Genetic analysis of Bacillus anthracis Sap S-layer protein crystallization domain

Candela, Thomas; Mignot, Tâm; Hagnerelle, Xavier; Haustant, Michel; Fouet, Agnès

doi:10.1099/mic.0.27832-0

Cited by 23 publications

(18 citation statements)

References 31 publications

(32 reference statements)

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“…Accordingly, csaB mutants, but not eag mutants, form long chains of bacilli (3; data not shown). Sap is synthesized as an 814-amino-acid precursor protein harboring an N-terminal signal peptide (residues 1 to 30), three SLH domains (residues 34 to 197) for which a structure has been reported (13), as well as a large C-terminal domain (residues 210 to 814) that promotes the crystallization of the S-layer protein (6). Predictions of secondary sequences suggest a high ␤-strand content in the C-terminal crystallization domain with the possible formation of a bacterial Ig-like domain 2 fold between residues 211 and 476 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

Kern

Theriot

et al. 2012

J Bacteriol

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The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surfacelayer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.

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Section: Resultsmentioning

confidence: 99%

Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

Kern

Theriot

et al. 2012

J Bacteriol

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“…Crystal Structure of the SLH Domains of Sap-The 814 amino acid Sap precursor harbors a N-terminal signal peptide (residues 1-30), three SLH domains (residues residues 34 -197) as well as a large C-terminal domain (residues 210 -814) that promotes the crystallization of the S-layer protein (25). The nucleotide sequence of the B. anthracis sap gene, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…B. anthracis can form S-layers from both extractable antigen 1 (EA1) and surface array protein (Sap) by tethering the SLH domains of these polypeptides to pyruvylated SCWP (20,22,23). C-terminal to the SLH domains, S-layer proteins encode crystallization domains, sequences predicted to enable subunit-subunit interactions within the S-layer (22,24,25). A simple model for S-layer assembly is that secreted subunits are recruited to the edge of an extant S-layer network via enthalpy-driven interactions between crystallization domains and are then tethered to the SCWP via the SLH domains (11).…”

mentioning

confidence: 99%

Structure of Surface Layer Homology (SLH) Domains from Bacillus anthracis Surface Array Protein

Kern

Wilton

Zhang

et al. 2011

Journal of Biological Chemistry

103

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Surface (S)-layers, para-crystalline arrays of protein, are deposited in the envelope of most bacterial species. These surface organelles are retained in the bacterial envelope through the non-covalent association of proteins with cell wall carbohydrates. Bacillus anthracis, a Gram-positive pathogen, produces S-layers of the protein Sap, which uses three consecutive repeats of the surface-layer homology (SLH) domain to engage secondary cell wall polysaccharides (SCWP). Using x-ray crystallography, we reveal here the structure of these SLH domains, which assume the shape of a three-prong spindle. Each SLH domain contributes to a three-helical bundle at the spindle base, whereas another ␣-helix and its connecting loops generate the three prongs. The inter-prong grooves contain conserved cationic and anionic residues, which are necessary for SLH domains to bind the B. anthracis SCWP. Modeling experiments suggest that the SLH domains of other S-layer proteins also fold into three-prong spindles and capture bacterial envelope carbohydrates by a similar mechanism.Surface layers (S-layers) 3 are para-crystalline sheets of protein, which self-assemble on the surface of microbial cells to form contiguous layers (1, 2). Most organisms that elaborate S-layers do so by abundantly producing and secreting a single protein species (3). Whether an organism produces an S-layer as a component of its envelope structure is assessed by electron microscopy of the cell surface (4). In this manner, species from nearly every branch of the Bacteria and Archaea have been discovered to produce S-layers (2). Proteins within S-layers fulfill variable functions in that they act either as a scaffold or enzyme in the bacterial envelope (5), promote nutrient diffusion or transport (6), or contribute to virulence by enabling microbial adhesion to infected host tissues (7).Most, but not all, S-layer proteins of bacteria share three tandem ϳ55 amino acid repeats of the Surface Layer Homology (SLH) domain (8 -10). Secreted proteins encoding three tandem SLH domains are tethered to the bacterial envelope by non-covalent interactions between the SLH domains and a secondary cell wall carbohydrate (11). SLH domains are remarkable for being both necessary and sufficient for the incorporation of chimeric proteins into S-layers (12, 13). The SbsC protein of Geobacillus stearothermophilus is an example for a class of protein that forms S-layers without SLH domains (14). SbsC binds to the secondary cell wall polysaccharide (SCWP) of G. stearothermophilus via its N-terminal domain, which consists of three triple-helical bundles that are connected by two contiguous helices (14). The N-terminal domain of SbsC has high similarity with S-layer proteins from G. stearothermophilus, Geobacillus kaustophilus, and Geobacillus tepidamans (14) and is not similar to proteins with SLH domains.The Gram-positive bacterium Bacillus anthracis is a rodshaped, spore-forming pathogen of mammalian hosts (15). The envelope of its vegetative forms is composed of a plasma membrane a...

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“…Sap is synthesized as an 814-amino-acid precursor protein harboring an N-terminal signal peptide (residues 1 to 30), three SLH domains (residues 34 to 197), and a large C-terminal domain (residues 210 to 814) that promotes the crystallization of the S-layer protein (4,36) (Fig. 6A).…”

Section: B Anthracis Slaq Is Required For S-layer Protein Secretionmentioning

confidence: 99%

“…While SecA2 and SlaP are required for efficient secretion of mCherry hybrids encompassing the signal peptide and SLH domains of Sap, SlaQ functions to promote secretion of mCherry hybrids that include the C-terminal crystallization domain. Work with S-layer proteins from several different bacteria, including B. anthracis Sap, demonstrated the propensity of crystallization domains to rapidly assemble into two-dimensional paracrystalline lattices (4,5,57). Factors that promote the assembly of S-layer proteins have not yet been reported.…”

Section: Figmentioning

confidence: 99%

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

Nguyen-Mau

Schneewind

et al. 2015

J Bacteriol

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Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. IMPORTANCES-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro.

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Genetic analysis of Bacillus anthracis Sap S-layer protein crystallization domain

Cited by 23 publications

References 31 publications

Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

Surface-Layer (S-Layer) Proteins Sap and EA1 Govern the Binding of the S-Layer-Associated Protein BslO at the Cell Septa of Bacillus anthracis

Structure of Surface Layer Homology (SLH) Domains from Bacillus anthracis Surface Array Protein

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

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