UBC Theses and Dissertations
Cloning, sequencing and physicochemical characterization of a hemagglutinin from Escherichia coli 09:H10:K99 Lutwyche, Peter
A mannose-resistant hemagglutinating (erythrocyte aggregating)protein was cloned from Escherichia coli 09:H10:K99. This hemagglutinin was determined to be different from the two mannose-resistant hemagglutinins that this strain was known to possess, F41 and K99, and was named Heat Resistant Agglutinin 1 (HRA1) on the basis of one of its physical properties. The HRA1 gene present on the recombinant plasmidpETE1 was localized and identified unambiguously by subcloning. The nucleotide sequence of the gene was determined and found to consist of a 792 base pair open reading frame coding for a protein of 29 kDa. This protein has a predicted prokaryotic N-terminal secretory signal sequence. The protein sequence derived from the genetic information showed no significant similarity to database protein sequences. Physical and chemical attempts to isolate HRA1 from the cell membrane met with limited success. N-terminal sequence analysis of a pronounced 25 kDa band present on polyacrylamide gels of crude membrane preparations of bacteria harbouring pETE1 correlated with the predicted N-terminal amino acid sequence of HRA1 after cleavage of the signal peptide. Four other open reading frames were found on the cloned DNA fragment. Three of the deduced proteins from these regions contained predicted signal sequences and membrane associated areas and one showed50 % identity with E.coli lipoprotein, suggesting that a region of DNA associated with outer membrane structure and function was cloned. Partition in various aqueous two-phase polymer systems showed that expression of HRA1 caused pronounced cell-surface changes in host bacteria. Viscometric analysis showed that the agglutination event mediated by HRA1 was less pronounced than that of F41. This was likely due to the fact that F41 is known to be an exposed high molecular weight multivalent adhesin, whereas evidence suggests that HRA1 is a monovalent molecule that is closely associated with the bacterial membrane.
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