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

More documents

Recommendations

Info

The two restriction enzyme digest patterns were very similar but did show some differences. The isolates which clustered with M protea (154,124.5ice3.744.794.214 and 54), showed the same clustering for both digests (Figs. 5.2a & b) indicating they were probably all the same species. Isolates 39 and 86 were probably also identical as they formed a single taxon with both restriction enzymes (Figs. 5.2a & b). Isolates 302 and 33 appeared to form a single taxon with Hpa II (Fig 5.2a), however, with Alu I they formed two separate taxa while still clustering together (Fig 5.2b), indicating that these two isolates had a higher similarity to each other than to any of the other isolates. The same situation was also shown by isolates 492 and 47. Isolate 494 appears as a direct outlier to the M protea taxon in both digest patterns indicating a distant relationship. Isolates 583 and 213 appear within the same group in both patterns, however, the degree of relatedness differs for the different enzymes. Using Alu I (Fig 5.2b) they clustered together with isolate 53 which when digested with Hpa II was found within the same group but at a different degree of relatedness (Fig 5.2a). Isolate 466 moved from an outlier of the 39/86 taxon using Hpa II (Fig 5.2a), to an outlier of the M protea taxon when using Alu I (Fig 5.2b). Isolate 732 shows a similar jump in position from a taxonomic grouping with the 302/33 taxon when using Hpa II (Fig 5.2a), to a grouping with the 39/86 taxon when using Alu I (Fig 5.2b). 5.2.2 -16S rRNA nucleotide sequencing PCR products were purified using the QlAquick PCR purification centrifugation protocol (QIAGEN), eluted in water and then sequenced directly (Chapter 2, section 2.9.6.4). The majority of the isolates provided approximately Ikb of nucleotide sequence directly from the PCR product. However, some of the PCR products failed to produce adequate sequence using this method; these sequences were cloned into the TOPO 2.1 vector (Chapter 2, section 2.9.6.3) using the TOPO TA cloning kit (Invitrogen). and sequenced using the M 13 forward and reverse primers. This provided better sequencing results for the remaining isolates. Isolates 494,213,492,5(ice3) and 466 proved difficult to clone and so these sequences have not be adequately sequenced, as time constraints did not allow further attempts. The available sequence data for the 16S rDNA gene was entered into the Genbank database (http: //wNti-w. ncbi. nlm. nih. ) and assigned an accession number (Table 5.1). 136
The closest published relative for each isolate was determined by comparison with the Genbank nucleotide database using the FASTA (http: //www. ebi. ac. uk) and BLAST (Basic Local Alignment Search Tools) search algorithms which compare multiple nucleotide sequences for sequence similarity and homology (Altschul et al., 1990). Isolates, 124,154,214,5ice3,54,744 and 794 were all 100% identical to Marinomonasprotea (AJ238597), confirming that these are all one species. Similarly isolates, 86 and 39 were 99.4% and 99.6% identical respectively to an unspeciated Pseudoalteromonas sp. (U85856); therefore it is probable that these two isolates were the same species. Isolates 33 and 302 were identified as an Antarctic seawater bacterium (AJ293825) and Pseudomonas fluorescens (AF228367) respectively. However, isolate 33 was also 97.8% related to an unspeciated Pseudomonas sp. (AB021318) and therefore shows a close relationship with isolate 302. Isolate 302 has been further identified as Pseudomonasfluorescens by culturing the bacterium on Pseudomonas agar F (fluorescens agar) and CFC agar (Table 5.2; Bridson, 1998). Isolate 33 was identified as a pseudomonad but did not produce fluorescein pigment. Pseudomonas agar F stimulates the production of fluorescein and reduces that of pyocyanin and/or pyorubin and therefore delineates between pseudomonads which produce fluorescein and those which produce pyocyanin and/or pyorubin. CFC agar selects for pseudomonads whilst inhibiting most other bacteria. Isolates 492 and 47 showed more than 99% similarity to Stenotrophomonas maltophilia (Ac. No. AJ293464). However, the sequence data for isolate 492 only comprised 408 bp and so were not sufficient to make a definitive identification (genus identification is supported by ARDRA, discussed in section 5.3.2.4). However. identification of isolate 47 was based on 1182 bp and was therefore sufficient for putati\ e speciation. Isolate 732 showed 99.5% similarity to Enterobacter agglomerans (AF348161) based on 1493 bp, which is nearly a complete gene sequence, it is therefore unlikely that this identification would be inaccurate. Isolate 53 was identified as 99.3% related to Idiomarina loihiensis, which is a hydrothermal vent bacterium. Isolate 494 was 100% related to an unidentified Sphingomonas sp (AF395031). However. this 137
Page 1 and 2:
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 and 56:
Larsemann Hills __ rp3rtrnent of th
Page 57 and 58:
Sampling trips took two days during
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
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
Page 115 and 116: 10 7 9 6 8 E ö 7 5 Co u c m c 40 w
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 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: Coefficient: Dice Algorithm: lPGMAA
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
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 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
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 - - - - - - - - - - - - - - -
show all

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

Create successful ePaper yourself

Delete template?

Save as template?