Molecular genetic studies of the surface layer of caulobacter crescentus : nucleotide sequencing and analysis of the regular surface array gene, and the effect of surface layer and other variables on the development of electroporation for the caulobacters

• Main Author: Gilchrist, Angus Robert
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/29849

The regular surface array gene (rsaA) of Caulobacter crescentus codes for the 130K surface layer (S-layer) protein. This S-layer protein forms a paracrystalline array which completely sheaths the bacterium and is comparable to those of many other bacteria. These studies investigate two aspects of the S-layer presented in two parts. In the first, the effect of S-layer on electroporation of Caulobacters is reported with the development of that technique for the transformation of Caulobacters. Many other interesting aspects to this procedure are also reported. The second part details the sequencing of the rsaA gene and includes a comprehensive review of the properties of the 130K S-layer protein as predicted from nucleotide sequence. A significant impediment to genetic analysis of Caulobacters has been the lack of a plasmid transformation procedure. High voltage electroporation-mediated plasmid transformation (electrotransformation) is unusually efficient with freshwater Caulobacters yielding up to 3 x 10⁸ transformants per µg of plasmid pKT230. Optimum conditions for electrotransformation of Caulobacters required a single pulse of high field strength and short duration. Changes in growth media to help adapt marine Caulobacters so that they would survive preparation for electroporation, along with a modification of the preparation regime, resulted in the electrotransformation of MCS6. Several genetic techniques were successfully applied using electroporation, including the direct introduction of ligation mixtures into bacteria, suicide mutagenesis, and the electrotransfer of plasmids from E. coli to Caulobacters. Presence of the S-layer greatly influenced electrotransformation. Caulobacters lacking S-layer protein were electrotransformed with approximately ten times greater efficiency than comparable strains with an S-layer. The nucleotide sequence of the 130K S-layer protein encoding regular surface array gene (rsaA) from Caulobacter crescentus CB15A was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature 130K protein and amino acid sequencing of retrievable peptides yielded two peptides: the first aligned with a region approximately two thirds of the way into the predicted amino acid sequence; the second peptide corresponded to the predicted carboxy terminus of the protein. Thus, no cleavage processing of the carboxy portion of 130K occurred during the export process and, with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted 130K amino acid profile was unusual, with small neutral residues predominating. With the exception of aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein with a predicted pi of 3.46. Secondary structure analysis did not predict any long stretches of regular structure. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted 130K sequence. However, 130K protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the 130K protein which was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in 130K may perform a similar function, since proper S-layer assembly and surface attachment requires calcium. 130K protein also shared some homology with ten other S-layer proteins with the surface array protein of Campylobacter fetus scoring highest. Codon usage for rsaA followed the strong codon bias exhibited by other C. crescentus genes. The reported DNA sequence increases the total known Caulobacter crescentus sequence by 30% and changes accepted codon usage frequencies. Manipulations of rsaA gene plasmid constructs, including subcloning of gene fragments and other routine genetic procedures indicated that many were toxic to Escherichia coli hosts, when presented within high copy number plasmids. The sequencing of the S-layer gene from a calcium independent mutant, CB15ACal0, and the background experimental history of this mutant are discussed. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate