AEM Accepted Manuscript Posted Online 27 January 2017 Appl ... · ñ óõ vµu }( P u]vv } v Z vµ ] v } ÁZ] Z Z Ç }v À Ç Á v ôì ](( v ]ooµ ] v ]v XdZ Æ v ] À oÇ µ ] o
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
Spore heat-activation requirements and germination responses correlate with sequences of germinant 1
receptors and with the presence of a specific spoVA2mob operon in food-borne strains of Bacillus subtilis 2
3
Antonina O. Krawczyk1,2, Anne de Jong1,2, Jimmy Omony1,2, Siger Holsappel1,2, Marjon H. J Wells-4
Bennik2,3, Oscar P. Kuipers1,2# and Robyn T. Eijlander1,2,3 5
6
Affiliations: 7
1. Laboratory of Molecular Genetics, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, the 8
Netherlands 9
2. Top Institute Food and Nutrition (TIFN), Nieuwe kanaal 9A, 6709 PA, Wageningen, the Netherlands 10
3. NIZO Food Research B.V., Kernhemseweg 2, 6718 ZB, Ede, the Netherlands 11
12
#Corresponding author: Oscar P. Kuipers, University of Groningen, Nijenborgh 7, 9747AG, Groningen, the 13
1. Scheldeman P, Pil A, Herman L, De Vos P, Heyndrickx M. 2005. Incidence and diversity of 543 potentially highly heat-resistant spores isolated at dairy farms. Appl Environ Microbiol 544 71:1480–1494. 545
2. Oomes SJCM, van Zuijlen ACM, Hehenkamp JO, Witsenboer H, van der Vossen JMBM, Brul 546 S. 2007. The characterisation of Bacillus spores occurring in the manufacturing of (low acid) 547 canned products. Int J Food Microbiol 120:85–94. 548
3. Lima LJR, Kamphuis HJ, Nout MJR, Zwietering MH. 2011. Microbiota of cocoa powder with 549 particular reference to aerobic thermoresistant spore-formers. Food Microbiol 28:573–582. 550
4. Berendsen EM, Zwietering MH, Kuipers OP, Wells-Bennik MHJ. 2015. Two distinct groups 551 within the Bacillus subtilis group display significantly different spore heat resistance 552 properties. Food Microbiol 45:18–25. 553
5. Scheldeman P, Herman L, Foster S, Heyndrickx M. 2006. Bacillus sporothermodurans and 554 other highly heat-resistant spore formers in milk. J Appl Microbiol 101:542–555. 555
6. Rosenkvist H, Hansen A. 1995. Contamination profiles and characterisation of Bacillus species 556 in wheat bread and raw materials for bread production. Int J Food Microbiol 26:353–363. 557
7. Setlow P. 2013. Summer meeting 2013 - when the sleepers wake: The germination of spores 558 of Bacillus species. J Appl Microbiol 15:1251–1268. 559
8. Setlow P. 2006. Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat and 560 chemicals. J Appl Microbiol 101:514–525. 561
9. Li L, Valenzuela-Martinez C, Redondo M, Juneja VK, Burson DE, Thippareddi H. 2012. 562 Inhibition of Clostridium perfringens spore germination and outgrowth by lemon juice and 563 vinegar product in reduced NaCl roast beef. J Food Sci 77:M598–603. 564
10. Velugoti PR, Bohra LK, Juneja VK, Thippareddi H. 2007. Inhibition of germination and 565 outgrowth of Clostridium perfringens spores by lactic acid salts during cooling of injected 566 turkey. J Food Prot 70:923–929. 567
11. Lovdal IS, Hovda MB, Granum PE, Rosnes JT. 2011. Promoting Bacillus cereus spore 568 germination for subsequent inactivation by mild heat treatment. J Food Prot 74:2079–2089. 569
12. Nerandzic MM, Donskey CJ. 2010. Triggering germination represents a novel strategy to 570 enhance killing of Clostridium difficile spores. PLoS One 5:e12285. 571
13. Behravan J, Chirakkal H, Masson A, Moir A. 2000. Mutations in the gerP locus of Bacillus 572 subtilis and Bacillus cereus affect access of germinants to their targets in spores. J Bacteriol 573 182:1987–1994. 574
14. Carr KA, Janes BK, Hanna PC. 2010. Role of the gerP operon in germination and outgrowth of 575 Bacillus anthracis spores. PLoS One 5:e9128. 576
15. Butzin XY, Troiano AJ, Coleman WH, Griffiths KK, Doona CJ, Feeherry FE, Wang G, Li YQ, 577 Setlow P. 2012. Analysis of the effects of a gerP mutation on the germination of spores of 578 Bacillus subtilis. J Bacteriol 194:5749–5758. 579
16. Paidhungat M, Setlow P. 2001. Localization of a germinant receptor protein (GerBA) to the 580 inner membrane of Bacillus subtilis spores. J Bacteriol 183:3982–3990. 581
17. Hudson KD, Corfe BM, Kemp EH, Feavers IM, Coote PJ, Moir A. 2001. Localization of GerAA 582 and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol 183:4317–4322. 583
18. Ross C, Abel-Santos E. 2010. The Ger receptor family from sporulating bacteria. Curr Issues 584 Mol Biol 12:147–158. 585
19. Paredes-Sabja D, Setlow P, Sarker MR. 2011. Germination of spores of Bacillales and 586 Clostridiales species: mechanisms and proteins involved. Trends Microbiol 19:85–94. 587
21. Christie G, Lowe CR. 2007. Role of chromosomal and plasmid-borne receptor homologues in 589 the response of Bacillus megaterium QM B1551 spores to germinants. J Bacteriol 189:4375–590 4383. 591
22. Li Y, Setlow B, Setlow P, Hao B. 2010. Crystal structure of the GerBC component of a Bacillus 592 subtilis spore germinant receptor. J Mol Biol 402:8–16. 593
23. Ramirez-Peralta A, Gupta S, Butzin XY, Setlow B, Korza G, Leyva-Vazquez M-A, Christie G, 594 Setlow P. 2013. Identification of new proteins that modulate the germination of spores of 595 Bacillus species. J Bacteriol 195:3009–3021. 596
24. Gupta S, Zhou KX, Bailey DMD, Christie G. 2015. Structure-function analysis of the Bacillus 597 megaterium GerUD spore germinant receptor protein. FEMS Microbiol Lett 362:fnv210. 598
25. Paidhungat M, Setlow P. 2000. Role of ger proteins in nutrient and nonnutrient triggering of 599 spore germination in Bacillus subtilis. J Bacteriol 182:2513–2519. 600
26. Setlow P. 2014. Germination of spores of Bacillus species: what we know and do not know. J 601 Bacteriol 196:1297–1305. 602
27. Berendsen EM, Boekhorst J, Kuipers OP, Wells-Bennik MHJ. 2016. A mobile genetic element 603 profoundly increases heat resistance of bacterial spores. ISME J 10:2633–2642. 604
28. Krawczyk AO, Berendsen EM, de Jong A, Boekhorst J, Wells-Bennik MHJ, Kuipers OP, 605 Eijlander RT. 2016. A transposon present in specific strains of Bacillus subtilis negatively 606 affects nutrient- and dodecylamine-induced spore germination. Environ Microbiol 18:4830–607 4846. 608
29. Griffiths KK, Zhang J, Cowan AE, Yu J, Setlow P. 2011. Germination proteins in the inner 609 membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster. Mol Microbiol 610 81:1061–1077. 611
30. Atluri S, Ragkousi K, Cortezzo DE, Setlow P. 2006. Cooperativity between different nutrient 612 receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by 613 alterations in the GerB receptor. J Bacteriol 188:28–36. 614
31. Yi X, Liu J, Faeder JR, Setlow P. 2011. Synergism between different germinant receptors in the 615 germination of Bacillus subtilis spores. J Bacteriol 193:4664–4671. 616
32. Luu S, Cruz-Mora J, Setlow B, Feeherry FE, Doona CJ, Setlow P. 2015. The effects of heat 617 activation on Bacillus spore germination, with nutrients or under high pressure, with or 618 without various germination proteins. Appl Environ Microbiol 81:2927–2938. 619
33. Fort P, Errington J. 1985. Nucleotide sequence and complementation analysis of a 620 polycistronic sporulation operon, spoVA, in Bacillus subtilis. Microbiology 131:1091–1105. 621
34. Alzahrani OM, Moir A. 2014. Spore germination and germinant receptor genes in wild strains 622 of Bacillus subtilis. J Appl Microbiol 117:741–749. 623
35. van der Voort M, Garcia D, Moezelaar R, Abee T. 2010. Germinant receptor diversity and 624 germination responses of four strains of the Bacillus cereus group. Int J Food Microbiol 625 139:108–115. 626
36. Velásquez J, Schuurman-Wolters G, Birkner JP, Abee T, Poolman B. 2014. Bacillus subtilis 627 spore protein SpoVAC functions as a mechanosensitive channel. Mol Microbiol 92:813–823. 628
37. Wang S, Faeder JR, Setlow P, Li Y. 2015. Memory of germinant stimuli in bacterial spores. 629 MBio 6:e01859-15. 630
38. Abhyankar W, Pandey R, Ter Beek A, Brul S, de Koning LJ, de Koster CG. 2015. Reinforcement 631 of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation. Food 632 Microbiol 45:54–62. 633
39. Hornstra LM, de Vries YP, Wells-Bennik MH, de Vos WM, Abee T. 2006. Characterization of 634 germination receptors of Bacillus cereus ATCC 14579. Appl Environ Microbiol 72:44–53. 635
40. Cooper GR, Moir A. 2011. Amino acid residues in the GerAB protein important in the function 636 and assembly of the alanine spore germination receptor of Bacillus subtilis 168. J Bacteriol 637 193:2261–2267. 638
41. Yi X, Setlow P. 2010. Studies of the commitment step in the germination of spores of Bacillus 639 species. J Bacteriol 192:3424–3433. 640
42. Berendsen EM, Krawczyk AO, Klaus V, de Jong A, Boekhorst J, Eijlander RT, Kuipers OP, 641 Wells-Bennik MHJ. 2015. Spores of Bacillus thermoamylovorans with very high heat 642 resistances germinate poorly in rich media despite the presence of ger clusters, but efficiently 643 upon non-nutrient Ca-DPA exposure. Appl Environ Microbiol 81:7791–7801. 644
43. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden 645 C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona 646 A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. 647
44. Segata N, Börnigen D, Morgan XC, Huttenhower C. 2013. PhyloPhlAn is a new method for 648 improved phylogenetic and taxonomic placement of microbes. Nat Commun 4:2304. 649
45. Letunic I, Bork P. 2016. Interactive tree of life (iTOL) v3: an online tool for the display and 650 annotation of phylogenetic and other trees. Nucleic Acids Res 44:W242-W245. 651
46. Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for eukaryotic 652 genomes. Genome Res 13:2178–2189. 653
47. Lechner M, Findeiss S, Steiner L, Marz M, Stadler PF, Prohaska SJ. 2011. Proteinortho: 654 detection of (co-)orthologs in large-scale analysis. BMC Bioinformatics 12:124. 655
49. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high 658 throughput. Nucleic Acids Res 32:1792–1797. 659
50. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial gene 660 identification with GLIMMER. Nucleic Acids Res 27:4636–4641. 661
51. Salzberg SL, Delcher AL, Kasif S, White O. 1998. Microbial gene identification using 662 interpolated Markov models. Nucleic Acids Res 26:544–548. 663
52. Baerends RJS, Smits WK, de Jong A, Hamoen LW, Kok J, Kuipers OP. 2004. Genome2D: a 664 visualization tool for the rapid analysis of bacterial transcriptome data. Genome Biol 5:R37. 665
53. Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A. 2015. The TOPCONS web server for consensus 666 prediction of membrane protein topology and signal peptides. Nucleic Acids Res 43:W401-667 W407. 668
54. Rost B, Yachdav G, Liu J. 2004. The PredictProtein server. Nucleic Acids Res 32:W321-W326. 669
55. Nicolas P, Mäder U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, Bidnenko E, Marchadier E, 670 Hoebeke M, Aymerich S, Becher D, Bisicchia P, Botella E, Delumeau O, Doherty G, Denham 671 EL, Fogg MJ, Fromion V, Goelzer A, Hansen A, Härtig E, Harwood CR, Homuth G, Jarmer H, 672 Jules M, Klipp E, Le Chat L, Lecointe F, Lewis P, Liebermeister W, March A, Mars RAT, 673 Nannapaneni P, Noone D, Pohl S, Rinn B, Rügheimer F, Sappa PK, Samson F, Schaffer M, 674 Schwikowski B, Steil L, Stülke J, Wiegert T, Devine KM, Wilkinson AJ, van Dijl JM, Hecker M, 675 Völker U, Bessières P, Noirot P. 2012. Condition-dependent transcriptome reveals high-level 676 regulatory architecture in Bacillus subtilis. Science 335:1103–1106. 677
56. Xiao Y, van Hijum SAFT, Abee T, Wells-Bennik MHJ. 2015. Genome-wide transcriptional 678 profiling of Clostridium perfringens SM101 during sporulation extends the core of putative 679 sporulation genes and genes determining spore properties and germination characteristics. 680 PLoS One 10:e0127036. 681
57. van der Meulen SB, de Jong A, Kok J. 2016. Transcriptome landscape of Lactococcus lactis 682 reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and 683 metabolism. RNA Biol 13:353-366. 684
58. de Jong A, van der Meulen S, Kuipers OP, Kok J. 2015. T-REx: Transcriptome analysis 685 webserver for RNA-seq Expression data. BMC Genomics 16:663. 686
59. Skinner ME, Uzilov A V, Stein LD, Mungall CJ, Holmes IH. 2009. JBrowse: a next-generation 687 genome browser. Genome Res 19:1630–1638. 688
60. Nagler K, Setlow P, Li Y-Q, Moeller R. 2014. High salinity alters the germination behavior of 689 Bacillus subtilis spores with nutrient and nonnutrient germinants. Appl Environ Microbiol 690 80:1314–1321. 691
61. Pandey R, Ter Beek A, Vischer NOE, Smelt JPPM, Brul S, Manders EMM. 2013. Live cell 692 imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat 693 stress quantitatively analyzed with SporeTracker. PLoS One 8:e58972. 694
62. Zhang P, Liang J, Yi X, Setlow P, Li Y-Q. 2014. Monitoring of commitment, blocking, and 695 continuation of nutrient germination of individual Bacillus subtilis spores. J Bacteriol 696 196:2443–2454. 697
63. Ghosh S, Zhang P, Li Y, Setlow P. 2009. Superdormant spores of Bacillus species have elevated 698 wet-heat resistance and temperature requirements for heat activation. J Bacteriol 191:5584–699 5591. 700
64. Zhou T, Dong Z, Setlow P, Li Y. 2013. Kinetics of germination of individual spores of 701 Geobacillus stearothermophilus as measured by raman spectroscopy and differential 702 interference contrast microscopy. PLoS One 8:e74987. 703
65. Foerster HF. 1983. Activation and germination characteristics observed in endospores of 704 thermophilic strains of Bacillus. Arch Microbiol 134:175–181. 705
66. Eijlander RT, de Jong A, Krawczyk AO, Holsappel S, Kuipers OP. 2014. SporeWeb: an 706 interactive journey through the complete sporulation cycle of Bacillus subtilis. Nucleic Acids 707 Res 42:D685-D691. 708
67. Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI, Rigden DJ. 2012. Genomic 709 determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-710 specific genes. Environ Microbiol 14:2870-2890. 711
68. Christie G, Lowe CR. 2008. Amino acid substitutions in transmembrane domains 9 and 10 of 712 GerVB that affect the germination properties of Bacillus megaterium spores. J Bacteriol 713 190:8009–8017. 714
69. Christie G, Götzke H, Lowe CR. 2010. Identification of a receptor subunit and putative ligand-715 binding residues involved in the Bacillus megaterium QM B1551 spore germination response 716 to glucose. J Bacteriol 192:4317–4326. 717
70. Li Y, Catta P, Stewart KA, Dufner M, Setlow P, Hao B. 2011. Structure-based functional 718 studies of the effects of amino acid substitutions in GerBC, the C subunit of the Bacillus 719 subtilis GerB spore germinant receptor. J Bacteriol 193:4143–4152. 720
71. Mongkolthanaruk W, Cooper GR, Mawer JS, Allan RN, Moir A. 2011. Effect of amino acid 721 substitutions in the GerAA protein on the function of the alanine-responsive germinant 722 receptor of Bacillus subtilis spores. J Bacteriol 193:2268–2275. 723
72. Li Y, Davis A, Korza G, Zhang P, Li Y, Setlow B, Setlow P, Hao B. 2012. Role of a SpoVA protein 724 in dipicolinic acid uptake into developing spores of Bacillus subtilis. J Bacteriol 194:1875–725 1884. 726
73. Vepachedu VR, Setlow P. 2007. Role of SpoVA proteins in release of dipicolinic acid during 727 germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme. J Bacteriol 728 189:1565–1572. 729
74. Perez-Valdespino A, Li Y, Setlow B, Ghosh S, Pan D, Korza G, Feeherry FE, Doona CJ, Li Y-Q, 730 Hao B, Setlow P. 2014. Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis 731 spores. J Bacteriol 196:2077–2088. 732
75. Vepachedu VR, Setlow P. 2007. Analysis of interactions between nutrient germinant 733 receptors and SpoVA proteins of Bacillus subtilis spores. FEMS Microbiol Lett 274:42–47. 734
76. Karshikoff A, Nilsson L, Ladenstein R. 2015. Rigidity versus flexibility: the dilemma of 735 understanding protein thermal stability. FEBS J 282:3899–3917. 736
77. Sauer DB, Karpowich NK, Song JM, Wang D-N. 2015. Rapid bioinformatic identification of 737 thermostabilizing mutations. Biophys J 109:1420–1428. 738
78. Rohl CA, Fiori W, Baldwin RL. 1999. Alanine is helix-stabilizing in both template-nucleated and 739 standard peptide helices. Proc Natl Acad Sci U S A 96:3682–3687. 740
79. López-Llano J, Campos LA, Sancho J. 2006. Alpha-helix stabilization by alanine relative to 741 glycine: roles of polar and apolar solvent exposures and of backbone entropy. Proteins 742 64:769–778. 743
85. Zeigler DR, Prágai Z, Rodriguez S, Chevreux B, Muffler A, Albert T, Bai R, Wyss M, Perkins JB. 755 2008. The origins of 168, W23, and other Bacillus subtilis legacy strains. J Bacteriol 190:6983–756 6995. 757
88. Zeigler DR. 2011. The genome sequence of Bacillus subtilis subsp. spizizenii W23: insights into 762 speciation within the B. subtilis complex and into the history of B. subtilis genetics. 763 Microbiology 157:2033–2041. 764
89. Nakamura LK, Roberts MS, Cohan FM. 1999. Relationship of Bacillus subtilis clades associated 765 with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and 766 Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49:1211–5. 767
90. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, 768 Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, 769 Bruschi C V, Caldwell B, Capuano V, Carter NM, Choi SK, Codani JJ, Connerton IF, Danchin A. 770 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 771 390:249–256. 772
91. Berendsen EM, Wells-Bennik MHJ, Krawczyk AO, de Jong A, van Heel A, Eijlander RT, Kuipers 773 OP. 2016. Draft genome sequences of 10 Bacillus subtilis strains that form spores with high or 774 low heat resistance. Genome Announc 4:e00124-16. 775
92. Caspers MPM, Schuren FHJ, van Zuijlen ACM, Brul S, Montijn RC, Abee T, Kort R. 2011. A 776 mixed-species microarray for identification of food spoilage bacilli. Food Microbiol 28:245–777 251. 778
93. Cazemier AE, Wagenaars SFM, ter Steeg PF. 2001. Effect of sporulation and recovery medium 779 on the heat resistance and amount of injury of spores from spoilage bacilli. J Appl Microbiol 780 90:761–770. 781
94. Kort R, O’Brien AC, van Stokkum IHM, Oomes SJCM, Crielaard W, Hellingwerf KJ, Brul S. 782 2005. Assessment of heat resistance of bacterial spores from food product isolates by 783 fluorescence monitoring of dipicolinic acid release. Appl Environ Microbiol 71:3556–3564. 784
95. Oomes SJCM, Brul S. 2004. The effect of metal ions commonly present in food on gene 785 expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance. Innov 786 Food Sci Emerg Technol 5:307–316. 787
Ta le 4. Va iatio s i a i o a id se ue es i ge i a t e epto su u its that oi ide ith spe ifi
ge i atio a d heat-a ti atio phe otypes of the i estigated B. subtilis st ai s. The a i o a id
esidue positio s a d p otei egio s a e as i ed a o di g to p otei se ue es a d st u tu e
p edi tio s fo the espe ti e su u its of B. su tilis . “e ue e a iatio s p ese t i Ge KB o Ge KC
su u its of st ai s B -B a d B that a e also p ese t i B a e u de li ed.
Phe otype St ai P otei Se ue e a iatio P otei egio s
No/poo espo se to
L-Ala
B TU-B-
Ge AA T/Q A T- , Q- ; T K; D/A E D- ; A-; E K
glo ula N- pos - a d C-te i i pos -
Ge AB F/V I F- ; V- ; I L; I V; V I; T ; I V
TM ; TM ; TM ; TM ; TM ; L /
Ge AC K T DIII β B Ge AC G C also i Ge AC-B ; K N DII, DIII α
B , B
Ge AB T “; M I TM ; TM Ge AC A/“ Δ A- ; “- ; K Δ; “ N; T I DI; DI; DII α ; DIII α
°C HA & st o g
espo se to L-Ala
B , B
Ge AA G “; “ L; F Y; L I* TM ; TM ; TM ; TM Ge AB A/T “ A- ; T- L /
Ge AC “/A F “- ; A- ; “ L** DI β ; DII li ke
Poo espo se to
AGFK High HA te p.
B - B , B , B
Ge BB “ N; K T L / Ge BC E D DIII
Ge KA V A; Q E; I “; V F; I L; F Y; K/D Q K- , D- ; Q K
glo ula N-te i i; L / ; L / ; L / ; TM ; glo ula C-te i i pos -
Ge KB A V; V I; I L TM ; L / ; TM
Ge KC A/T V A- ; T- ; V A; A D; M I; T “; Y F; F L; N D
“ig al se ue e pos - ; DI; DI α ; DII β ; DII β ; DIII β ; DIII β
Ge KD M I TM “t o g
espo se to AGFK
despite high HR
B
Ge BC N K DIII α Ge KA V G; L M; P Q TM ; L / ; TM Ge KB D E OL Ge KC “ Y; T K; R W DI β ; DII; DII
E pla atio s: α – alpha heli ; β – eta st a d; Δ – deletio ; DI, DII, DIII – do ai I, II a d III, espe ti el ; L / – loop et ee t a s e a e heli es u e a d ; TM – t a s e a e heli ; T/Q A T- , Q- - T p ese t
i st ai s a d Q p ese t i st ai s at positio su stituted A i the st ai s that sho espe ti e phe ot pe et . * L F utatio i Ge AA auses st o g ge i atio defe t i L-ala i e . **“ esidue e hi its % o se atio a o g the se e Ge BC ho ologs f o B. su tilis, Ba illus a yloli uefa ie s, Ba illus pu ilis, Ba illus lausii a d Ba illus e eus ; Δ - i Ge BC auses loss of espo se to AGFK .