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2.9.3. Amplified ribosomal DNA restriction analysis ............... 43 2.9.3.1. PCR amplification of 16S rRNA gene ........................ 43 2.9.3.2. Restriction endonuclease digestion of 16S rDNA fragment ................................................... 45 2.9.4. Computer enhanced phenetic analysis of ARDRA patterns 2.9.5. Denaturing gradient gel electrophoresis ........................ ...... 45 46 2.9.5.1. Collection of bacterial community ........................ 46 2.9.5.2. Genomic extraction for DGGE ........................ 46 2.9.5.3. Nested V3 region PCR amplification for DGGE ...... 50 2.9.5.4. Denaturing gradient gel electrophoresis ............... 51 2.9.6. Sequencing of bacterial 16S rDNA ................................. 52 2.9.6.1. Purification of 16S rDNA PCR products ............... 52 2.9.6.2. Purification of PCR products by gel extraction ...... 53 2.9.6.3. PCR product cloning .......................................... 53 2.9.6.4. Sequencing reaction .......................................... 54 2.10. Protein extraction and antifreeze protein assessment technique 2.10.1. Total cellular protein extraction ................................. ...... 55 55 2.10.2. `SPLAT' analysis ................................................... 56 Chapter 3: BIOLOGICAL AND PHYSICO-CHEMICAL CHARACTERISTICS OF THE STUDY LAKES 3.1. Introduction 3.2. Results ..................................................................... ..................................................................... ..................................................................... 58 5 8 59 3.2.1. Summer sampled lakes .......................................... 59 3.2.1.1. Physico-chemical characteristics ........................ 60 3.2.1.1.1. Ice cover .......................................... 60 3.2.1.1.2. Temperature .......................................... 60 3.2.1.1.3. Salinity .......................................... 60 3.2.1.2. Inorganic nutrients .......................................... 61 3.2.1.2.1. Nitrate (N03-N) and nitrite (N02-N) ...... 61 3.2.1.2.2. Ammonium (NH4-N) and SRP (P04-P) ...... 61
3.2.1.2.3. DOC and chlorophyll a ........................ 61 3.2.2. Focus study lakes ................................................... 65 3.2.2.1. Physico-chemical characteristics ........................ 65 3.2.2.1.1. Ice cover .......................................... 65 3.2.2.1.2. Temperature .......................................... 65 3.2.2.1.3. Salinity .......................................... 65 3.2.2.2. Inorganic nutrients .......................................... 70 3.2.2.2.1. Nitrate 3.2.2.2.2. Nitrite 3.2.2.2.3. Ammonium .......................................... .......................................... .......................................... 70 70 70 3.2.2.2.4. Soluble reactive phosphate ........................ 70 3.2.2.2.5. Dissolved organic carbon ........................ 71 3.2.2.3. Biological characteristics 3.2.2.3.1. Chlorophyll a ................................. ................................. 77 77 3.2.2.3.2. Bacterial cell concentrations ............... 77 3.2.2.3.3. HNAN abundance ................................. 77 3.2.2.3.4. PNAN abundance ................................. 78 3.3. Discussion ..................................................................... 81 3.3.1. Summer sampled lakes .......................................... 81 3.3.2. Detailed study lakes ................................................... 88 3.3.2.1. Ace Lake ................................................... 88 3.3.2.2. Pendant Lake ................................................... 97 3.3.2.3. Triple Lake .................................................. 102 3.3.2.4. Deep Lake and Club Lake ................................ 106 3.3.3. Oval Lake ........................................................... 111 3.4. Conclusions .................................................................... 112 Chapter 4: ANTIFREEZE PROTEIN ANALYSIS ................................ 114 4.1. Introduction .................................................................... 114 4.1.1. Disadvantages of the SPLAT assay 115 4.1.2. Development of the high-throughput AFP analysis protocol
Page 1 and 2: COLD ADAPTATION STRATEGIES AND DIVE
Page 3 and 4: , 71 "If you march your Winter Jour
Page 5: 1.6.3. Molecular typing techniques
Page 9 and 10: 5.3.2.5. The Sphingomonas group, is
Page 11 and 12: LIST OF FIGURES Figure I. I. Diagra
Page 13 and 14: Figure 3.17a Mean bacterial abundan
Page 15 and 16: LIST OF TABLES Table 1.1. Types of
Page 17 and 18: List of Abbreviations AFP AFGP AFLP
Page 19 and 20: ACKNOWLEDGEMENTS This study was fun
Page 21 and 22: Chapter 1: INTRODUCTION 1.1 - Tempe
Page 23 and 24: 1999 ; Hand & Burton, 1981) or iden
Page 25 and 26: Cold adaptation is dependant on the
Page 27 and 28: not at 25°C is a function of the a
Page 29 and 30: The most prolific literature on col
Page 31 and 32: process known as thermal hysteresis
Page 33 and 34: dominant, role in the ice-growth in
Page 35 and 36: explanation for this diversity is t
Page 37 and 38: 1.5.3.5 - Membrane bound solute tra
Page 39 and 40: een well established (Russell & Nic
Page 41 and 42: & Bowman, 2001; Labrenz & Hirsch, 2
Page 43 and 44: The reaction mix contains both deox
Page 45 and 46: gene mixtures and hence can be used
Page 47 and 48: displaying a high number of common
Page 49 and 50: AACGTCGAAA AACCTCGAAA (Organism A)
Page 51 and 52: particular set of environmental par
Page 53 and 54: Lake Maximum Depth (m) Approximate
Page 55 and 56: Larsemann Hills __ rp3rtrnent of th
Page 57 and 58:
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
Page 131 and 132:
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
Page 201 and 202:
contain contaminants. If the profil
Page 203 and 204:
(Section 6.3.2.4.3 ) could not be r
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most dominant species in the commun
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study was characterised as M protea
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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
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
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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
Page 251 and 252:
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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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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