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Analysis of the s-layer transporter mechanism and smooth lipopolysaccharide synthesis in caulobacter crescentus Awram, Peter Alan


C. crescentus is a Gram-negative bacterium that possesses an hexagonal array called the S-layer that covers the entire outer surface of the bacterium. This array is composed of an estimated 60 000 copies of the 98 kDa protein RsaA. RsaA secretion is directed by a C-terminal secretion signal located in the last 82 amino acids of the protein. Once RsaA is secreted from the cell, it assembles into the S-layer and attaches to the outer membrane via a specific species of smooth lipopolysaccharide (S-LPS). The mechanisms required for the secretion of RsaA and the synthesis of the S-LPS were examined in this thesis. Tn5 mutagenesis of wildtype C. crescentus demonstrated the presence of two genes, rsaD and rsaE, 3' of the rsaA gene that were required for transport of RsaA. These genes were isolated and are capable of complementing the Tn5 mutations 3' of RsaA in trans. The resulting proteins of rsaD and rsaE belong to the type I secretion family that uses three components: an ATP Binding Cassette-transporter (RsaD), a Membrane Fusion Protein (RsaE) and an outer membrane protein (OMP), to secrete proteins through both membranes of Gramnegative bacteria. The OMP, RsaF, of the Rsa system was found by screening the partial Caulobacter genome sequence for sequence identity to other type I OMPs. The gene for RsaF is found 5 kb 3' of rsaE. Deletion of the N-terminus or C-terminus of RsaF prevents the Rsa secretion mechanism from functioning. The secretion of the S-layer subunits in a number of other Caulobacter species was also examined. A partial ORF from FWC27 with 44.6% identity to RsaA was isolated. In addition, the ABC-transporter components from FWC6, FWC8 and FWC39 were isolated. These components were >95% identical to RsaD. These results were used to explore the evolutionary relationships between the different Caulobacter species. Eighteen Tn5 mutations resulting in the inability of the S-layer to attach to the surface of the bacterium were also isolated. Southern blot analysis demonstrated that twelve of these insertions were linked to the Rsa transporters. The Tn5 insertion points were isolated and sequenced allowing identification of several putative genes involved in S-LPS synthesis from the Caulobacter genome sequence. A total of twelve open reading frames (ORFs) were found by Tn5 mapping and two more were found 3' of rsaE. Six of these putative genes may code for proteins involved in the synthesis of sugar residues including five that make perosamine. Five of the genes appear to be glycosyltransferases involved in forming the linkages between sugar residues in the O-antigen. One of the genes appears to be a repressor, while the remaining genes are unidentified. These data suggest that the major component of the O-antigen is perosamine and that a number of different linkages are made between the perosamine residues.

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