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Chapter 5: BACTERIAL MOLECULAR TYPING
5.1 -
Introduction
Bacterial isolates that were shown to be antifreeze protein (AFP) active (Chapter
4) were characterised using a polyphasic taxonomic approach. combining amplified
ribosomal DNA restriction analysis (ARDRA) with 16S rRNA molecular sequencing.
These molecular phenetic and phylogenetic techniques were used to identify the isolates
that show AFP activity, to enable an understanding of the taxonomic distribution of
Antarctic bacterial AFP activity.
Characterisation of the isolates was performed using ARDRA, in which
endonucleases restriction of the 16S rRNA gene was used to delineate phenetic
relationships between bacterial isolates. The 16S rRNA gene is highly conserved,
approximately 1500bp long and contains sub-species specific variations (Vaneechoutte et
al., 1995; Vila et al., 1996; Rademaker & de Bruijn, 1997; Jawad et al., 1998). ARDRA
was used because it is a well established, rapid and repeatable technique which enables
sub-species identification between isolates (Vaneechoutte et al., 1995, Rademaker & de
Bruijn, 1997) and therefore could determine the phenetic relatedness of the AFP active
species, down to the sub-species level.
Sequencing of the 16S rRNA gene was performed using the chain-termination
method (Chapter 2, section 2.9.6.4; Sanger et al., 1977; Brown, 1996). The unidentified
sequence can then be compared against 16S rRNA sequences from known species and a
percentage similarity can be calculated to identify the isolate. By comparing the sequence
of an unidentified isolate with those of a number of similar identified sequences,
it is
possible to infer phylogenetic relationships between the sequences. These relationships
can be represented in a phylogenetic tree (Priest & Austin, 1995), which is used to
graphically represent a possible evolutionary pathways which link the represented species
to the unidentified isolate.
In the current study all 19 AFP active isolates were characterised using ARDRA
and molecular sequencing of the 16S rRNA gene to ascertain if AFP activity is present
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