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arisen in very few species due to chance mutation. During this study AFP activity has
been shown to occur in strains of common bacterial species, such as Pseudomonas
fluorescens (Chapter 5), which is known to inhabit household refrigeration systems
(Dodd, pers. com. ). It is assumed that the P. flourescens isolated in Antarctica is a
separate strain from that isolated from refrigeration systems, but it suggests that AFP
activity might not be a rare condition, but in fact a cold induced protein that might occur
in all psychrophilic bacteria. It is indeed possible that all of the Antarctic isolated bacteria
may be genotypically AFP positive, but the conditions under which they were cultured
may not have been those necessary for AFP induction. If this is correct, then there could
be a very high proportion of false negatives within the 866 bacteria cultivated. There
could also be bacteria which do express the protein under the culture conditions but the
protein is degraded or inactivated in someway during protein extraction or during the
AFP analysis itself. However, there is no evidence to suggest this. Isolates known to have
activity, e. g. Marinomonas protea, do show activity using these conditions and these
assays. As AFP from other sources (e. g. fish, insects, plants) are known to be extremely
variable in structure (Yeh & Feeney, 1996; Davies & Sykes, 1997; Smaglik, 1998), it is
possible that the conditions used to assess Antarctic bacteria during the current study,
may have been ineffective at identifying activity of certain types of AFP. More detailed
analysis will have to be performed to isolate the proteins and, subsequently, the genes
which code for them, to help assess whether AFP activity is present within these putative
non-active isolates, using specific oligonucleotide probes which identify specific
conserved regions of the AFP gene. To date there has been no known successful
published work on a bacterial AFP gene and, therefore, it is unknown as to whether there
are conserved regions.
AFPs have shown an ability to alter the structure of ice crystals. Barrett (2001)
suggests that AFPs alter the ice growth morphology significantly. producing bypy'rimidal
crystallites and columnar spicules. The visualisation required to observe crystal shapes
was not performed for the current study due to time constraints. It is suggested that AFPs
bind to the bypyrimidal planes. so that when crystal growth occurs close to the hN steresis
temperature (lowered freezing point), it occurs along the c-axis of the crystal (Grandum
et al.. 1999). Therefore, AFPs bind to a particular face of the embryonic ice crystal and
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