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conferenceseries.com
Volume 7, Issue 6 (Suppl) J Bacteriol Parasito
ISSN: 2155-9597 JBP, an open access journal Microbiology 2016
November 28-29, 2016
November 28-29, 2016 Valencia, Spain
7th World Congress on
Microbiology
Tine Descamps et al., J Bacteriol Parasitol 2016, 7:6 (Suppl)
Transposon-mutagenesis based identification of virulence factors of Paenibacillus larvae
Tine Descamps, Lina De Smet, Paul De Vos and Dirk C de Graaf
Ghent University, Belgium
Paenibacillus larvae, is an etiological agent of American Foulbrood, a deadly disease of the European Honey Bee (Apis mellifera).
American Foulbrood is the most important bacterial honey bee disease but relatively little is known of its virulence. In recent
years, however, some virulence factors have been identified. In our research, the goal is to identify virulence factors of the pathogen
in an unbiased way. This is accomplished by EZ-Tn5 transposon mutagenesis. Using the EZ-Tn5 transposome complex (Epibio),
a library of random knock-out transformants was created. The virulence of these transformants was tested using infection assays.
Spores were fed to 1st instar honey bee larvae, which were further reared in vitro. Of the 158 transformants, only 93 were able to
sporulate. Preliminary tests with these 93 transformants rendered 15 with atypical virulence compared with the wild type. These
15 transformants were fully tested using three independent infection groups with 30 larvae each. Statistical test confirmed that
7 transformants had a mortality rate that was significantly lower than the wild type. Identification of the interrupted genes was
done using rescue cloning. The genomic DNA was sheered by three restriction enzymes, blunt ended by T4 DNA polymerase and
circulized by T4 DNA ligase. Since the transposon carried R6Kγori and kanamycin resistance marker, selection of plasmids carrying
the transposon could be done by cloning into pir E. coli. In a final step, the regions flanking the transposon were sequenced to identify
the knocked out gene.
Biography
Tine Descamps has completed her Master’s degree in Biochemistry and Biotechnology in 2013 and is currently pursuing PhD in the Laboratory for Molecular
Entomology and Bee Pathology (L-MEB) at Ghent University, Belgium.