The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-12112003-115308 |
| Date | 11 December 2003 |
| Creators | Chaban, Bonnie |
| Contributors | Ziola, Barry, Xiao, Wei, Hemmingsen, Sean M., Deneer, Harry |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-12112003-115308/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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