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coli 0157: H7 by semi-automated fluorescent AFLP analysis. Microbcs and Infection. 2,107-113. ZUBER, H. (1988). Temperature adaptation <strong>of</strong> lactate dehydrogenase. Structural, functional and genetic aspects. Biophysical Chemistry. 29,171-179. 24
APPENDICES Al. Media Tryptic Soya Agar (TSA) Tryptic Soya Broth (Sigma) Agar (Difco) Sterile RO Water 30 g 15 g 1L Yeast Malt Agar (YMA) Yeast Extract (Sigma) Malt (Sigma) Peptone (Sigma) Dextrose (Sigma) Agar (Difco) Sterile RO Water 3g 3g 5g 10 g 20 g 1L Seawater Aizar (SWA Yeast Extract (Sigma) Peptone (Sigma) Salt (Coral Life) Agar (Difco) Sterile RO Water 1g 1g 38 g 15 g 1L '/2 Seawater Agar (1/2 SWA Yeast Extract (Sigma) Peptone (Sigma) Salt (Coral Life) Agar (Difco) Sterile RO Water 1g 1g 19 g 15 g 1L For each recipe the corresponding liquid media was made without the agar. For each recipe the corresponding cryo-preservation liquid medium was made containing 10% glycerol (Sigma), which was added after incubation time immediately prior to freezing. A2 Buffers and Reagents Protein Extraction Buffer (PEB) 25 mM Tris (Tris{hydroxymethyl}amino-methane)/HC1 pH 7.0 1 mM EDTA (Ethylenediamine-tetraacetic acid) 1 mM PMSF* (Phenylmethylsulphonyl fluoride) from a 100mM stock in ethanol. 2 µg ml-1 Pepstatin A* from a 2.5mg stock in methanol. 235
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COLD ADAPTATION STRATEGIES AND DIVE
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, 71 "If you march your Winter Jour
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1.6.3. Molecular typing techniques
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3.2.1.2.3. DOC and chlorophyll a ..
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5.3.2.5. The Sphingomonas group, is
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LIST OF FIGURES Figure I. I. Diagra
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Figure 3.17a Mean bacterial abundan
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LIST OF TABLES Table 1.1. Types of
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List of Abbreviations AFP AFGP AFLP
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ACKNOWLEDGEMENTS This study was fun
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Chapter 1: INTRODUCTION 1.1 - Tempe
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1999 ; Hand & Burton, 1981) or iden
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Cold adaptation is dependant on the
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not at 25°C is a function of the a
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The most prolific literature on col
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process known as thermal hysteresis
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dominant, role in the ice-growth in
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explanation for this diversity is t
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1.5.3.5 - Membrane bound solute tra
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een well established (Russell & Nic
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& Bowman, 2001; Labrenz & Hirsch, 2
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The reaction mix contains both deox
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gene mixtures and hence can be used
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displaying a high number of common
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AACGTCGAAA AACCTCGAAA (Organism A)
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particular set of environmental par
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Lake Maximum Depth (m) Approximate
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Larsemann Hills __ rp3rtrnent of th
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Sampling trips took two days during
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Sigma, Sigma-Aldrich Company Ltd.,
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oxidized and the sample then conver
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sample. An appropriate volume was l
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min to ensure complete extension of
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Figure 2.3 - Lane delineation image
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Figure 2.6b - Molecular weights are
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RNA 41F (5'-GCT CAG ATT GAA CGC TGG
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I min. The spin column was removed
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compiled using Seqman and any erron
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ecorded and related to the level of
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information regarding the cold adap
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50ppt to 186ppt. Thirteen were sali
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M N N 't M M \p \p 00 kf) M kf) bA
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as would normally be expected, as b
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1444 6ý L CC vý O Q O O NO u 'c z
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5 - 250 3 245 240 G (, -3 235 a 0-
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3.2.2.2.5 - Dissolved organic carbo
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d 14 12 10 14 12 7 10 8 6 4 2 0 00/
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SRP ('g L- 05 10 15 20 2 3 Depth4 5
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3.2.2.3 - Biological characteristic
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a 0 Bacterial Abundance (x 106 ml-1
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3.3 - Discussion 3.3.1 - Summer sam
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to high surface water temperatures
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chemocline (Rankin et al., 1999). M
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a 250 200 150 100 50 70 60 50 40 ý
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The temperature (Fig 3.2a) through
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et al., 1999). No melt out was obse
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increase in ammonium is probably du
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10 7 9 6 8 E ö 7 5 Co u c m c 40 w
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main pigments were chlorophylls a a
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Inorganic nitrogen (NO2, NO3 and NH
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744 -- 7 644 6 19 ü SE 400-- 4 2s
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salinity decreased from July to Sep
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a Bacterial abundance (x 106 1.1) 0
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8 ö a N Ö oý L _> Q O O 4 0 rn O
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undoubtedly true of Club Lake. The
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suggested that input of bacteria an
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Pendant Lake is an unstratified, sa
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non-AFP active peptides from inhibi
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. ter rý :. 'ý Figure 4.2 -Fryka
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-ý- ---- ,. r. __-_-. ___. r. ýý
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4.2 - Results 4.2.1 - Sampling and
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SPLAT score. It has been shown that
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3'E t ý _d M! .PJ r rn 15 3.5 4 3
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4.3 - Discussion Of the 866 bacteri
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arisen in very few species due to c
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Chapter 5: BACTERIAL MOLECULAR TYPI
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Fig 5.1 - Photographs of ARDRA gel
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Coefficient: Dice Algorithm: lPGMAA
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The closest published relative for
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identification was made based on 15
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Isolate number Closest phylogenetic
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e included due to its small sequenc
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5.3.2 - Identification of the AFP a
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explains the fluctuation in related
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contamination was lower as the PCR
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seen to a certain extent in the ARD
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American Type Culture Collection (A
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Phylogenetic cluster alignment of t
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et al., 1994) and the Antarctic SIM
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Chapter 6: BACTERIAL COMMUNITY ANAL
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6.2.1 - Detection of AFP active iso
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using the same PCR protocol as orig
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Figure 6.1 - Denaturing gradient ge
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6.2.2 - Temporal and spatial variat
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Figure 6.4c - Gel I image showing b
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Coefficient: Dice Algorithm: UPGMA
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etween Om and 2m was obviously the
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All PCR-based rRNA molecular techni
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6.3.2.3 - Cellular lysis and DNA pu
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hybrids should be equal. However, S
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contain contaminants. If the profil
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Page 205 and 206: most dominant species in the commun
Page 207 and 208: study was characterised as M protea
Page 209 and 210: 11 species) showed AFP activity. Th
Page 211 and 212: that AFP activity could have evolve
Page 213 and 214: 7.2 - Future Work The 19 AFP active
Page 215 and 216: REFERENCES ABYZOV, S. S. (1993). Mi
Page 217 and 218: BELL, E. M. and LAYBOURN-PARRY, J.
Page 219 and 220: Symposium on Environmental Biogeoch
Page 221 and 222: Research. 5,477-493. CLESCERI, L. S
Page 223 and 224: DRICKAMER, K. (1999). C-type lectin
Page 225 and 226: antifreeze proteins from Atlantic s
Page 227 and 228: FRANZMANN, P. D. and DOBSON, S. J.
Page 229 and 230: GRIFFITH, M., ALA, P., YANG. D. S.
Page 231 and 232: IVANOVA, E. P., KIPRIANOVA, E. A.,
Page 233 and 234: Antarctic Ecosystems. G. A. Llano (
Page 235 and 236: Howard-Williams and I. Hawes). Balk
Page 237 and 238: W. (1998). Rep-PCR-mediated genomic
Page 239 and 240: putida strains from Antarctica. Mic
Page 241 and 242: NICHOLS, D., BOWMAN, J., SANDERSON,
Page 243 and 244: PEARCE, D. A. and BUTLER, H. G. (20
Page 245 and 246: (1999). Palaeohydrological modellin
Page 247 and 248: tracers of biogeochemical processes
Page 249 and 250: R. G. Landes Company, Austin, Texas
Page 251 and 252: partial 16S rDNA sequencing. Applie
Page 253: YABUUCHI, E., WANG, L., ARAKAWA, M.
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