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Isolate Closest relative (EMBL prokaryotic nucleotide Lake of isolation Depth of isolation Date of isolation database, using FASTA search engine) 124 Marinomonas protea Ace Lake 8m 13/11/00 154 Marinomonas protea Ace Lake 4m ]3/11/00 214 Marinomonas protea Ace Lake 2m 131/11/00 5ice3 Marinomonas protea Pendant Ice Core 50cm from ice/water interface 07/09/00 54 Marinomonas protea Pendant Lake 4m 13/11/00 744 Marinomonas protea Pendant Ice Core 794 Marinomonas protea Pendant Ice Core 50cm from ice/water interface Ice/Water interface 13/11/00 13/11/00 39 Pseudoalteromonas sp. Ace Lake 4m 14/01/00 86 Pseudoalteromonas sp. Ace Lake 4m 14/01/00 302 Pseudomonasfluorescens Triple Lake Om 23/01/00 33 Antarctic seawater Triple Lake 2m 17/07/00 bacterium 53 Idiomarina loihiensis Triple Lake 8m 07/09/00 732 Enterobacter agglomerans Ace Lake 2m 04/03/00 47 Stenotrophomonas Ace Lake 6m 14/01/00 maltophilia 492 Stenotrophomonas Ace Lake Om 15/07/00 maltophilia 583 Psychrobacter sp. Triple Lake Om 07/09/00 494 Sphingomonas sp Pendant Lake Om 13/11/00 213 Halomonas sp. Triple Lake 4m 07/09/00 466 Bacillus sp. * Oval Lake Om 04/02/00 Table 5.4 - Isolate number with closest relative from the EMBL prokaryotic nucleotide database using the FASTA search engine, lake of isolation and the depth in the water column or ice core of isolation. *This identification is extremely tenuous. 144
5.3.2 - Identification of the AFP active isolates 5.3.2.1 - The Marinomonas protea group, 124,154,214,54, -44,794 and 5 icC3. Both the M protea taxon (which comprises isolates 124,154,214,54,744,794 & 5ice3 ) and the Pseudoalteromonas sp. taxon (comprising isolates 86 and 39) have a robust phenetic relationship when compared using ARDRA, demonstrated by the fact that the grouping remains the same using the 2 different restriction enzymes. Based on subsequent 16S rRNA analysis it can be stated that these isolates are identical species, 124,154,214,5ice3,54,744 and 794, they are all Marinomonas protea (Fig 5.4). This species was first isolated from Ace Lake (Vestfold Hills, Antarctica) in 1996 (Mills. 1999). However, these bacterial strains were not all isolated from Ace Lake (Table 5.4). Although three of these isolates, 124,154 and 214 were isolated from various depths in Ace Lake on the 13th November 2000, the rest were isolated from Pendant Lake on the same date except 5ice3 which was isolated in September 2000. Three of these isolates came from the Pendant Lake ice cores (5ice3,744 & 794). These lakes were sampled throughout the year 2000 and yet the majority of AFP active M. protea was isolated on the 13th November 2000. This suggests that M. protea is more abundant within the bacterial population around late winter/early summer and hence is more readily isolated (Dang & Lovell, 2002). However, it is also possible that experimental procedure could have caused such an anomaly, for example through unintentional preferential culturing of M. protea. Until the remainder of the bacterial isolates, which were isolated from the lakes of the Vestfold Hills for the current study, are characterised to species level it will be impossible to determine whether the dominance of M. protea during the November sampling period for AFP active bacteria is an experimental artefact or represents actual variation in bacterial dominance. Two bacterial isolates (AF277471 & AF277469) closely related (97%) to M. protea, have also been isolated from the sea ice microbial communities (SIMCO, Brown and Bowman, 2001), and the majority of Marinomonax species are found directly associated with the marine environment (Van Landschoot and De Ley, 1983) suggesting that the genus could have evolved from a marine based organism, which is supported by the fact that both Ace and Pendant Lakes are marine 145
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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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Page 117 and 118: main pigments were chlorophylls a a
Page 119 and 120: Inorganic nitrogen (NO2, NO3 and NH
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Page 123 and 124: salinity decreased from July to Sep
Page 125 and 126: a Bacterial abundance (x 106 1.1) 0
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Page 129 and 130: undoubtedly true of Club Lake. The
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Page 133 and 134: Pendant Lake is an unstratified, sa
Page 135 and 136: non-AFP active peptides from inhibi
Page 137 and 138: . ter rý :. 'ý Figure 4.2 -Fryka
Page 139 and 140: -ý- ---- ,. r. __-_-. ___. r. ýý
Page 141 and 142: 4.2 - Results 4.2.1 - Sampling and
Page 143 and 144: SPLAT score. It has been shown that
Page 145 and 146: 3'E t ý _d M! .PJ r rn 15 3.5 4 3
Page 147 and 148: 4.3 - Discussion Of the 866 bacteri
Page 149 and 150: arisen in very few species due to c
Page 151 and 152: Chapter 5: BACTERIAL MOLECULAR TYPI
Page 153 and 154: Fig 5.1 - Photographs of ARDRA gel
Page 155 and 156: Coefficient: Dice Algorithm: lPGMAA
Page 157 and 158: The closest published relative for
Page 159 and 160: identification was made based on 15
Page 161 and 162: Isolate number Closest phylogenetic
Page 163: e included due to its small sequenc
Page 167 and 168: explains the fluctuation in related
Page 169 and 170: contamination was lower as the PCR
Page 171 and 172: seen to a certain extent in the ARD
Page 173 and 174: American Type Culture Collection (A
Page 175 and 176: Phylogenetic cluster alignment of t
Page 177 and 178: et al., 1994) and the Antarctic SIM
Page 179 and 180: Chapter 6: BACTERIAL COMMUNITY ANAL
Page 181 and 182: 6.2.1 - Detection of AFP active iso
Page 183 and 184: using the same PCR protocol as orig
Page 185 and 186: Figure 6.1 - Denaturing gradient ge
Page 187 and 188: 6.2.2 - Temporal and spatial variat
Page 189 and 190: Figure 6.4c - Gel I image showing b
Page 191 and 192: Coefficient: Dice Algorithm: UPGMA
Page 193 and 194: etween Om and 2m was obviously the
Page 195 and 196: All PCR-based rRNA molecular techni
Page 197 and 198: 6.3.2.3 - Cellular lysis and DNA pu
Page 199 and 200: hybrids should be equal. However, S
Page 201 and 202: contain contaminants. If the profil
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Page 205 and 206: most dominant species in the commun
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Page 209 and 210: 11 species) showed AFP activity. Th
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Page 213 and 214: 7.2 - Future Work The 19 AFP active
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REFERENCES ABYZOV, S. S. (1993). Mi
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BELL, E. M. and LAYBOURN-PARRY, J.
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Symposium on Environmental Biogeoch
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Research. 5,477-493. CLESCERI, L. S
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DRICKAMER, K. (1999). C-type lectin
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antifreeze proteins from Atlantic s
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FRANZMANN, P. D. and DOBSON, S. J.
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GRIFFITH, M., ALA, P., YANG. D. S.
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IVANOVA, E. P., KIPRIANOVA, E. A.,
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Antarctic Ecosystems. G. A. Llano (
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Howard-Williams and I. Hawes). Balk
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W. (1998). Rep-PCR-mediated genomic
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putida strains from Antarctica. Mic
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NICHOLS, D., BOWMAN, J., SANDERSON,
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PEARCE, D. A. and BUTLER, H. G. (20
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(1999). Palaeohydrological modellin
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tracers of biogeochemical processes
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R. G. Landes Company, Austin, Texas
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partial 16S rDNA sequencing. Applie
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YABUUCHI, E., WANG, L., ARAKAWA, M.
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APPENDICES Al. Media Tryptic Soya A
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"D C 'O I. .. r .. r 6 rr E , 4mo .
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Ö ON N M 1 O - N 'zt "o l'" 00 C N
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1 T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
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1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
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1 1 1 1 I 1 + I I 1 I 1 1 I 1 1 1 I
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1 1 1 1 1 1 1 I I 1 I 1 I 1 1 I T I
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Cl M 'tt tn '. p t- 00 O\ N M O - (
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d' O - (N M 'nt V) \O N. 00 O> N M
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1 1 T 1 1 1 T 1 1 1 1 1 1 1 1 1 1 1
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e O --ý N M t Vn 1,0 [-- 00 O> N M
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