LIFE AT THE LIMITS - EXTREME ENVIRONMENTS "HOW CAN THEY SURVIVE?"

COMPARATIVE MICROBIAL GENOMICS ANALYSIS LIFE AT THE LIMITS - EXTREME ENVIRONMENTS "HOW CAN THEY SURVIVE?" Group 5 Kanokwan Inban Lakkhana Khanhayuwa Parwin Tantayapirak

Introduction 2

Source: http://pathmicro.med.sc.edu/fox/evol.gif 3

The Archaea are a group of single-celled microorganisms. They have no cell nucleus or any other organelles within their cells. 4

Initially, archaea were seen as extremophiles that lived in harsh environments, such as hot springs and salt lakes, but they have since been found in a broad range of habitats, such as soils, oceans, and marshlands. The archaea exploit a much greater variety of sources of energy than eukaryotes: ranging from familiar organic compounds such as sugars, to using ammonia, metal ions or even hydrogen gas as nutrients. 5

Sulfolobus solfataricus 98/2 Sulfolobus islandicus Y.G.57.14 Metallosphaera sedula DSM 5348 Staphylothermus marinus F1 Sulfolobus islandicus L.S.2.15 Nitrosopumilus maritimus SCM1 Sulfolobus solfataricus P2 Pyrobaculum arsenaticum DSM 13514 Thermofilum pendens Hrk 5 Pyrobaculum islandicum DSM 4184 Sulfolobus islandicus L.D.8.5 Sulfolobus islandicus M.16.4 Pyrobaculum aerophilum str. IM2 Hyperthermus butylicus DSM 5456 Crenarchaeota 6

Thermoproteus neutrophilus V24Sta Aeropyrum pernix K1 Sulfolobus islandicus M.14.25 Sulfolobus islandicus M.16.27 Pyrobaculum calidifontis JCM 11548 Sulfolobus tokodaii str. 7 Ignicoccus hospitalis KIN4/I Sulfolobus islandicus Y.N.15.51 Sulfolobus acidocaldarius DSM 639 Desulfurococcus kamchatkensis 1221n Aciduliprofundum boonei T469 Natronomonas pharaonis DSM 2160 Methanosaeta thermophila PT Halorubrum lacusprofundi ATCC 49239 Crenarchaeota Euryarchaeota 7

Sulfolobus Archaea Crenarchaeota Thermoprotei Sulfolobales Sulfolobaceae Yellowstone Na9onal Park Ecology: terrestrial volcanic hot springs with op1mum growth occurring at ph 2-3 + temperature of 75-80 o C + sulfur present acidic environment Genome Structure: circular chromosome that consists of 2,992,245 bp (S. ilandicus) Descrip3on and Significance: - grow either lithoautotrophically or chemoheterotrophically by oxidizing sulfur. - TCA cycle system similar to mitochondria of eukaryotes. Mount St. Helens ** oxidize hydrogen sulfide to sulfate intracellularly used to treat industrial waste water 8

Desulfurococcus Archaea Crenarchaeota Thermoprotei Desulfurococcales Desulfurococcaceae Ecology: - deep- sea thermal vents and subterranean hot springs - op1mal temperature for growth is 85ºC. - anaerobic archaea Descrip3on and Significance: - spherical, 0.5-15 microns in diameter. - cells are surrounded by an interes1ng protein subunit envelope. - have an interes1ng lasce- like protein structure cell covering - one long flagella 3D image of lasced protein on Desulfurococcus' cell envelope 9

Aeropyrum Archaea Crenarchaeota Thermoprotei Desulfurococcales Desulfurococcaceae hyperthermophile ** All species isolated from these environments had been strictly anaerobic, that is, un1l the discovery of Aeropyrum!!! Ecology: solfotaric vent at Kodakara- jima Island in Kyusyu, Japan. - temperatures ranging between 90 to 95 o C, ph 7.0, and a salinity of 3.5% - aerobic archaeon Genome Structure: circular chromosome Descrip3on and Significance: - spherical in shape and are 0.8 to 1.2 microns in diameter - pathways that allow A. to be aerobic gene in the TCA cycle (Krebs' cycle) coding for alpha- ketoglutarate dehydrogenase was not present 10

Hyperthermus Archaea Crenarchaeota Thermoprotei Desulfurococcales Pyrodic9aceae hyperthermophile Ecology: - sulfur reducing archaeon that grows between 95 and 106 C and at ph of 7.0. forms H 2 S - sea floor of a hot, solfataric habitat on the coast of São Miguel Island in Azores, Portugal Descrip3on and Significance: - one of the most thermophilic archaea isolated so far (106 C) 11

Thermoproteus Archaea Crenarchaeota Thermoprotei Thermoproteales Thermofilaceae hyperthermophile Ecology: acidic hot springs and water holes op1mal growth temperature is 85C. Anaerobes; autotrophic sulfur reduc1on. Genome Structure: Total genome length is 16000 nt, and the DNA is double- stranded and circular Descrip3on and Significance: - rod- shaped and reproduce by developing branches on the end of the cell which grow into individual cells. - mo1le by flagella Acidic hot spring, typical environment of Thermoproteus 12

Natronomonas Archaea Euryarchaeota Halobacteria Halobacteriales Halobacteriaceae Ecology: aerobic, extremely haloalkaliphilic archaeon. - grows op1mally in 3.5M NaCl and at ph 8.5, - sensi1ve to high magnesium concentra1ons Genome Structure: genome of Natronomonas pharaonis consists of three circular replicons. - chromosome which is 2,595,221 bp in length, - a typical haloarchaeal 131- kb plasmid, and - a unique mul1copy 23- kb plasmid Its choromosome has a high G + C content (63.4%) ** a high propor1on of acidic amino acids (average 19.3%) is found in the proteins of N. pharaonis which results in low isoelectric points (average pi 4.6) adap1ve features of haloarchaea to survive in their hypersaline environment 13

Methodology 14

1. Downloading Genomes and Prediction of Genes 2. Finding RNA genes and building a 16s rrna tree 3. Genome Atlases and BLAST matrices 4. Pan- Core- genome plots and BLAST atlases 15

1. Downloading Genomes and Prediction of Genes 2. Finding RNA genes and building a 16s rrna tree 3. Genome Atlases and BLAST matrices 4. Pan- Core- genome plots and BLAST atlases 16

Genome GC content Aciduliprofundum boonei T469 39.16% Aeropyrum pernix K1 56.31% Desulfurococcus kamchatkensis 1221n 45.34% %GC Content Halorubrum lacusprofundi ATCC 49239 66.72% Hyperthermus butylicus DSM 5456 53.74% Ignicoccus hospitalis KIN4/I 56.52% Metallosphaera sedula DSM 5348 46.23% Methanosaeta thermophila PT 53.55% Natronomonas pharaonis DSM 2160 63.44% Nitrosopumilus maritimus SCM1 34.17% Pyrobaculum aerophilum str. IM2 51.36% Pyrobaculum arsenaticum DSM 13514 55.05% Pyrobaculum calidifontis JCM 11548 57.15% Pyrobaculum islandicum DSM 4184 49.60% Staphylothermus marinus F1 35.73% Sulfolobus acidocaldarius DSM 639 36.71% Sulfolobus islandicus L.D.8.5 35.25% Sulfolobus islandicus L.S.2.15 35.11% Sulfolobus islandicus M.14.25 35.10% Sulfolobus islandicus M.16.27 35.01% Sulfolobus islandicus M.16.4 35.00% Sulfolobus islandicus Y.G.57.14 35.39% Sulfolobus islandicus Y.N.15.51 35.29% Sulfolobus solfataricus 98/2 35.83% Sulfolobus solfataricus P2 35.79% Sulfolobus tokodaii str. 7 32.79% Thermofilum pendens Hrk 5 57.67% Thermoproteus neutrophilus V24Sta 59.91% 17

Gene Count Genome Count number of genes genbank refseq prodigal Different Aciduliprofundum_boonei_T469.proteins.fsa 1544-1568 - Aeropyrum_pernix_K1.proteins.fsa 1700 1700 1716 16 Desulfurococcus_kamchatkensis_1221n.proteins.fsa 1471 1471 1461 10 Halorubrum_lacusprofundi_ATCC_49239.proteins.fsa 3560 3560 3647 87 Hyperthermus_butylicus_DSM_5456.proteins.fsa 1602 1602 1842 240 Ignicoccus_hospitalis_KIN4_I.proteins.fsa 1434 1434 1491 57 Metallosphaera_sedula_DSM_5348.proteins.fsa 2256 2256 2407 151 Methanosaeta_thermophila_PT.proteins.fsa 1696 1696 1818 122 Natronomonas_pharaonis_DSM_2160.proteins.fsa 2843 2820 2846 26 Nitrosopumilus_maritimus_SCM1.proteins.fsa 1795 1795 1947 152 Pyrobaculum_aerophilum_str_IM2.proteins.fsa 2605 2603 2674 71 Pyrobaculum_arsenaticum_DSM_13514.proteins.fsa 2298 2299 2528 229 Pyrobaculum_calidifontis_JCM_11548.proteins.fsa 2149 2149 2312 163 Pyrobaculum_islandicum_DSM_4184.proteins.fsa 1978 1978 2246 268 Staphylothermus_marinus_F1.proteins.fsa 1570 1570 1666 96 Sulfolobus_acidocaldarius_DSM_639.proteins.fsa 2223 2223 2373 150 Sulfolobus_islandicus_LD85.proteins.fsa 2949-3159 - Sulfolobus_islandicus_LS215.proteins.fsa 2738 2737 3115 378 Sulfolobus_islandicus_M1425.proteins.fsa 2609 2608 2911 303 Sulfolobus_islandicus_M1627.proteins.fsa 2657 2656 2987 331 Sulfolobus_islandicus_M164.proteins.fsa 2736 2735 2876 141 Sulfolobus_islandicus_YG5714.proteins.fsa 2905 2903 3089 186 Sulfolobus_islandicus_YN1551.proteins.fsa 2900 2900 3323 423 Sulfolobus_solfataricus_98_2.proteins.fsa 2679-3036 - Sulfolobus_solfataricus_P2.proteins.fsa 2994 2977 3355 378 Sulfolobus_tokodaii_str_7.proteins.fsa 2826 2825 3041 216 Thermofilum_pendens_Hrk_5.proteins.fsa 1876 1876 1941 65 Thermoproteus_neutrophilus_V24Sta.proteins.fsa 1966 1966 2052 86 refseq-prodigal 18

Gene Length Genome genbank refseq prodigal Mean StdDev Min Max Mean StdDev Min Max Mean StdDev Min Max Aciduliprofundum_boonei_T469.proteins.fsa 399 364.2 80 1184 219 116.45 63 410 296 142.95 124 578 Aeropyrum_pernix_K1.proteins.fsa 219 116.45 63 410 - - - - 225 256.71 54 820 Desulfurococcus_kamchatkensis_1221n.proteins.fsa 228 243.95 37 878 228 243.95 37 878 329 109.8 123 504 Halorubrum_lacusprofundi_ATCC_49239.proteins.fsa 311 214.58 65 597 311 214.58 65 597 252 181.1 33 642 Hyperthermus_butylicus_DSM_5456.proteins.fsa 212 132.33 93 517 212 132.33 93 517 263 178.69 89 687 Ignicoccus_hospitalis_KIN4_I.proteins.fsa 289 176.21 92 653 289 176.21 92 653 213 125.66 64 441 Metallosphaera_sedula_DSM_5348.proteins.fsa 341 206.74 76 698 341 206.74 76 698 342 319.17 92 1152 Methanosaeta_thermophila_PT.proteins.fsa 360 113.41 139 532 360 113.41 139 532 313 140.12 130 524 Natronomonas_pharaonis_DSM_2160.proteins.fsa 245 162.29 66 592 245 162.29 66 592 306 232.86 57 747 Nitrosopumilus_maritimus_SCM1.proteins.fsa 252 235.41 55 820 252 235.41 55 820 257 257.87 32 889 Pyrobaculum_aerophilum_str_IM2.proteins.fsa 268 202.07 73 794 268 202.07 73 794 214 182.66 44 615 Pyrobaculum_arsenaticum_DSM_13514.proteins.fsa 334 120.26 102 501 334 120.26 102 501 355 430.09 52 1437 Pyrobaculum_calidifontis_JCM_11548.proteins.fsa 278 109.99 132 459 278 109.99 132 459 221 132.93 46 371 Pyrobaculum_islandicum_DSM_4184.proteins.fsa 242 146.29 79 482 242 146.29 79 482 255 135.52 42 440 Staphylothermus_marinus_F1.proteins.fsa 243 117.49 101 439 243 117.49 101 439 313 162.4 103 606 Sulfolobus_acidocaldarius_DSM_639.proteins.fsa 324 151.09 143 561 324 151.09 143 561 192 93.98 65 391 Sulfolobus_islandicus_LD85.proteins.fsa 244 119.07 46 477 - - - - 260 166.18 55 518 Sulfolobus_islandicus_LS215.proteins.fsa 262 190.12 42 690 262 190.12 42 690 251 148.16 42 420 Sulfolobus_islandicus_M1425.proteins.fsa 257 130.72 132 530 257 130.72 132 530 246 98.53 79 429 Sulfolobus_islandicus_M1627.proteins.fsa 209 130.89 72 417 209 130.89 72 417 330 184.11 114 627 Sulfolobus_islandicus_M164.proteins.fsa 239 145.51 52 451 239 145.51 52 451 147 115.62 44 378 Sulfolobus_islandicus_YG5714.proteins.fsa 341 237.72 38 754 341 237.72 38 754 224 141.89 85 554 Sulfolobus_islandicus_YN1551.proteins.fsa 287 125.86 103 505 287 125.86 103 505 226 156.61 64 469 Sulfolobus_solfataricus_98_2.proteins.fsa 227 132.43 47 425 - - - - 215 164.89 61 469 Sulfolobus_solfataricus_P2.proteins.fsa 313 131.46 138 537 313 131.46 138 537 184 108 39 376 Sulfolobus_tokodaii_str_7.proteins.fsa 273 122.54 145 511 273 122.54 145 511 172 91.44 75 339 Thermofilum_pendens_Hrk_5.proteins.fsa 333 131.8 118 608 333 131.8 118 608 288 210.34 106 802 Thermoproteus_neutrophilus_V24Sta.proteins.fsa 269 214.15 91 799 269 214.15 91 799 315 342.02 74 1168 19

1. Downloading Genomes and Prediction of Genes 2. Finding RNA genes and building a 16s rrna tree 3. Genome Atlases and BLAST matrices 4. Pan- Core- genome plots and BLAST atlases 20

Sulfolobus sp. 16s rrna tree Pyrobaculum sp. 21

1. Downloading Genomes and Prediction of Genes 2. Finding RNA genes and building a 16s rrna tree 3. Genome Atlases and BLAST matrices 4. Pan- Core- genome plots and BLAST atlases 22

Thermoproteus neutrophilus V24Sta Sulfolobus sp. Pyrobaculum sp. BLAST Matrices 23

1. Downloading Genomes and Prediction of Genes 2. Finding RNA genes and building a 16s rrna tree 3. Genome Atlases and BLAST matrices 4. Pan- Core- genome plots and BLAST atlases 24

Pan- Core genome plots 25

Pan- Core genome plots 26

THANK YOU 27