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Bacterial Genbank Size of sequenced Closest relative (EMBL prokaryote Isolate accession 16S rDNA nucleotide database, using FASTA Number number fragment (bp) search engine) 494 na 153 Sphingomonas sp. Accession number AF395031 (100%) 213 na 179 Halomonas sp. Acc. No. ABO 16049 (97.2%) 302 AY092072 1370 Pseudomonas fluorescens Acc. No. AF228367 (99.92%) 33 AY092073 1370 Antarctic Seawater Bacterium Acc. No. AJ293825 (99.927%) 53 AY092077 1381 Idiomarina loihiensis Acc. No. AF288370 (99.348%) 86 AY092080 962 Pseudoalteromonas sp. Acc. No. U85856 (99.476%) 39 AY092074 1376 Pseudoalteromonas sp. Acc. No. U85856 (99.636%) 732 AY092079 1493 Enterobacter agglomerans Acc. No. AF348161 (99.512%) 492 AY092076 408 Stenotrophomonas maltophilia. Acc. No. AJ293464 (99.020%) 47 AY092075 1182 Stenotrophomonas maltophilia Acc. No. AJ293466 (99.833%) 583 AY092078 814 Psychrobacter sp. Acc. No. AJ272303 (94.711%) 124 AY092065 886 Marinomonas protea, Acc. No. AJ238597 (100%) 154 AY092066 1372 Marinomonas protea, Acc. No. AJ238597 (100%) 214 AY092067 972 Marinomonas protea, Acc. No. AJ238597 (100%) 5ice3 AY092068 277 Marinomonas protea, Acc. No. AJ238597 (100%) 54 AY092069 965 Marinomonas protea, Acc. No. AJ238597 (100%) 744 AY092070 1019 Marinomonas protea, Acc. No. AJ238597 (100%) 794 AY092071 970 Marinomonas protea. Acc. No. AJ238597 (100%) 466 na 567 Bacillus aquamarinus Acc. No. AF202056 (87.223%) Table 5.1 Original isolate - number, Genbank accession number, the size of the sequenced 16S rDNA fragment used for identification and the closest relative identified by FASTA search of the EMBL prokaryote nucleotide database. na = not available, these sequences were not entered into the Genbank database due to their shortness of length. Isolate 466 shows only 87% similarity to Bacillus aquamarinus, thus although included here it is not considered to be an accurate identification. 138
identification was made based on 153 bp of sequence and therefore the identification is considered to be of low confidence. Isolate 583 showed a 94.7% similarity to an unspeciated Psychrobacter sp. (AJ272303). Isolates 213 had a sequence similarity with an unspeciated Halomonas sp. (AB016049) of 97.2% from a sequence length of 179bp. Isolate 466 showed no close similarity to any sequence in the database for the 567bp entered. Therefore, isolate 466 will require further investigation to provide an accurate identification. Growth media Isolate 302 Isolate 33 Fluorescens Agar + - CFC Agar + + Table 5.2 - Growth results for suspected pseudomonad species isolates on pseudomonad selective agar and fluorescein selective agar. 5.2.3 - Phylogenetic tree 16S rDNA sequences for each of the 19 AFP active bacterial isolates from this study were aligned and analysed against the electropherograms from the sequencing results (see chapter 2). The consensus sequences for each of the 19 isolates were then compared against the Genbank nucleotide database and the nearest known phylogenetic relatives for each isolate were determined (Table 5.3). Only 13 of the isolates had sufficient sequence length for analysis, which was determined as being over 800 bp of contiguous sequence. Therefore, isolates 494 (153bp), 213 (179bp), 492 (408bp), 466 (567bp) and 5ice3 (277bp) could not be included in the alignment. Also removed was isolate 47 as although it comprised 1182bp of sequence, that sequence was not contiguous, it included a 300bp `gap' in the middle and so was not of sufficient contiguous length for alignment. An 887 bp consensus sequence for each of the remaining 13 isolates were re-aligned with the sequences of the nearest known relatives using PileUp (GCG package), which aligns the sequences using progressive pairwise alignment. This was the shortest contiguous sequence length in the analysis. The same 887 bp region was used for all isolates to enable an accurate phylogeny to be calculated. A total of 18 published species were aligned (Table 5.3) these included species that Nt ere putatively related to the 6 isolates which could not be included in the alignment. Isolate 494 was identified as Sphingomonas sp, which is the only identification belonging to a 139
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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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Page 109 and 110: The temperature (Fig 3.2a) through
Page 111 and 112: 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
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: The closest published relative for
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 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
Page 203 and 204: (Section 6.3.2.4.3 ) could not be r
Page 205 and 206: most dominant species in the commun
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11 species) showed AFP activity. Th
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that AFP activity could have evolve
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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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. + + + 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 - - - - - - - - - - - - - - -
show all

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