spectrometry, demonstrating that the heterologous secretion and anchoring of a protein of more than 200 kDa was efficient. The anchoring to the cell-wall seems to be more efficient when the LPXTG motif of PrtS was used instead of the S-layer motif of PrtH. CONCLUSIONS: We demonstrated S. thermophilus LMD-9 could heterologously secrete a high molecular weight protein and probably covalently anchor it to the cell-wall.

Dataset Information

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.

Publications

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor.

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