ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

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`"-ýý la m1a1C11Ca t 0 20 kin \MV Loewe . '" r Ma sif., i ý", ff-Ol manning Massif. 'NICICod Massifi -%A 71°S ", Lake Padock , 67°W 69°W Figure 2.1c -A map of the epishelf lake, Beaver Lake, MacRobertson Land (modified from Laybourn-Parry et al., 2001b) 36
Sampling trips took two days during July, due to the lack of daylight (approx 3 hours) in the winter season, whilst in early summer (September and November) trips only took one day due to an increase in the number of hours of daylight per day. 2.3 - Water sample processing On return to the laboratory, samples were stored at 1 °C until all initial processing was completed. Initially 500mL aliquots were taken for inorganic nutrient analysis and then frozen (-20°C) for ammonia analysis. Aliquots (IOmL) were taken for bacterial and flagellate counts; these were fixed in buffered glutaraldehyde to a final concentration of 2% (Appendix A2) and stored at 1 °C in the dark. For each sample, 120mL aliquots were vacuum-filtered through a 0.2µm mixed cellulose esters filter (nitrate and acetate, Millipore) and the filter was stored in TE buffer (Appendix A2) and frozen at -70°C for denaturing gradient gel electrophoresis (DGGE) analysis. Exactly 1L of the water was filtered through a 47mm GF/C (Whatman) glass microfiber filter, which was stored at -70°C for subsequent chlorophyll a analysis. The remaining water was filtered through a 47mm GF/F (Whatman, heated at 450°C for -5 hours) microfiber filter. The filtrate was decantered into a 21 polypropylene bottle (Acid washed, 10% HC1), 25m1 of which was dispensed into a sterile universal tube and frozen at -20°C for dissolved organic carbon analysis. The remainder of the GF/F filtrate was frozen at -20°C until inorganic chemical analysis was carried out. 2.4 - Culturing and maintenance of isolates Microorganisms were cultured from lake water onto various non-selective solid media. The inoculation procedure was as follows: l00µ1 of lake water was pipetted onto four different agar media and spread using a flame sterilised glass spreader. Incubation temperature and nutrients were used which would allow rapid growth whilst providing similar conditions to the environment from which they were isolated. This prevented the chances of adaptive radiation that may cause the loss of AFP activity in a previously active bacterium. The four types of agar used were tryptic soya agar, yeast agar, seawater agar (3.8% salinity) and'/2 seawater agar (1.9% salinity) (TSA, YSA, SWA and 1/2 SWA respectively, Appendix Al). For the ice cores, a 4cm thick cross section of ice was taken from the ice-water interface and from 50-54cm from the ice-water interface. Each section was cut with a sterilized ice saw blade and then sterilized by flaming the exterior surface 37
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COLD ADAPTATION STRATEGIES AND DIVE
Page 3 and 4:
, 71 "If you march your Winter Jour
Page 5 and 6: 1.6.3. Molecular typing techniques
Page 7 and 8: 3.2.1.2.3. DOC and chlorophyll a ..
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: Larsemann Hills __ rp3rtrnent of th
Page 59 and 60: Sigma, Sigma-Aldrich Company Ltd.,
Page 61 and 62: oxidized and the sample then conver
Page 63 and 64: sample. An appropriate volume was l
Page 65 and 66: min to ensure complete extension of
Page 67 and 68: Figure 2.3 - Lane delineation image
Page 69 and 70: Figure 2.6b - Molecular weights are
Page 71 and 72: RNA 41F (5'-GCT CAG ATT GAA CGC TGG
Page 73 and 74: I min. The spin column was removed
Page 75 and 76: compiled using Seqman and any erron
Page 77 and 78: ecorded and related to the level of
Page 79 and 80: information regarding the cold adap
Page 81 and 82: 50ppt to 186ppt. Thirteen were sali
Page 83 and 84: M N N 't M M \p \p 00 kf) M kf) bA
Page 85 and 86: as would normally be expected, as b
Page 87 and 88: 1444 6ý L CC vý O Q O O NO u 'c z
Page 89 and 90: 5 - 250 3 245 240 G (, -3 235 a 0-
Page 91 and 92: 3.2.2.2.5 - Dissolved organic carbo
Page 93 and 94: d 14 12 10 14 12 7 10 8 6 4 2 0 00/
Page 95 and 96: SRP ('g L- 05 10 15 20 2 3 Depth4 5
Page 97 and 98: 3.2.2.3 - Biological characteristic
Page 99 and 100: a 0 Bacterial Abundance (x 106 ml-1
Page 101 and 102: 3.3 - Discussion 3.3.1 - Summer sam
Page 103 and 104: to high surface water temperatures
Page 105 and 106: 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
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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
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contain contaminants. If the profil
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(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
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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
Page 287 and 288:
gj a - - - - - - - - - - - - - - -
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