S-Layer Anchoring and Localization of an S-Layer-Associated Protease in
            <i>Caulobacter crescentus</i>

Ford, Matthew J.; Nomellini, John F.; Smit, John

doi:10.1128/jb.01690-06

Cited by 48 publications

(52 citation statements)

References 44 publications

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“…The native sapA and rsaA genes were inactivated by gene replacement with an internal deletion (sapA) or an amber mutation (rsaA353B). The gene replacements and chromosomal insertion of repBAC were accomplished using pK18mobsacB (41), as described previously (18). Strain JS 1021 is a NA1000 derivative containing only the repBAC insertion (27).…”

Section: Methodsmentioning

confidence: 99%

“…Secretion and subsequent self-assembly require calcium (35), and it is assumed that the RTX repeat domain (characteristic of type I secreted proteins) that is located adjacent to the C-terminal secretion signal is responsible for at least some of the calcium interaction; whether there is a second domain to complete subunit-subunit interactions is unknown. The S layer is attached to the outer membrane (OM) surface by interaction of an N-terminal attachment domain of approximately 200 amino acids (18) with the fraction of lipopolysaccharide (LPS) that is modified with an O-polysaccharide (3,52).…”

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Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

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The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

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Section: Methodsmentioning

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Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

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“…This was done by subcloning the rsaA/protein G construct as an EcoRI-HindIII segment into pMOBSacB and performing gene replacement procedures as previously described (12). Protein G display was confirmed by immunofluorescence microscopy using Alexa Fluor 488-coupled secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

“…JS4022 (26) is further modified by mutation of sapA, which eliminates degradation of heterologous insertions in RsaA, and the introduction of repBAC, which allows replication of the p4A shuttle plasmid vector in Caulobacter (12). p4A containing a variant of rsaA with a BamHI site inserted at a position corresponding to amino acid 723 and a version further derived to contain domain 1 of CD4 have been described (27).…”

Section: Methodsmentioning

confidence: 99%

“…A typical cell has approximately 40,000 copies of this protein monomer (25,33,34). We have demonstrated that it is feasible to insert large genetic segments within the S-layer gene while maintaining all functional aspects: secretion, assembly (crystallization), and cell surface attachment of the resulting recombinant protein, as well as consequent high density presentation of the inserted peptide (12,17,25,26). S-layer expression/display of CD4 (domain 1) and MIP1␣ has successfully generated recombinant caulobacters that can inhibit HIV infection (27).…”

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Enhanced Neutralization of HIV by Antibodies Displayed on the S-Layer of Caulobacter crescentus

Duval

Lewis

Nomellini

et al. 2011

Antimicrob Agents Chemother

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Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods. Here, we demonstrate that the bacterium Caulobacter crescentus and its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed on Caulobacter was as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD4 binding (CD4i) was codisplayed with CD4, there was significant enhancement in HIV-1 neutralization. These results suggest that caulobacters displaying anti-HIV antibodies offer a distinct improvement in the use of antibodies as microbicides. Furthermore, these reagents can specifically evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, C. crescentus, which can be easily produced and chemically stabilized at low cost, is well suited for engineering as an effective platform, offering an inexpensive way to produce and deliver HIV-1-specific microbicides.

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S‐Layers, Microbial, Biotechnological Applications

Egelseer¹,

Ilk²,

Pum³

et al. 2009

Encyclopedia of Industrial Biotechnology

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Crystalline bacterial cell surface layers (S‐layers), a unique self‐assembly system optimized during billions of years of biological evolution, are one of the most commonly observed cell, envelope structures of prokaryotes. Although self‐assembly of molecules is an ubiquitous strategy of morphogenesis in nature, research in the area of molecular nanotechnology, nanobiotechnology, and biomimetics are only beginning to exploit its potential for the functionalization of surfaces and interfaces as well as for the production of biomimetic membranes and encapsulation systems. In this context, S‐layers fulfill key requirements for controlled assembly of supramolecular materials. As S‐layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications in nanobiotechnology depend on the ability of isolated native S‐layer proteins and S‐layer fusion proteins incorporating functional sequences to self‐assemble into monomolecular crystalline arrays in suspension, on a great variety of solid substrates, and on various lipid structures, including planar membranes and liposomes. S‐Layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological molecules enabling innovative approaches for the controlled ‘bottom‐up’ assembly of functional supramolecular structures and devices as required for life‐ and nonlife science applications.

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S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus

Cited by 48 publications

References 44 publications

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

Enhanced Neutralization of HIV by Antibodies Displayed on the S-Layer of Caulobacter crescentus

S‐Layers, Microbial, Biotechnological Applications

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