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deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Archives of Microbiology. 164.165-172. MUYZER, G. and SMALLA, K. (1998). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek. 73.127-141. MYERS, R. M., FISCHER, S. G., LARMAN, L. S. and MANIATIS. T. (1985). Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research. 13, 3131-3145. MYLVAGANAM, S. and DENNIS, P. P. (1992). Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui. Genetics, 130,399-410. NAGANUMA, T. (2000). Microbes on the edge of the global biosphere. Uchu Scihutsu Kagaku. 14,4,323-331. NAJM, I. & MARCINKO, J. (1995). Impact of Analytical Methodology and Water Quality on TOC Analytical Result. Proceedings of 1995 Water Quality Technology Conference, 71-77. NARINX, E., BAISE, E. and GERDAY, C. (1997). Subtilisin from psychrophilic Antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Engineering. 10.11.1271- 1279. NEEFS, J. -M., VAN DE PEER, Y., HENDRIKS, L. and DE WACHTER, R. (1990). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 18, 2237-2317. NICHOLAS, K. B. and NICHOLAS, H. B. Jr. (1997). Genedoc: A tool for editing and annotating multiple sequence alignments. Distributed by the Author. NICHOLS, D. S., NICHOLS, P. D. and McMEEKIN. T. A. (1993). Polyunsaturated fatty acids in Antarctic bacteria. Antarctic Science. 5,2,149-160. NICHOLS, D. S., BROWN. J. L., NICHOLS. P. D. and McMEEKIN. T. A. (1997). Production of eicosapentaenoic and arachidonic acids by an Antarctic bacterium: response to growth temperature. FEMS Microbiology Letters. 152.349- 354. 220
NICHOLS, D., BOWMAN, J., SANDERSON, K., MANCUSO NICHOLS. C.. LEWIS. T., McMEEKIN, T. and NICHOLS, P. D. (1999). Developments with Antarctic microorganisms: culture collections, bioactivity screening, taxonom\ . PUFA production and cold-adapted enzymes. Current Opinion in Biotechnolog -. I0. 240-246. NICOLAISEN, M. H. and RAMSING, N. B. (2002). Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. Journal of Microbiological Methods. 1618, In Press. NOBEL, U., ENGELEN, B., FELSKE, A., SNAIDR, J., WIESHUBER, A., AMANN, R. I., LUDWIG, W. and BACKHAUS, H. (1996). Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. Journal of Bacteriology. 178,19,5636-5643. NOBEL, U., GARCIA-PICHEL, F., CLAVERO, E. and MUYZER, G. (2000). Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient. Environmental Microbiology. 2.2,217- 226. OBATA, H., ISHIGAKI, H. KAWAHARA, H. and YARMADE. K. (1998). Purification and characterization of a novel cold-regulated protein from an ice-nucleating bacterium, Pseudomonasfluorescens KUIN-1. Biosciences, Biotechnology and Biochemistry. 62,11,2091-2097. OGASAWARA, N., NAKAI, S. and JOSHIKAWA, H. (1994). Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Research, 1,1-14. OLSEN, G. J., LANE, D. J., GIOVANNONI, S. J., PACE, N. R. and STAHL. D. A. (1986). Microbial ecology and evolution: a ribosomal RNA approach. Annual Review of Microbiology. 40.337-365. OPPENHEIMER, C. H. (1970). Temperature. In: Microbial Ecologn'. Volume I. pp. 47- 361. (Ed. 0. Kinne). Wiley, New York. OVREAS, L., FORNEY, L.. DAAE, F. L. and TORSVIK, V. (1997). Distribution of bacterioplankton in meromictic lake Saelenvannet. as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S 1
Page 1 and 2:
COLD ADAPTATION STRATEGIES AND DIVE
Page 3 and 4:
, 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
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
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: putida strains from Antarctica. Mic
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 and 254: YABUUCHI, E., WANG, L., ARAKAWA, M.
Page 255 and 256: APPENDICES Al. Media Tryptic Soya A
Page 257 and 258: "D C 'O I. .. r .. r 6 rr E , 4mo .
Page 259 and 260: . + + + I I I I I I + I + + I I I I
Page 261 and 262: Ö ON N M 1 O - N 'zt "o l'" 00 C N
Page 263 and 264: 1 T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Page 265 and 266: 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Page 267 and 268: 1 1 1 1 I 1 + I I 1 I 1 1 I 1 1 1 I
Page 269 and 270: 1 1 1 1 1 1 1 I I 1 I 1 I 1 1 I T I
Page 271 and 272: Cl M 'tt tn '. p t- 00 O\ N M O - (
Page 273 and 274: d' O - (N M 'nt V) \O N. 00 O> N M
Page 275 and 276: 1 1 T 1 1 1 T 1 1 1 1 1 1 1 1 1 1 1
Page 277 and 278: + + i i i i i i " i " . + + + + " +
Page 279 and 280: e O --ý N M t Vn 1,0 [-- 00 O> N M
Page 281 and 282: FT ce + I I r. to \, O t- 00 O\ N M
Page 283 and 284: kn 110 r- 00 Oý N M O - N M Itt vn
Page 285 and 286: , It N M "t N M Iýt N M M M M Oý
Page 287 and 288: gj a - - - - - - - - - - - - - - -
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