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(AF288370), I. abyssalis (AF052740), I. zobellii (AF052741). I. baltica (AJ440214)) it was possible to identify the regions of the 16S rRNA gene which showed the greatest level of differentiation between species,. These were identified as bp 424-472. bp 1114- 1123 and bp 1242-1257 (Escherichia coli numbering). These regions of variation were all included within the 16S rDNA sequenced from isolate 53. Therefore, further characterisation of this species is necessary to determine its complete identification and discern whether this is a highly cold adapted strain of Idiomarina loihiensis or a different species of Idiomarina. 5.3.2.9 - The Halomonas sp group, isolate 213. Isolate 213 could not be definitely identified due to the lack of 16S rRNA sequence; only 179bp were retrieved for this particular isolate. However, those 179 bp did identify the bacterial strain as belonging to the genus Halomonas (Table 5.1). The ARDRA analysis clustered isolate 213 into a group with isolates 53 (Idiomarina loihiensis), 583 (Psychrobacter sp. ), 732 (Enterobacter agglomerans), 39 and 86 (Pseudoalteromonas sp. ). The relationship between these isolates varied with the two different restriction enzymes. Although isolate 213 could not be utilized for direct assessment in the phylogenetic alignment (Fig 5.4) the species Halomonas variablis (AJ306893) was included and indeed it did show a relationship with the genera Psychrobacter, Idiomarina, Pseudoalteromonas, Marinomonas and Enterobacter (Fig 5.4). Halomonas showed a closer grouping with Marinomonas in the phylogenetic tree (Fig 5.4), however, the estimated statistical likelihood of the occurrence of this grouping was 53%. Therefore there is a possibility that the Halomonas sp and the Arctic seawater bacterium grouping could of clustered with the Psychrobacter, Idiomarina, Pseudoalteromonas, Enterobacter Glade if a more robust branching pattern was calculated, however time constraints would not allow for this. The genus Halomonas has been readily identified as ubiquitous in the marine habitat (Kaye & Baross, 2000, Maruyama et al., 2000; Teske et al.. 2000; Sass et al.. 2001; Ivanova et al.. 2002), and has shown particular abundance in the deep-sea habitat and hydrothermal vents (Kaye & Baross, 2000; Teske et al.. 2000). The Halomona. s genus has also been identified in lakes of the Vestfold Hills (Bowman et al., 2000; James 156
et al., 1994) and the Antarctic SIMCO (Brown & Bowman; 2001). However, it has never been isolated from Triple Lake, Vestfold Hills, which demonstrates such an extreme hypersaline and cold environment, and it may therefore be a novel species or strain. Bowman et al.. (2000) did demonstrate a 16S rRNA clone from the sediments of Deep Lake (the most hypersaline lake in the Vestfold Hills) which showed a close relationship with Halomonas subglaciescola. This indicates not only that Halomonas species can exist in extremely saline and cold environments, but also that Halomonas species have been isolated from the lakes of the Vestfold Hills, thus increasing the likelihood of the identification of isolate 213 as a member of the genus Halomonas. The identification of isolate 213 as Halomonas sp is therefore not unexpected, but complete sequencing of the 16S rRNA gene would be necessary for this identification to be definitive. 5.3.2.10 - Isolate 466 Isolate 466 is shown using ARDRA analysis to be a distant relative of the ;t protea grouping this was demonstrated in both enzymatic digestions, but was more obvious in the Alu I digestion, where the phenetic similarity was -75%. Due to this assessment it was considered that it is possible that this strain does belong to the gamma Proteobacteria, however, sequence assessment of the 153bp 16S rRNA gene fragment for isolate 466 demonstrated a very weak relationship (87.2%) with Bacillus aquamarinus (AF202056) which is part of the Bacillus / Clostridium group, which has been noted in Antarctic lakes (Brambilla et al., 2001) and psychrophilic species have been noted in deep-sea sediments (Rüger et al., 2000), and while it is a marine based species (Yoon et al., 2001) the sequence similarity is to small to realistically consider it as a possibility. More conclusively isolate 466 was Gram negative whereas members of the genus Bacillus are Gram positive, and hence it is extremely unlikely that it could be related to this genus. It is thus possible that the sequence obtained is the result of PCR contamination. Hence no definitive identification of isolate 466 can be given at this point: further work was required to identify this isolate, but as it had a low level of AFP activity it was considered to be superfluous and so was not pursued further. 157
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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
Page 125 and 126: a Bacterial abundance (x 106 1.1) 0
Page 127 and 128: 8 ö a N Ö oý L _> Q O O 4 0 rn O
Page 129 and 130: undoubtedly true of Club Lake. The
Page 131 and 132: suggested that input of bacteria an
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 and 164: e included due to its small sequenc
Page 165 and 166: 5.3.2 - Identification of the AFP a
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: Phylogenetic cluster alignment of t
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
Page 203 and 204: (Section 6.3.2.4.3 ) could not be r
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
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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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. + + + I I I I I I + I + + I I I I
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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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+ + i i i i i i " i " . + + + + " +
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e O --ý N M t Vn 1,0 [-- 00 O> N M
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FT ce + I I r. to \, O t- 00 O\ N M
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kn 110 r- 00 Oý N M O - N M Itt vn
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, It N M "t N M Iýt N M M M M Oý
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gj a - - - - - - - - - - - - - - -
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