crispr direct repeat sequences olivia ho-shing 22 november 2009

CRISPR Direct Repeat Sequences

Olivia Ho-Shingolhoshing@davidson.edu

22 November 2009

QUESTIONS: • Do different halophile species share similar direct repeat sequences? • Can direct repeats indicate phylogeny? • Is there any structure to the direct repeats for some potential function?

• 21 – 37 bp in length• Surround spacers that may contain viral sequences• Not palindromic, some dyad symmetry

----GACTAC----CTG----GTAGTC----

degenerate DR

CRISPR Finder

H. mukohataei CRISPR 1_12

H. californiaeH. denitrificansH. mediterraneiH. mucosumH. sinaiiensisH. sulfurifontisH. volcaniiH. vallismortis*

H. mukohataei direct repeats match hits in: • Haloarcula marismortui• Halorhabdus utahensis• Natronomonas pharaonis

Do different halophile species share similar direct repeat sequences?

CLUSTAL 2.0.12 multiple sequence alignment

Sulfurifontis_17_1 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30Denitrificans_10_x2 ----------GCTTCAATCCCACAAGGGTTCGTCTGAAAC---------- 30Mukohataei_2_1 ----------GCTTCAATCCCACAAGGGTCCGTCTGAAAC---------- 30Sinaiiensis_116_1 ----------GCTTCAATCCCACATGGGTTCGTCTGAAAC---------- 30Californiae_86_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGTAAC---------- 30Utahensis_1_1 ----------GCTTCAACCCCACGAGGGTCCGTCTGAAAC---------- 30Marismortui_1_2 ----------GCTTCAACCCCACAAGGGTCCGTCTGAAAC---------- 30Californiae_86_2 ----------GCTTCAACCGCCCAAGGGTCCGTCTGAAAC---------- 30Mediterranei_5_x3 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mucosum_17_1 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAACC--------- 31Mediterranei_13_x2 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mucosum_10_x4 ----------GCTTCAACCCAACTAGGGTTCGTCTGTAAC---------- 30Mukohataei_1_12 ----------GTTTCAGACGGACCCTTGTGGGATTGAAGC---------- 30Californiae_65_1 ----------GTTTCAGACGGACCCTTGGGCGGTTGAAGC---------- 30Mucosum_4_x4 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30Mucosum_11_1 ----------GTTACAGACGAACCCTAGTTGGGTTGAAGC---------- 30Californiae_108_1 ----------GTTACAGACGGACCCTCGTGGGGTTGAAGC---------- 30Denitrificans_5_x3 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Npharaonis_1_1 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Volcanii_16_x2 ----------GTTTCAGACGAACCCTTGTGGGGTTGAAGC---------- 30Sulfurifontis_12_2 ---------AGTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 31Sinaiiensis_116_2 ----------GTTTCAGACGAACCCTTGTGGGATTGAAGC---------- 30Volcanii_72_2 ---------GGTTTCAGACGAACCCTTGTGGGTTTGAAGC---------- 31Sulfurifontis_21_1 ----------GTTTCAGACGAACCCTTGTTGGGTTGAAGT---------- 30Sulfurifontis_26_1 ----------GTTTCAATC---CCGTTCTGGGTTTCTACCGCATCGCGAC 37Sinaiiensis_46_1 ---------------AACCAGAGCGAACAGGGACCACC------------ 23Sulfurifontis_19_1 GTCGCGATGCGGTAGAAAC---CCAGAACGGGATTGAAAC---------- 37Sulfurifontis_26_2 GTCGCAGGGCAATAGAAAC---CCAGAACGGGATTGAAAC---------- 37Npharaonis_1_4 GTCGAGACGGACTGAAAAC---CCAGAACGGGATTGAAAC---------- 37Sulfurifontis_13_1 ------------CCGACACCGACGGCGACGGTCTCGACGACGG------- 31Californiae_37_1 -----------CTTGTCCTTGACCTCGGTCGTCTTGTCTTT--------- 30

Do different halophile species share similar direct repeat sequences?

H. sinaiiensis 116-1

H. sinaiiensis 116-2

H. sinaiiensis 46-1

N. pharaonis 1-1

N. pharaonis 1-4

H. californiae 86-1H. californiae 86-2

H. californiae 65-1

H. californiae 108-1

H. californiae 37-1

H. mukohataei 1-12

H. mukohataei 2-1

H. mediterranei 13(x2)H. mediterranei 5(x3)

H. mucosum 10(x4)

H. mucosum 17-1

H. mucosum 11-1

H. mucosum 4(x4)

H. denitrificans 10(x2)H. denitrificans 5(x3)

H. volcanii 16(x2)

H. volcanii 72-2

H. sulfurifontis 17-1H. sulfurifontis 19-1

H. sulfurifontis 26-2

H. sulfurifontis 13-1

H. sulfurifontis 26-1

H. sulfurifontis 12-2

H. sulfurifontis 21-1

ClustalW alignment grouped by speciesDo different halophile species share similar direct repeat sequences?

Do different halophile species share similar direct repeat sequences?

H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei

H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis

Can direct repeats indicate phylogeny?Phylogram based on direct repeat sequences

H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei

H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis

Can direct repeats indicate phylogeny?Phylogram based on 16S rRNA sequences

H. californiaeH. denitrificansH. marismortuiH. mediterraneiH. mucosumH. mukohataei

H. sinaiiensisH. sulfurifontisH. utahensisH. volcaniiN. pharaonisH. vallismortis

Is there any structure to the direct repeats for some potential function?Characterizing a halophile consensus direct repeat sequence

G T T T C A A A C G A A C C [AC] [GT] G G T G G G T T T G A A [AG] C

Is there any structure to the direct repeats for some potential function?Comparing consensus halophile sequence to other species

CLUSTAL 2.0.12 multiple sequence alignment

Consensus_Halophile -GTTTCAAACGAACCCGGGTGGGTTTGAAAC-------- 30Hmarismortui_2 -GCTTCAACCCCACAAGGGTCCGTCTGAAAC-------- 30NostocPCC_6 -GTTTCCATCCCCGTGAGGGGTA--AAGGAATTAAAAC- 35NostocPCC_12 -GTTTCCATCCCCGTGAGGGGTA--AGAGATTAAAAAC- 35NostocPCC_14 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37NostocPCC_15 -GTTTCAATCCCTGATAGGGATTTTTGTTAGTTAAAAC- 37Rxylan_2 -GTTTCAATCCCTTATAGGTAGGCTCAAAAC-------- 30Ecoli_4 -CGGTTTATCCCCGCTGGCGCGGGGAACTC--------- 29Ecoli_5 --GGTTTATCCCCGCTGGCGCGGGGAACAC--------- 28Hmarismortui_C1_2 ---GGCGGTCCCTGTTCGCTCTGGTT------------- 23NostocPCC_2 GTTACTTACCATCACTTCCCCGCAAGG-GGATGGAAAC- 37NostocPCC_18 -CTTTCAACCCTCCCATTACTGGAAGGAGGGTTGCAACG 38NostocPCC_7 -GTTTTAATTCCTTTACCCCT-CACGG-GGATGGAAAC- 35NostocPCC_8 GTTTCTATTAACACA-AATCCCTATCAGGGATTGAAAC- 37NostocPCC_17 -GTTGCAACACCATATAATCCCTATTAGGGATTGAAAC- 37Rxylan_3 --TACCAGGCGTGGATCTTGCCCTCGGACAC-------- 29

Is there any structure to the direct repeats for some potential function?

Conclusions

• Halophile direct repeats are similar to each other (significant e-values) – swapping, functionality

• Interesting triplet motifs in dyad symmetry of direct repeats– Binding site for CRISPR-associated proteins?– Folding site for siRNA?

• Direct repeats may be more indicative of phylogeny in a larger more widespread group of species