Short communicationIn vivo and in vitro surface display of heterologous proteins on Bacillus thuringiensis vegetative cells and spores
Introduction
Bacterial surface display is based on the fusion of recombinant proteins to an anchor protein that immobilizes the fusion protein onto the cell surface of the bacteria [1], [2]. This technique has been used in a variety of procedures such as the development of live vaccines [3], construction and screening of protein libraries [4], whole cell biocatalysis [5], and development of environmental bioadsorbents [6].
Many bacterial peptidoglycan hydrolases (PGHs) contain the lysin motif (LysM), the tandem repeat domain that can bind to the peptidoglycan molecules of various bacteria [7]. These PGHs can be employed as anchors in cell-surface display systems. To date, several heterologous proteins have been displayed on Lactococcus lactis cells [8], [9] and Bacillus thuringiensis cells [10] using this strategy.
The spore-forming bacterium B. thuringiensis is advantageous for cell-surface display due to its rigid cell-wall structure, distinctive biosynthesis and biopesticidal capabilities. The surface display of some functional proteins on this bacterium such as antimicrobial protein, chitinase and antigens, among others, is a promising approach that can be extended to agricultural or biotechnological applications. However, only a few cell-surface display systems have been exploited in B. thuringiensis vegetative cells [10], [11], [12] or spores [13] to date. Further, despite its potential, an anchoring protein that can confer a dual immobilization activity for both vegetative cells and spores has not been revealed so far.
Previously, a surface display system was developed in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-β-N-acetylglucosaminidase (Mbg), as the anchor protein to target two foreign proteins onto vegetative cells of this bacterium [10]. In the current study, the N-terminal domain of Mbg anchor can be employed to direct protein incorporation onto the surface of vegetative cells and spores in vivo, as well as the vegetative cells/spores of two other Bacillus strains through the immobilization in vitro. The surface localizations and surface display efficiencies of recombinant proteins were first analyzed using green fluorescence protein (GFP) as reporter. Then the system was further applied to display a bacterial laccase onto the vegetative cells and spores of B. thuringiensis and other Bacillus bacteria in vivo and in vitro.
Section snippets
Bacterial strains, culture conditions and spore preparation
Escherichia coli DH5α (TaKaRa Bio Inc.) and ER2566 (New England Biolabs, Inc.) were respectively used for constructing recombinant plasmids and for recombinant protein expression. A plasmid-free B. thuringiensis BMB171 was used as the host strain for the surface display of recombinant proteins. The collected MGSC (Wuhan, China) wild-type strains B. thuringiensis YBT-1520, Bacillus subtilis BF7658, and Bacillus cereus AS1.229 were used as the target strains for in vitro immobilization of
In vivo immobilization of Mbgn-GFP and Mbgn-WlacD fusion proteins onto B. thuringiensis BMB171 vegetative cells and spores
The putative peptidoglycan hydrolase Mbg (Mbgn) can direct GFP and a mutated bacterial laccase WlacD on the surface of B. thuringiensis vegetative cells [10]. To investigate whether this system can be applied to display GFP and WlacD onto spores, the N-terminal domain (Mbgn) of Mbg was used as the surface anchoring motif to construct and express the Mbgn-GFP and Mbgn-WlacD fusion proteins. The recombinant plasmids pMB164 and pMB316 respectively containing mbgn-gfp and mbgn-wlacD fusion genes
Conclusion
The N-terminal domain of a putative peptidoglycan hydrolase (Mbgn) from B. thuringiensis can be used successfully as a surface-anchoring motif to immobilize the heterologous proteins GFP and WlacD onto vegetative cells and spores. The targeted proteins also retained their biological activity. The Mbgn-mediated in vivo and in vitro immobilization of target proteins on both vegetative cells and spores implies that this system can be used throughout the life cycle of engineered B. thuringiensis.
Acknowledgement
This work was supported by grants from the National Natural Science Foundation of China, item nos. 30670054, 40830527, and 31070111.
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