Dataset Information

Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display.

ABSTRACT: Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a ?-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inactivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were proportional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B. subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusions were distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also appeared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium. Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed on the surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS, 47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Characterization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system. This system may have various biotechnological applications.

SUBMITTER: Liew PX

PROVIDER: S-EPMC3256611 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display.

Liew Pei Xiong PX Wang Christopher L C CL Wong Sui-Lam SL

Journal of bacteriology 20111021 1

Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a β-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as t ...[more]

PMID: 22020651

Similar Datasets

Project description: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.

Project description:BackgroundSubtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot.ResultsIn this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed "online" reporter strains to efficiently monitor the dynamics of subtilin biosynthesis.ConclusionsWithin this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

Project description:5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.

Project description:UnlabelledIn Bacillus subtilis, the dl-endopeptidase LytE is responsible for lateral peptidoglycan hydrolysis during cell elongation. We found that σ(I)-dependent transcription of lytE is considerably enhanced in a strain with a mutation in ltaS, which encodes a major lipoteichoic acid (LTA) synthase. Similar enhancements were observed in mutants that affect the glycolipid anchor and wall teichoic acid (WTA) synthetic pathways. Immunofluorescence microscopy revealed that the LytE foci were considerably increased in these mutants. The localization patterns of LytE on the sidewalls appeared to be helix-like in LTA-defective or WTA-reduced cells and evenly distributed on WTA-depleted or -defective cell surfaces. These results strongly suggested that LTA and WTA affect both σ(I)-dependent expression and localization of LytE. Interestingly, increased LytE localization along the sidewall in the ltaS mutant largely occurred in an MreBH-independent manner. Moreover, we found that cell surface decorations with LTA and WTA are gradually reduced at increased culture temperatures and that LTA rather than WTA on the cell surface is reduced at high temperatures. In contrast, the amount of LytE on the cell surface gradually increased under heat stress conditions. Taken together, these results indicated that reductions in these anionic polymers at high temperatures might give rise to increases in SigI-dependent expression and cell surface localization of LytE at high temperatures.ImportanceThe bacterial cell wall is required for maintaining cell shape and bearing environmental stresses. The Gram-positive cell wall consists of mesh-like peptidoglycan and covalently linked wall teichoic acid and lipoteichoic acid polymers. It is important to determine if these anionic polymers are required for proliferation and environmental adaptation. Here, we demonstrated that these polymers affect the expression and localization of a peptidoglycan hydrolase LytE required for lateral cell wall elongation. Moreover, we found that cell surface decorations with teichoic acid polymers are substantially decreased at high temperatures and that the peptidoglycan hydrolase is consequently increased. These findings suggest that teichoic acid polymers control lateral peptidoglycan hydrolysis by LytE, and bacteria drastically change their cell wall content to adapt to their environment.

Dataset Information

Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display.

Publications

Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets