ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

5.4 -
Conclusions
The ARDRA patterns demonstrated putative relationships between the isolates
but did not show a robust pattern; there was fluctuation between the relatedness of
isolates and their position within the dendrogram. This is probably due to the way the
DNA fingerprints produced from the different restriction sites for the different enz mes
cluster in the analysis. DNA sequencing was used to identify the bacterial taxa.
Phylogenetic assessment using the 16S rRNA gene sequences for the bacterial isolates
showed a more similar pattern of relatedness between the isolates to the Alu I ARDRA
pattern, suggesting that this pattern may be a more accurate representation of the
relatedness of the isolates. The 19 bacterial isolates that demonstrated AFP activity from
the 866 isolates cultured and screened for activity were found predominantly within the y-
Proteobacteria, with one isolate from the a-Proteobacteria. To date, all characterised AFP
active bacteria have been shown to belong to the y-Proteobacteria (Sun et al., 1991;
Duman & Olsen, 1993; Mills, 1999) except Rhodococcus erythropolis which belongs to
the Actinobacteria (High G+C Gram-positive class of the phylum Firmicutes: Duman &
Olsen, 1993) suggesting that either there is a phylogenetic relationship for AFP proteins
within the y-Proteobacteria or that there is a bias towards AFP activity assessment of y-
Proteobacteria through their psychrophilic marine and lacustrine environmental isolation.
This shows a relatively close phylogenetic relatedness for AFP activity, suggesting that
the gene for the antifreeze protein has evolved in only one limited eubacterial phylum.
However, y- and a-Proteobacteria are considered to be the dominant eubacterial groups
within the Antarctic marine and aerobic lacustrine systems. Therefore, if environmental
factors are selecting for these groups based on halotolerant and psychrotrophic function,
then it is possible that AFP would evolve only in these groups. However, selective
culturing techniques should not be ruled out. Other dominant groups within the Antarctic
ecosystems e. g. the order Verrucomicrobiales, the class Actinobacteria, the
Clostridium/Bacillus
subphylum of the Gram positives and the Coophage-
Flavobacterium-Bacteroides
phylum, are not represented within the characterised AFP
strains. It is therefore possible that the culturing techniques used may have inadvertently
selected for Proteobacteria. Further discussion of this will be presented in Chapter 7.
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