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4.2.3 - High-throughput AFP assay The assay was applied to all 866 isolates from the Antarctic lake environments. Of these, 187 (21.6%) proved to be positive for AFP activity by this assay. Activit\ ww as demonstrated by a reduction in transparency of the protein extracts when compared with a 30% sucrose solution without protein extract. The samples could not be quantified or qualified for level of activity as with a `SPLAT' assay, however, protein extracts were marked as either active or non-active. Those with reduced transparency (increased opacity) or colouration which were difficult to confirm were classed as positive, and those extracts with refraction equal or less than the negative control (30% sucrose) were classed as negative. The 187 bacteria strains, which were identified as positive using the HTAP assay, are shown in table 4.1. These isolates were returned to England and analysed using the `SPLAT' assay to confirm AFP activity within the bacteria. Of the 187 bacteria identified as AFP active using the HTAP assay, only 12 were Gram positive. 4.2.4 - SPLAT assay The 187 bacterial isolates from which positive protein extracts (using the HTAP assay) were extracted, were brought back to Colworth (Unilever) and assessed for activity using the `SPLAT' assay. Nineteen of the bacterial isolates were confirmed to have some level of AFP activity within their total cellular extracts (Table 4.2), which equates to 10.2% of those isolates showing a positive protein extract using the HTAP assay. Ten of these 19 isolates showed a SPLAT score of 4 or 5 (Section 2.10.2). indicating high AFP activity, comparable with pure fish AFP III protein (1 mg mU 1) or total cellular protein extracts of Marinomonasprotea (Mills, 1999). The rest of the samples had an activity of 3. Samples with an activity of less than 3 were considered to have insufficient activity for further analysis. AFP active bacteria with a score >3 were isolated from Ace Lake, Triple Lake, Pendant Lake, Pendant Ice cores and Oval Lake (Table 4.2 and Fig. 4.5). All AFP active bacteria, as confirmed by the SPLAT assay of total cellular protein extracts, were Gram negative. The concentrations of protein extracts, prepared from each putative AFP active isolate, were assessed using the `SPLAT' analysis and were calculated using the BioRad Protein Kits (BioRad, see Chapter 2 section 2.10.1). Figure 4.6 shows that there was no direct relationship between total cellular protein concentration used for anale sis and 122
SPLAT score. It has been shown that activity level is affected by AFP concentration. However, the AFP active quotient of these samples does not appear to be affected by the total cellular protein concentration. Oval 5% e 2T6riPl% Ace 42 Pendant (ice) Pendant 16% 11% Figure 4.5 - Percentages of `SPLAT' assay confirmed AFP active bacteria isolated from each lake. 4.2.5 - Crystal morphology The majority of the AFP positive protein samples produced small round crystal morphologies, as can be seen in figures 4.1 and 4.7. This morphology is consistent with crystal morphologies seen in SPLAT analysis, as crystals are formed and recrystalise after crash-cooling in a liquid medium. The interface between ice and water is molecularly rough and the transport of water molecules to the growing crystal surface is limited by diffusion, therefore circular discs often form (Whitworth & Petrenko, 1999). Isolate number 494 had a SPLAT activity of between 4 and 5 and produced small round crystals as far as could be ascertained (Fig. 4.7). 123
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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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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
Page 107 and 108: a 250 200 150 100 50 70 60 50 40 ý
Page 109 and 110: The temperature (Fig 3.2a) through
Page 111 and 112: et al., 1999). No melt out was obse
Page 113 and 114: increase in ammonium is probably du
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Page 117 and 118: main pigments were chlorophylls a a
Page 119 and 120: Inorganic nitrogen (NO2, NO3 and NH
Page 121 and 122: 744 -- 7 644 6 19 ü SE 400-- 4 2s
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: 4.2 - Results 4.2.1 - Sampling and
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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 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
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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
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gj a - - - - - - - - - - - - - - -
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