Linker Mutagenesis of the Caulobacter crescentus S-Layer Protein
As a part of its life cycle, the gram-negative bacterium Caulobacter crescentus exhibits a characteristic morphological switch between a sessile stalked cell and a flagellated dispersive or swarmer cell. During this complex differentiation process however, both cell types continue to elaborate a paracrystalline S-layer of hexagonal organization. The S-layer is composed of a single 98 kDa secreted protein (RsaA) noncovalently attached to other protein monomers and to the surface of the outer membrane; the latter interactions may be mediated by calcium ions and a specific Slayer associated molecule present in the outer membrane. Because RsaA is a secreted protein which interacts with itself as well as other molecules present in the outer membrane, multiple functional regions are expected to exist within the protein including those involved in secretion, calcium binding, outer membrane attachment and formation of the core and connectivity regions of the S-layer. Despite this expectation, analysis of the translated nucleotide sequence of the rsaA gene has not revealed possible functional regions of the protein beyond the existence of a probable calcium binding region. Similarly, N-terminal amino acid sequencing and sequencing of C-terminal peptide fragments derived from the S-layer protein have shown that no N- or C-terminal processing of the protein occurs, providing few clues to the mechanism of secretion. However, gene fusion studies have shown that the first 35–52 amino acids of the RsaA N-terminus can direct reporter proteins to the periplasm. (For a review of the present state of knowledge surrounding the C. crescentus S-layer, see Bingle et al., this book).
KeywordsOuter Membrane Inclusion Body Formation Swarmer Cell Caulobacter Crescentus Cover Amino Acid
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