Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus The GenBank accession number for the NA1000 rsaADEF and gmd, per, wbqA, wbqR, wbqX and wbqZ sequences reported in this paper is AF06235.

Awram, Peter; Smit, John

doi:10.1099/00221287-147-6-1451

Cited by 46 publications

(30 citation statements)

References 43 publications

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“…The x axis of the HPLC spectrum is the elution time (in minutes); the y axis is the absorbance. unable to anchor the exported protein to its lipopolysaccharide layer (43,52). Electron microscopy images also showed that the peptidoglycan layer of the ⌬sll1213 strain was less electron dense than in the ⌬sll1951 strain or the Synechocystis wild type (Fig.…”

Section: Discussionmentioning

confidence: 92%

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Trautner

Vermaas

2013

Journal of Bacteriology

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Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negativestain transmission electron microscopy, a ϳ30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the ⌬sll1951 strain. Instead, the ⌬sll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the ⌬sll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the ⌬sll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the ⌬sll1951 strain was similar to that of the wild-type strain, the viability of the ⌬sll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.

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Section: Discussionmentioning

confidence: 92%

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Trautner

Vermaas

2013

Journal of Bacteriology

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“…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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confidence: 99%

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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“…Mutations (8) and truncations (9) in the RsaA extreme N terminus lead to an S-layer shedding phenotype. A species of smooth lipopolysaccharide (SLPS), whose O side chain is composed at least in part of N-acetylperosamine, is found on the outer membrane of C. crescentus (2). This SLPS is at least involved in S-layer anchoring, since strains deficient in O side chain biogenesis shed S-layer (2), (40).…”

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confidence: 99%

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

2007

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The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunitsubunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first ϳ225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first ϳ225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.

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Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus The GenBank accession number for the NA1000 rsaADEF and gmd, per, wbqA, wbqR, wbqX and wbqZ sequences reported in this paper is AF06235.

Cited by 46 publications

References 43 publications

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

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

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

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