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The proteome of spore surface layers in food spoiling bacteria

Abhyankar, W.R.

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Citation for published version (APA):Abhyankar, W. R. (2014). The proteome of spore surface layers in food spoiling bacteria

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3 In Pursuit of Protein Targets: Proteomic

characterization of Bacterial Spore Outer Layers.

Wishwas Abhyankar, Abeer H. Hossain, André Djajasaputra, Patima Permpoonpattana, Alexander Ter Beek, Henk L. Dekker, Simon M. Cutting, Stanley Brul, Leo J. de Koning,

Chris G. de Koster

Published in Journal of Proteome Research, 2013, 12, 4507−4521

Supplementary material can be found at

http://pubs.acs.org/doi/suppl/10.1021/pr4005629

Abstract Bacillus cereus, responsible for food poisoning and Clostridium difficile, causative agent of Clostridium difficile-associated diarrhoea (CDAD) are both spore forming pathogens involved in food spoilage, food intoxication and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins we identified 11 superfamily domains common to the identified proteins from two Bacilli and a Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.

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Introduction

Studies with Bacillus subtilis have provided good insights in the sporulation and germination processes of spores. However, studies of more pathogenic species like aerobic or facultatively anaerobic Bacillus cereus and strictly anaerobic Clostridium difficile are needed to gain more insights in the spore structure and germination characteristics of these spores. The B. cereus group (also known as B. cereus sensu lato), is comprised of several closely related species: Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus [1]. B. cereus is mainly known as a food-borne pathogen causing two types of gastrointestinal diseases: emesis and diarrhea [2]. B. cereus related emesis is caused by the cereulide toxin, which once produced remains stable upon enzyme, heat or acid treatment. Diarrhea, on the other hand, is caused by enterotoxins haemolysin BL (Hbl), non-haemolytic enterotoxin (Nhe) and cytotoxin K (CytK) [2, 3] that are produced by the vegetative cells. B. cereus strains have also been reported in few cases to be associated with non-gastrointestinal diseases including: respiratory tract infections, endophthalmitis, central nervous system infections, gas gangrene-like infections, cutaneous infections, endocarditis, osteomyelitis and urinary tract infections [4]. Spore forming Clostridium spp. (e.g. Clostridium botulinum, Clostridium tetani), like Bacilli, are also known for the infections and diseases caused by them. Pathogenic C. difficile can colonize the intestinal tracts of humans and other mammals [5, 6]. Routine treatments containing use of antibiotics such as Metronidazole, Vancomycine have led to emergence of resistant strains [7, 8] and thus prolonged treatment with such antibiotics can result in C. difficile overgrowth and can lead to diseases ranging from diarrhea to life-threatening pseudomembranous colitis, especially in immunocompromised people [9, 10]. C. difficile is reported to be a continuously evolving species [11, 12] and in recent times the organism has emerged as one of the major causes of nosocomial diarrhea (Clostridium difficile-associated diarrhea; CDAD). CDAD is mainly caused by the secretion of two cytotoxic, enterotoxic and proinflammatory toxins known as toxin A (TcdA) and toxin B (TcdB) [13]. CDAD is particularly problematic to treat and to avert because of the robust endospores that can persist and be easily transferred, person-to-person, in a hospital environment and thus the morbidity and mortality rates have been increasing in recent years. Endospores, are metabolically dormant multi-layered cellular forms which upon germination, lose their protective external layers and resume vegetative growth [14]. In B. subtilis the outermost thick concentric proteinaceous layers - the inner and the outer spore coat [15] - aid in resistance against variety of environmental assaults. Additionally in spores of many Bacillus and Clostridium species surrounding the coats an external loosely-fitting, hydrophobic, glycosylated, balloon-like layer - the exosporium - is present, which assists in adherence to the surfaces [16]. Though the cortex layer from spores might have evolved from the cell walls homologous to those of vegetative cells, the proteinaceous coat is quite unique. The immense resistance of the coat to attack by microorganisms and by free enzymes is unmatched in the bacterial domain. The wide variety of germination signals amongst the closely related species suggests that

Spore surface layer proteins from B. cereus & C. difficile

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germination receptor assembly is a relatively later event in the evolution of the spore structure as the ancestral Bacilli and Clostridia moved to different niches after their divergence about 2.3 billion years ago [17]. Albeit the evolutionary distance, the ability to form spores and the overall sporulation process are conserved in these organisms with significant differences still existing both in the regulation of spore formation and in the spore structure [18]. Thus it is imperative to study the spore coat and exosporium layers from the spores of these two organisms in order to obtain detailed knowledge about the spore structure as well as to design quick and simple techniques for detection and thereby facilitating eradication of spores form the environment. Previous extensive research has led to the identification of up to 70 different proteins from the spore coat layers in B. subtilis. The exosporium is a relatively less studied layer, possibly due to the difficulties in obtaining its large quantities, and is reported to comprise of at least 25 proteins as studied from B. cereus and B. anthracis spores [15, 19-21]. Mostly these studies have focused on the soluble fraction of proteins. Our “gel-free” method [22] allowed us for the first time to focus on the insoluble protein fraction from the spore coat, which makes up to 30% of the proteins [15], and is characterized by extensive inter-protein cross-linking and thus is difficult to analyze using conventional PAGE gels. We have showed that our gel-free method is comprehensive in isolating and identifying proteins from the insoluble protein fraction of B. subtilis spore coats with identification of 19 new proteins. Here, we have extended our method to B. cereus and C. difficile to identify protein targets for early and rapid detection of spores and have characterized for the first time the insoluble proteome of the coat and exosporium of B. cereus ATCC 14579 spores and of the coat layers of C. difficile 630 spores. Recently, McKenney et al. [23] mentioned that the coat morphogenetic proteins may be the targets for evolutionary adaptation for spore formers. Therefore to obtain a universal set of spore coat and exosporium proteins we also assessed different spore formers for the conservation of the spore surface proteins that were identified from B. subtilis [22], B. cereus and C. difficile in our studies. Results and Discussion

Spore preparation

The harvested B. cereus ATCC 14579 spore crop contained >95% purified spores and for the C. difficile strain 630 the sporulation efficiency was noted to be ~75%. In contrast to the B. cereus spores, the presence of exosporium in harvested C. difficile spores was inconsistent and if present only remnants of the exosporium were seen under the phase contrast microscope, as observed previously [24]. Spore peptidoglycan analysis

Protein isolation and extensive washing of the pellets with salt reduced the amount of cortex muramic acid below1% compared to the intact spores. However the final reduced, alkylated pellet unexpectedly showed a slight increase in the amount of muramic acid. This could be due to the release of sugar moieties post-digestion. Lysozyme

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treatment did not further lower the muramic acid content of the pellet material. Deglycosylation of the reduced and alkylated pellet did significantly reduce the muramic acid content to values below 0.05% (results not shown). In conclusion, the final pellets used for proteomic analysis hardly contained any cortex muramic acid contamination. Conserved Domain Search

Amongst all the identified proteins from both species, 148 different superfamily domains including 22 distinct Domains of Unknown Function (DUFs) were identified by a domain searching tool from NCBI (see Supplementary Table 1). The hydrolases and peptidases identified from B. cereus ATCC 14579 and C. difficile 630 in this study might be important for spore germination and spore structure. Amongst the proteins identified from C. difficile, a preference towards proteins with catalase-domains, ferritin-like domains and metal-binding domains that might have a role in resistance towards oxidative stress (see below), was observed. There were 11 superfamily domains that emerged as common to the identified proteins from B. subtilis 168[22], B. cereus ATCC 14579 and C. difficile 630 (Figure 1).

Figure 1. Venn diagramme of conserved superfamily domains in the identified spore coat & exosporium proteins from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630. A total of 148 superfamily domains were assigned to the identified proteins from the three species indicated. The numbers correspond to the superfamily domains from the identified proteins. The eleven superfamily domains common to the three organisms are listed in the box.

Protein identification and Orthologue evaluation i.e. PSI-BLAST analysis

Orthologues are genes (in different species) evolved from a common ancestral gene by speciation. This suggests that orthologues retain the same function in the course of evolution. Evolutionary pressure however is not equal on all residues of a protein. For instance, buried residues at an active site or at a binding site are generally more conserved than residues in loops. Also evolutionary pressure may force addition or deletion of gene segments from the genomes leading to different gene and protein sizes. Orthologue identifications has been done by various different approaches amongst which a Best-Reciprocal BLAST Hit (BBH) or Bi-directional Best BLAST hit (BDBH) approach has


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been considered the best[25]. However, this approach has its own flaws in the sense that only one-to-one orthologous pairs are found, for duplicated genes or paralogues only a single hit is found. The PSI-BLAST search tool helps in identification of regions of importance (not variable) and gives them more weight in subsequent comparisons. The PSI-BLAST tool may also detect subtle relationships between proteins that are distant structural or functional homologues. Such relationships are often not detected by a simple BLAST search. Our current study focused for the first time on the insoluble fraction and led to the identification of total 100 proteins from the spore coat and exosporium layers of B. cereus ATCC 14579 and 54 proteins from the spore coat layers of C. difficile 630 (Table 1 & 2). These proteins were then used for orthologue evaluation using PSI-BLAST search (see Supplementary Tables 2, 3, 4 and 5). As mentioned above, due to the possibility of various sizes of proteins, we assigned the sequence identity threshold at 30% and an E-value of 0.005 was the default value chosen. The experimentally identified proteins with conserved orthologues in two Bacilli and a Clostridium species analyzed by us are listed in Table 3. As seen proteins CotJC, DacF, SpoIVA and YisY were identified experimentally from all the three organisms. For further comparative analysis, we selected five Bacillus species (11 different strains) and four Clostridium species (total 13 different strains) available on the Genolist database (http://genolist.pasteur.fr/GenoList). Proteins SafA (BC_4420), CotE (BC_3770), YhcN (BC_4419), YusW (BC_0212), CotD (BC_1560), YxeE (BC_3534) and others (Table 1) are among the proteins identified only from coat + exosporium (fraction 1) and coat (fraction 2) confirming their localization in the spore coat while proteins InA (BC_1284), CalY (BC_1279) and BC_1591 were among those identified only from fraction 1 & exosporium (fraction 3). As seen there was a considerable overlap between fractions 2 & 3 id est between spore coat and exosporium fractions implying two possibilities - either these proteins may be spread across both the layers or the removal of the exosporium layer was inefficient. Five proteins - BC_0944, SodF (BC_1468), YppG (BC_1559), BC_2237 and BclB (BC_2382) - were identified only by a semi-trypsin enzyme search. In case of C. difficile 630, CdeC [26] (CD1067), CD1581, CotA (CD1613), CotB (CD1511), CotJB1/CotCA (CD0597), CotJC1/CotCB (CD0598), CotJC2/CotD (CD2401), CotE (CD1433), CotF (CD0596), CotJB2 (CD2400), Rbr (CD2845), SipL (CD3567), were among the high scoring proteins with > 10 peptides identified per protein. In total, from the identified C. difficile proteins 24 proteins have been identified previously [27] from the whole spore protein extracts. We did not study a separate exosporium fraction for C. difficile 630 as the stability of the exosporial layer may depend on the spore preparation method as well as their storage and in our sample the presence of exosporium was inconsistent possibly due to its loss during spore harvest. (see Materials & Methods).

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Table 1. Identified spore coat and exosporium proteins from Bacillus cereus ATCC 14579.

a Gene name a Uniprot ID

a Protein description a Mass (Da)

Spore coat & Exosporium

Spore coat Exosporium

bFDR= 1.77% FDR= 1.62% FDR= 1.99% cScore cno.

pept Score no.

pept Score no.

pept (I) Proteins involved in spore coat morphogenesis and other known spore coat proteins

BC_4420 (safA) d Q812R2 SpoVID-dependent spore coat assembly factor SafA 65354 1286 46 2357 70

BC_2872 (cotX) Q81CA3 Spore coat protein X 16345 1145 28 1288 32 216 5

BC_2030g Q81EF0 Spore coat protein G 24013 1141 36 1309 41 471 30

BC_0389 (cotB) Q81IJ7 Spore coat protein B 19768 1109 67 2763 100 524 23

BC_3770 (cotE) d Q81A24 Spore coat protein E 20615 971 37 122 5 BC_0390 (cotB) Q81IJ6 Spore coat protein B 17339 744 29 1563 39 328 11

BC_4419 (yhcN) d Q817V9 Putative uncharacterized protein 24526 672 21 628 21 BC_1222 Q81GH8 Spore coat protein Y 17596 486 19 729 21 5668 188

BC_5056 d Q815S6 Collagen adhesion protein 34657 383 19 1427 37 BC_2874 (cotX) Q81CA1 Spore coat protein X 21850 306 12 293 12 36 1 BC_1509 (spoIVA) Q81FR0 Stage IV sporulation protein A 55636 242 11 BC_4640 (ytfJ) d Q817B7 Putative uncharacterized protein 13968 237 10 81 3 BC_1560 (cotD) d Q81FM0 Spore coat protein D 15393 214 10 565 16 BC_0212 (yusW) d Q81IY1 Putative uncharacterized protein 17725 122 3 204 6

BC_4075 (dacF) d Q819B1 D-alanyl-D-alanine carboxypeptidase 44032 85 3 150 5

BC_1279 (cotN) Q81GC8 Spore coat-associated protein N 21848 79 3 BC_1245 Q81GF8 Putative uncharacterized protein 15287 39 2 BC_0822 (cotJB) Q81HI6 CotJB protein 10523 45 1 BC_0821 (cotJC) Q81HI7 CotJC protein 21694 37 1 BC_0823 (cotJA) Q81HI5 CotJA protein 8475 30 1 BC_3534 (yxeE) d Q81AM8 IG hypothetical 17193 16298 21 1 33 1 BC_2095 (ytfJ) d Q81E93 Putative uncharacterized protein 15087 120 5 32 1 BC_0063 (yabP) Q81J89 Putative uncharacterized protein 11627 45 1 BC_2677 Q81CR9 L-alanyl-D-glutamate peptidase 31439 41 1 BC_0047 (yabG) Q81JA4 Sporulation-specific protease

YabG 33219 23 1 BC_1559(yppG)f Q81FM1 Spore coat protein 22228 23 1

(II) Spore coat proteins likely to play a role in spore resistance

BC_4047 (cotα) Q819D8 Putative uncharacterized protein 14344 519 24 680 28 310 18

BC_4639 d Q817B8 Thiol peroxidase 18055 158 8 48 2 BC_1391 (yqfX) d Q81G20 Putative uncharacterized protein 13457 187 5 69 1 BC_2099 (yqfX) d Q81E89 Putative uncharacterized protein 12239 26 1 67 1 BC_4774 (yisY) d Q816P9 Non-heme chloroperoxidase 30166 54 1 62 1

(III) Exosporium proteins likely to be involved in attachment to surfaces

BC_1218 (exsY) Q81GI1 Spore coat protein Y 16761 640 26 1447 46 2274 106

BC_2493 (exsK) Q81D85 Putative uncharacterized protein 13556 1128 26 1372 34 10874 236

BC_3712 (bclC) Q812Y5 Hypothetical Membrane Spanning Protein 75838 673 37 483 21 820 30

BC_1221 (bxpB) Q813V0 Exosporium basal layer protein 17390 425 19 448 21 1775 65


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pept Score no.

pept Score no.

pept BC_3547 Q81AL6 Cell surface protein 97913 199 5 713 16 848 32

BC_2374 (exsFB) Q813L4 Hypothetical Membrane Spanning Protein 17496 141 6 88 3 164 5

BC_2382 (bclB)f Q81DI4 Putative uncharacterized protein 39119 50 1

BC_2639 d Q81CV2 Cell surface protein 521640 80 1 29 1 BC_3345 Q81B46 Collagen-like triple helix repeat

protein 77508 116 2

BC_2569 Q81D14 Collagen triple helix repeat protein 53405 73 3 24 1 85 2

BC_2149 (bxpA) d Q81E43 Putative uncharacterized protein 32484 47 1 412 8 (IV) Exosporium proteins possibly involved in pathogenicity

BC_1284 (inA) e Q81GC3 Immune inhibitor A 85713 920 41 137 5

BC_2267 Q81DT6 Putative uncharacterized protein 21872 274 11 433 14 261 10

BC_2266 Q81DT7 Putative uncharacterized protein 20239 41 2 144 4 42 2

BC_1281 (calY) e Q81GC6 Cell envelope-bound metalloprotease (Camelysin) 21801 409 18 21 1

(V) Spore coat proteins involved in spore germination

BC_3607 (yaaH) d Q81AG3 Spore peptidoglycan hydrolase (N-acetylglucosaminidase) 48167 1185 54 1235 41

BC_5391 (gerQ) d Q814N4 Putative uncharacterized protein 16090 587 21 227 8 BC_0264 (alr1) Q81IT5 Alanine racemase 1 43789 382 22 678 18 1393 47

BC_2889 (iunH) Q81C90 Inosine-uridine preferring nucleoside hydrolase 36552 306 11 580 20 1797 61

BC_2207 Q81DY9 Sporulation-specific N-acetylmuramoyl-L-alanine amidase 35924 129 8

BC_5390 (cwlJ) d Q814N5 Cell wall hydrolase cwlJ 16467 93 5 45 3 BC_4319 (gpr) Q818E2 Germination protease 40450 90 3 BC_1591 e Q81FJ1 Putative uncharacterized protein 58965 87 2 5360 170

BC_3552 (iunH) Q81AL1 Inosine-uridine preferring nucleoside hydrolase 34507 27 1

BC_2752 (ypeB) Q813I5 Spore germination protein 50109 81 2 BC_2753 (sleB) d P0A3V0 Spore cortex-lytic enzyme 28297 66 2 32 1

(VI) Other putative spore coat and/or exosporium proteins

BC_0987 Q81H38 Putative uncharacterized protein 14633 736 33 1800 39 878 30

BC_0996 Q81H29 Putative uncharacterized protein 15501 536 23 725 31 366 11

BC_5135 (eno) d Q815K8 Enolase 46400 516 16 95 4 BC_p0002 d Q814F0 Putative uncharacterized protein 17989 333 11 97 4 BC_2858 Q81CB5 Putative uncharacterized protein 9626 266 12 232 7 52 1

BC_2426 Q81DE1 Putative uncharacterized protein 26265 263 16 365 21 139 5

BC_2026 Q81EF3 Oligopeptide-binding protein oppA 63075 237 11 BC_1613 d Q81FH4 Zn-dependent hydrolase 62004 193 8 187 5 BC_4410 (yajC) d Q817W7 Protein translocase subunit YajC 9486 132 6 72 2 BC_0337 d Q814A8 Hypothetical Membrane Spanning

Protein 14189 126 8 151 9 BC_3195 Q813E6 Hypothetical Cytosolic Protein 15025 113 7 74 3 109 5

BC_3787 Q81A08 Zinc protease 48972 112 3 BC_3786 Q81A09 Zinc protease 49288 112 2 BC_3586 Q81AI2 Oligopeptide-binding protein oppA 63759 104 3

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pept Score no.

pept Score no.

pept BC_3133 Q81BM0 Putative hydrolase 42323 98 3 BC_1424 Q81FY8 Ferredoxin--nitrite reductase 60770 97 3 BC_3986 d Q812V3 Hypothetical Cytosolic Protein 9170 95 3 61 2 BC_4480 (tig) d Q812Q9 Trigger factor 47316 88 4 35 1 BC_0344 (rocA) d Q81IP0 1-pyrroline-5-carboxylate

dehydrogenase 56418 84 4 23 1 BC_2375 Q81DJ0 Putative uncharacterized protein 9489 70 2 BC_0825 Q81HI3 Putative uncharacterized protein 30284 65 1 BC_2745 Q81CL1 Putative uncharacterized protein 43460 62 3 BC_1029 d Q81GZ8 IG hypothetical 18063 33466 57 4 25 1 BC_3090 d Q81BR0 Putative uncharacterized protein 18565 44 4 48 2 BC_5181 d Q815H3 UPF0145 protein BC_5181 11143 42 1 51 1 BC_3992 Q819I6 Putative uncharacterized protein 12700 30 1 44 2 22 1

BC_2969 Q81C15 Putative uncharacterized protein 12769 30 1 BC_2481 Q81D93 Putative uncharacterized protein 12069 28 1 BC_1456 Q81FV9 Putative uncharacterized protein 16272 27 2 BC_1708 Q81F89 Putative uncharacterized protein 25183 27 1 BC_3977 Q819J9 Zn-dependent hydrolase 62055 27 1 BC_3515 d Q813A3 Hypothetical Glycosyltransferase 82417 24 1 38 1 BC_0395 Q81IJ1 Metal-dependent hydrolase 23560 23 1 BC_3784 Q81A11 IG hypothetical 16623 9209 195 5 BC_4387 Q817Y7 Putative uncharacterized protein 4832 58 1 BC_3582 (yodI) Q81AI6 Putative uncharacterized protein 14601 213 4 25 1

BC_1334 Q813U4 Hypothetical Exported Protein 28367 106 1 BC_2878 Q81C97 Putative uncharacterized protein 29564 42 2 66 4

BC_0263 Q81IT6 Putative uncharacterized protein 38513 42 1 BC_1468 (sodF)f Q81FV0 Superoxide dismutase 26798 29 1 BC_0944 f Q81H77 Putative uncharacterized protein 61227 28 1

BC_2237f Q81DW2 Putative uncharacterized protein 74111 26 1

BC_2427 Q81DE0 Putative uncharacterized protein 34256 26 1 aDetails obtained from Uniprot database (www.uniprot.org/). bPeptide False Discovery Rate. cMASCOT MudPIT score & total number of identified peptides over three biological replicates. d Proteins identified only from coat + exosporium & coat fractions. eProteins identified only from coat + exosporium & exosporium fractions. fProteins identified only be semitrypsin search. g Phosphorylation sites identified by the error-tolerant MASCOT search. None of the other proteins were identified with modifications.

We divided the identified proteins from both species into six different categories based on their possible functions as indicated by their descriptions in the database - (1) proteins involved in spore coat morphogenesis and other known spore coat proteins; (2) spore coat proteins with a possible role in spore resistance; (3) exosporium proteins likely to be involved in attachment to surfaces; (4) exosporium proteins possibly involved in pathogenicity; (5) spore coat proteins possibly involved in spore germination and (6) other putative spore coat and/or exosporium proteins.


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Table 2. Identified spore proteins from Clostridium difficile 630.

a Gene name a

Uniprot ID


Spore coat bFDR= 1.72%

cScore cno. pept

(I) Proteins involved in spore coat morphogenesis and other known spore coat proteins

CD1067(cdeC)d, e Q18AS2 Putative uncharacterized protein 46845 6392 173

CD1581 e Q186D6 Putative uncharacterized protein 20043 2572 103

CD1613 (cotA)d, e Q186G8 Putative uncharacterized protein 34834 1680 50

CD2401 (cotJC2/cotD) d, e Q181Y5 Spore coat peptide assembly protein CotJC 2 21497 1231 39

CD1433 (cotE) d, e Q18BV5 Putative bifunctional protein: peroxiredoxin/chitinase 82019 957 46

CD3567 (sipL) e Q181G7 Putative phage cell wall hydrolase 59373 869 44

CD0598 (cotJC1/cotCB) d, e Q189E4 Spore-coat protein 21577 823 38

CD2400 (cotJB2) e Q181Y6 Spore coat peptide assembly protein CotJB 2 10663 722 16

CD0597 (cotJB1/cotCA) d, e Q189E5 Spore coat peptide assembly protein 10756 389 15

CD1511 (cotB) d ,e Q18C29 Putative uncharacterized protein 35056 370 16

CD0596 (cotF) d, e Q189D6 Putative uncharacterized protein 8844 336 13

CD2399 e Q181Y3 Putative uncharacterized protein 8247 169 5

CD2598 Q182T7 Putative oligosaccharide deacetylase 29780 70 2

CD2629 (spoIVA) e Q182W3 Stage IV sporulation protein A 55539 55 6

CD3569 (yabG) Q181G9 Sporulation-specific protease 32137 33 1

CD0213 (cotF) Q18CV2 Putative spore coat protein 10927 31 3

(II) Spore coat proteins likely to play a role in spore resistance

CD2845 (rbr) e Q183T4 Rubrerythrin 22603 373 12

CD2864 (yisY) e Q183V0 Putative hydrolase 30520 243 13

CD1524 Q18C45 Putative rubrerythrin 20319 126 9

CD1623 Q186H7 Putative oxidoreductase 94987 110 5

CD0825 (rbr) e Q18A24 Rubrerythrin 20829 64 3

CD0116 Q18CK8 Putative ferredoxin/flavodoxin oxidoreductase,alpha subunit 39290 106 4

CD0117 Q18CK7 Putative ferredoxin/flavodoxin oxidoreductase,beta subunit 27031 61 2

CD1567 (cotG) d, e Q186C1 Putative manganese catalase 25329 45 3

CD0176 Q18CR6 Putative oxidoreductase, NAD/FAD binding subunit 45889 38 2

(III) Exosporium proteins likely to be involved in attachment to surfaces

CD0332 (bclA1)e Q18D69 Putative exosporium glycoprotein 68114 85 5

(IV) Exosporium proteins possibly involved in pathogenicity

-- -- -- -- -- -- --

(V) Spore coat proteins involved in spore germination

CD0551 (sleC) e Q188Z5 Spore cortex-lytic enzyme pre-pro-form 47426 227 10

CD3102 Q184U1 Putative peptidase, M20 family, peptidase V related 53995 29 2

(VI) Other putative spore coat and/or exosporium proteins

CD3032 Q184M0 Putative pyridoxal phosphate-dependent transferase 47977 332 18

CD3620 Q181M3 Putative uncharacterized protein 14256 232 11

CD1133 Q18AZ2 Putative uncharacterized protein 23857 217 10

CD3580 Q181I4 Putative uncharacterized protein 29071 157 5

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a Gene name a

Uniprot ID


Spore coat bFDR= 1.72%

cScore cno. pept

CD0855 (oppA) Q18A51 ABC-type transport system, oligopeptide-family extracellular solute-binding protein 58649 126 9

CD3664 Q181R8 Putative aminotransferase 44997 119 7

CD1536 Q18C55 Ferredoxin--NADP(+) reductase subunit alpha 33353 94 3

CD3522 Q181C2 Putative uncharacterized protein 50689 91 4

CD1291 (dacF) e Q18BF4 D-alanyl-D-alanine carboxypeptidase 42194 90 3

CD2808 Q183P2 Putative uncharacterized protein 23705 87 1

CD1463 Q18BY4 Putative uncharacterized protein 17988 68 5

CD3652 Q181Q6 Putative peptidase, M1 family 54828 62 5

CD3170 (eno) e Q181T5 Enolase 46289 67 3


CD2865 Q183U9 Putative bacterioferritin 20747 54 3

CD3613 Q181L4 Putative uncharacterized protein 17425 42 3

CD2431 Q182B6 Putative nitrite/sulphite reductase 58684 40 1

CD0115 Q18CK1 Putative 4Fe-4S ferredoxin, iron-sulfur binding domain protein, delta subunit 8140 40 2

CD1622 Q186H8 Putative uncharacterized protein 23461 39 1

CD3232 Q17ZX7 UPF0597 protein CD630_32320 45787 39 1

CD3457 Q180V4 Putative uncharacterized protein 15859 37 2

CD1063.1 e Q18AR2 Putative uncharacterized protein 8221 35 2

CD0894 Q18A86 Putative iron-dependent hydrogenase 56384 32 1


CD0279 Q18D22 Putative uncharacterized protein 14922 24 1

CD2477 e D5Q2G9 Putative uncharacterized protein 36070 22 1 aDetails obtained from Uniprot database (www.uniprot.org/). bPeptide False Discovery Rate. cMASCOT MudPIT score & total number of identified peptides over three biological replicates. dRenamed in the previous studies[24, 26, 28]. eProteins identified previously from the whole spore protein extract[27]. 1. Proteins involved in spore coat morphogenesis and other known spore coat proteins.

In the previous studies in B. subtilis different morphogenetic proteins important for the morphogenesis of the spore coat were assigned. These proteins include, starting from the cortex and moving towards the outer coat, SpoVM, SpoIVA, SpoVID, SafA and CotE. For the biogenesis of the inner coat SpoIVA and SafA are important while CotE plays a pivotal role in outer coat development [15, 23]. In our current study we identified CotE (BC_3770), SafA (BC_4420) and SpoIVA (BC_1509) from B. cereus ATCC 14579 and SpoIVA (CD2629) as well as SipL (CD3567, recently identified [29] morphogenetic protein) from C. difficile 630. The CotE orthologue of B. subtilis was not found by protein sequence comparison in the C. difficile 630 proteome. However CD1433 renamed as CotE [24], was identified. Orthologues of other morphogenetic proteins i.e. SpoVID, SpoVM were not identified but for these two proteins it is important to keep in mind that


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Table 3. Spore coat and/or exosporium proteins identified in our studies from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630.

Spore coat and/or exosporium proteins identified in our studies and having conserved orthologues (> 30% protein identity) in the three spore formers. A letter Y indicates experimental identification of the proteins. Proteins CotJC, DacF, SpoIVA and YisY were identified experimentally from all the three organisms. they are mostly identified in the soluble fraction of spores from B. subtilis [30, 31]. In B.subtilis, coat proteins CotC, CotG and CotU are known to be important for the coat structure wherein cross-linking among these proteins plays a major role [32-35]. Genes for CotC, CotG, CotS, CotT and CotW are not present in the B. cereus genome [15]. Likewise, orthologues of SpoVID, SpoVM, CotC, CotG, CotT, CotU and CotW are not observed in the C. difficile genome indicating a possible difference in the spore structure. Spore coat G (BC_2030) identified in our study should not be confused with CotG from B. subtilis as it is 89% identical to the highly phosphorylated exosporium protein ExsB from B. anthracis. The phosphorylated region of BC_2030 is 96% identical to the region identified from ExsB from B. anthracis [36]. Identical to B. anthracis ExsB [36], we identified Threonine residues at positions 76, 79, 82, 105, 114, 117, 129 from BC_2030 as phosphorylated in our error-tolerant MASCOT search. Identified protein CD2399 is a paralogue of CD0596 (renamed as CotF [28]) also identified in the current study. Both these proteins contain a CotJA superfamily domain of spore proteins and thus could function as the CotJA orthologues. Two copies of each, spore coat B (BC_0389 and BC_0390), spore coat Y (BC_1218 and BC_1222) and spore coat X (BC_2872 and BC_2874), are present in the genome of B. cereus ATCC 14579, and these were all identified in this study. Considering the Bacillus species in which the orthologues of these proteins are found and based on the fact that B. subtilis has an orthologue of one of each duo (as predicted from the GenoList database), it is possible that these proteins are

Protein Bacillus cereus ATCC 14579 Clostridium difficile 630 Bacillus subtilis 168

YabG Y Y YabP Y YdbD Y Y CotJC Y Y Y CotJB Y Y SpoIVA Y Y Y SleB Y Y RnjA Y YmfH Y YmfF Y DacF Y Y Y Gpr Y Tig Y YtfJ Y Y YtjP Y YisY Y Y Y Eno Y Y CwlJ Y Y

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localized either in the spore coat, the exosporium or in the basal layer of the exosporium. For instance, protein spore coat Y (BC_1218) is in fact a close orthologue of exosporium morphogenetic protein BAS1141 or ExsY from B. anthracis [37]. Another structural protein identified in our study was spore coat protein D (BC_1560), which is an orthologue of CotD - an inner coat protein in B. subtilis identified prominently from the soluble fraction. This indicates a possible structural difference amongst B subtilis and B. cereus spore coats. From B. cereus, we also identified protein BC_4419, an orthologue of YhcN from B. subtilis (< 30% sequence identity and thus left out of the orthologue data (Supplementary Table 2)) that contains a Spore_YhcN_YlaJ motif. This motif is reported to be found in lipoproteins present in spores and not in vegetative cells. In B. subtilis 168 spores YhcN is suggested to be located in the inner membrane or integument of the spore [38]. Although the expression of YhcN in B. subtilis is controlled by the transcription factor σG (expressed late in the forespore during sporulation [39]) due to the signal sequence carried by the protein it may be identified in the insoluble coat fraction. The collagen adhesion protein (BC_5056) contains a TQxA domain and is conserved amongst the B. cereus group. TQxA domain containing proteins have been reported to be associated with LPXTG-containing sortase target domains. Additionally, LPXTG proteins are located at the cell wall peptidoglycan [40]. It is plausible that BC_5056 is in the spore coat or in the integument region connecting cortex and the coat. Another interesting protein is the L-alanyl-D-glutamate peptidase BC_2677, an orthologue of CwlK which is thought to be involved in cell wall peptidoglycan hydrolysis in B. subtilis [41]. This protein has a VanY domain that is present in proteins involved in cell wall biosynthesis. Additionally, BC_2677 also has SH3 superfamily domain. Proteins belonging to the SH3 family are involved in the formation of multi-protein complex assemblies [42]. This is an essential feature in spore coat build-up and this protein could be an interesting candidate for studying structural built-up in B. cereus spores. Superoxide dismutase (SodA) also plays an important role in spore coat assembly in B. subtilis by mediating protein cross-linking. We did not identify the enzyme in B. cereus ATCC 14579. By a semitrypsin enzyme search with MASCOT we identified SodF (BC_1468) from the spore coat fraction. The superoxide dismutase (CD1631) from the genome of C. difficile was not identified in our study but, to our surprise, PSI-BLAST with one iteration did not predict orthologue for SodA either. In fact CD1631 is more identical to SodF identified from exosporium fractions previously (Supplementary Table 5). 2. Spore coat proteins with a role in spore resistance.

CotA from B. subtilis is known to play a role in UV-resistance of spores. Interestingly CotA from B. subtilis has orthologues present in C. botulinum A ATCC 19397, C. botulinum A ATCC 3502 and C. botulinum strain A Hall but not in any other species and/or strains considered for orthologue evaluation (Supplementary Table 4). On the other hand CD1613 (renamed as CotA) suggested to be involved in assembly of the outer coat in C. difficile 630 spores[28] was identified. The newly identified protein BC_4047 is an orthologue of the outer coat protein Cotα (BAS3957) in B. anthracis


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Sterne and contains a DUF3915 domain of unknown function. There are three Cysteine residues at the amino terminus (first 20 residues) and similar to the case in B. anthracis spores [43], these are likely to be cross-linked. The absence of Cotα is reported to affect visual appearance of the outer spore coat as well as the chemical resistance and sensitivity properties of the spores towards phenol, chloroform, and hypochlorite [43]. In anaerobic organisms defense against oxidative stress is of prime importance. Since spores are important in the dissemination of the clostridial species it is likely that proteins with ability to scavenge the oxidative radicals are present in the outermost layers of the spores. This is also evident from the identified catalases (CotJC1/CotCB (CD0598), CotG (CD1567), ferritin like proteins, ruberythrins (CD0825, CD1524, CD2845) and oxidoreductases (CD0117, CD0176, CD1623) from C. difficile spores (Supplementary Table 1). These proteins may play a dual role - one in structural built-up of spores coat layers by mediating dityrosine cross-linking among proteins and/or second in resistance against oxidative stress [44, 45] or they may have some other hitherto unknown functions. For instance, in addition to the radical scavenging activity, the oxidoreductase CD1623 also has a predicted β-lactamase domain and thus might be important in antibiotic resistance. 3. Exosporium proteins likely to be involved in the attachment to the surfaces.

The exosporium layer has been less extensively studied than the spore coat. BclA from B. anthracis is one of the few exosporium proteins that is well characterized and is an important structural component of the hair-like nap in exosporium [46, 47]. We could not identify this protein (accession no. HM071986.1[16]) in our B. cereus exosporium preparations but proteins BC_3345 & BC_2569 (56% and 58% identical to HM071986.1 respectively) and BclC (BC_3712) were identified in this study. These Bcl-family proteins, in general, form an extended rod-like structure in the regions that possess the (Gly-X-Y)n motifs, which is characteristic for a triple helix, rod-like structure formation [48]. A recent study [49] showed that the BC_2569 protein, similar to BclA, contains a potential NTD attachment motif (-INPDLLGPTLPAI-) necessary to dock this protein in the basal layer of the exosporium. From C. difficile, CD0332 (BclA1) was identified which indicates that the presence of the exosporium in spores was inconsistent in our study. As opposed to the predictions of algorithms used by databases such as KEGG (www.genome.jp/kegg/), orthologues for Bcl family of proteins were not identified to be conserved by the PSI-BLAST analysis by a single PSI-BLAST iteration from any other clostridial species considered in this study. Recently other exosporium proteins BxpA, BxpB, BclB [19, 50, 51] have also been identified in B. anthracis. Protein BxpA (BC_2149) has an amino acid sequence unusually rich in glutamine and proline [52]. Interestingly, VrrA protein in B. anthracis is also rich in glutamine and proline residues which are suggested to be involved in cross-linking of protein subunits [53]. BxpB is an exosporium basal layer protein in B. anthracis Sterne [50] and its orthologue BC_1221 in B. cereus ATCC 14579 was identified in the current study. In B. anthracis BxpB has a paralogue ~80% identical, named ExsFB (BAS2303) and the identified protein BC_2374 is an orthologue of ExsFB.

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Both BC_1221 & BC_2374 are 75% identical to BxpB (BAS1144) and ExsFB (BAS2303) as shown by a Clustal Omega alignment tool. This suggests that BC_1221 and BC_2374 may be localized in the basal layer of the B. cereus ATCC 14579 exosporium in providing attachment sites for BclA-like proteins. In bxpB mutants of B. anthracis, the function of BxpB could not be complemented with BAS2303, implying a different function for BAS2303 in B. anthracis spores[50]. Orthologues for BxpA (BC_2149), BxpB (BC_1221) and ExsFB (BC_2374) are well conserved in the B. cereus group. Protein BC_2493 is an orthologue of exosporium protein ExsK identified from the exosporium fraction of B. anthracis spores (BA_2554) in a study of Redmond et al.[54] Identified cell surface proteins BC_2639 and BC_3547 (523.5 kDa and 97.9 kDa, respectively) contain multiple unknown domains named DUF11 and non-specific domains called B_ant_repeat domains. These B_ant_repeat domain containing proteins are shown to be encoded by one, two or three very conserved large genes in the B. cereus group [55]. These genes are expressed in the last developmental time waves during sporulation [56]. Although a function is unknown it has been reported that these proteins may have a similar function to that of proteins that form ribbon-like appendage structure on the exosporium of Clostridium taeniosporum spores [55]. These proteins denoted P29a and P29b in C. taeniosporum are smaller in size than the proteins we identified, but the size may be extremely variable between species. These appendage-like structures may be common in spores of Bacilli and of Clostridia [57] and may facilitate spore dissemination, assist in spore nutrition during formation, or have no function and result from a deranged metabolism [58]. 4. Exosporium proteins possibly involved in pathogenicity.

Since the exosporium is the outermost layer of the spores in many species, it is the initial point of contact with the host cells and tissues. Therefore, in addition to providing unique adherence and hydrophobic properties to spores, it may express molecules that play a significant role in spore attachment and pathogenicity. The BclA protein is suggested to be the immunodominant epitope on the surface of Bacillus anthracis spores and contains a C-terminal TNF-like domain [47, 59]. Though not required for pathogenesis [60] BclA may mediate internalization of spores by host cells via the C1q/TNF-like domain. Putative uncharacterized proteins BC_2266 and BC_2267, identified in our study both possess a domain belonging to the TNF superfamily and has a weak homology with the C1q domain. The C1q domain has been reported to play a role in spore attachment and in host entry mechanisms where spores are suggested to bind to the gC1qR receptors on the host cell via some unidentified molecules, in a Ca2+ dependent manner [61]. Thus, BC_2266 and BC_2267 could very well be mediators involved in a spore attachment and host entry process. Tertiary structure modeling with Phyre2 tool for both molecules showed that they have 99-100% homology with TNF in the region from amino acids 70 to 150. About 79% to 84% of the sequence could be modeled with over 90% confidence. Further studies are required to understand the precise location and the function (e.g. using animal models) of these two proteins.


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5. Spore coat proteins involved in spore germination.

While the spores of Bacillus and Clostridium species can survive for years in the dormant state, they can be rapidly converted to a metabolically active state via the process of spore germination and outgrowth. A variety of small molecules can trigger spore germination preliminary by binding to the germination receptors in the inner membrane. L-alanine is known to be a potent germinant of B. cereus spores, whereas D-alanine inhibits germination. Alanine racemase (Alr1; BC_0264) by converting L-alanine to its stereoisomer thereby preventing germination [62] and the inosine-uridine preferring nucleoside hydrolase (IunH; BC_2889) by converting the germinant inosine to D-ribose could play a role in preventing germination[63]. Once germination is triggered, the coat needs to be broken down and peptidases identified in this study (Supplementary Table 3) may be involved in germination. Subsequent spore outgrowth requires that the spore cortex is degraded immediately once germination is triggered. Cortex lytic enzymes (CLEs) like CwlJ and SleB play an important role in cortex degradation and are located in the B. subtilis spore coat. In B. cereus ATCC 14579 we identified SleB (BC_2753) and CwlJ (BC_5390). Identified protein BC_5391 is an orthologue of GerQ/YwdL that is involved in proper localization of the cortex lytic enzyme CwlJ in the B. subtilis spore coat [64]. However, in other studies GerQ/YwdL and CwlJ were identified in the exosporium fraction of B. cereus ATCC 10876 and B. anthracis spores, respectively [52, 65]. Another enzyme identified from the insoluble fraction in this study and reported to be involved in spore cortex degradation [66] during spore germination was YaaH orthologue BC_3607. Newly identified BC_1591 contains a predicted pectate lysase domain, which is related to the family of glycosyl hydrolase proteins and may therefore be involved in peptidoglycan modification, degradation or synthesis. In addition there were also other hydrolases identified in our study that can be involved in spore germination (Supplementary Table 3). From C. difficile the predominant cortex lytic enzyme SleC (CD0551) was identified in this study. Of further interest, the transcriptomic data of Dembek and colleagues [67] shows that genes cd0115, cd0116, cd0117, cd0176, cd0279, cd0587, cd0825, cd0855, cd1133, cd1524, cd1536, cd1622, cd1623, cd3232, cd3664 are down-regulated while genes cd0213, cd1511, cd1613, cd2845, cd3567 are up-regulated upon initiation of germination. It is noteworthy that genes cd0115, cd0116, cd0117, cd0176, cd1536, cd1623 (all encoding oxidoreductases) and cd0825, cd1524, cd2845 (all encoding ruberythrins) may play a role in energy-dependent germination [68] of Clostridium spores. We identified all these gene products and thus it could be worthwhile to study their role in spore germination. 6. Other putative spore coat and/or exosporium proteins.

Another newly identified protein in our study could be an important target for the B. cereus group of spore formers. It is an orthologue of BAS5303 in B. anthracis Sterne [69] and BA5699 in B. anthracis Ames, identified previously in the exosporium fraction in the Ames strain [52] and also shown to be immunogenic in animal models [69, 70]. BC_0996 could therefore also be located in the exosporium of B. cereus ATCC 14579,

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although its function remains unclear. Protein BC_0987 is much less conserved in the B. cereus group and could very well be a candidate marker for the B. cereus spores. There were many peptidases and hydrolases identified (Supplementary Table 1 & 2) which we could not assign to any particular category and were classified as putative coat and or exosporium proteins. The plasmid encoded BC_p0002 was found to be unique to the B. cereus ATCC 14579 strain. Notably, this protein might have a distant relation (20% sequence identity, E-value 1.8) with the envelope glycoprotein GP120 (residues 193 - 372) from Human Immunodeficiency Virus (HIV). The highly abundant [27] and cysteine-rich (~9%) protein CD1067, recently shown to be important for exosporium assembly [26], was identified and could very well be a potential marker for this species. Another abundant protein CD1581 was identified as well and is uniquely present in C. difficile 630. Identified protein CD3613 belongs to the Stay-green (SGR) protein family. The SGR family is conserved in plants and is involved in chlorophyll degradation. Distant SGR homologs are also present in algae and, unexpectedly, in species of Bacillus and Clostridium, but not in other bacterial genomes [71]. However, the exact role of these proteins in bacterial species remains to be studied. Protein CD3652 from C. difficile 630 has a characteristic GluZincin protease domain. The GluZincin family (thermolysin-like peptidases or TLPs) includes several zinc-dependent metallopeptidases that contain His-Glu-X-X-His and Glu-X-X-X-Asp motifs as part of their active site. Interestingly, the light-chain (LC, 448 a.a from the N-terminus of the protein) of the potent neurotoxin BoNT/A from C. botulinum also has this domain [72]. Previous studies have confirmed that zinc-binding plays an essential role in the catalytic activity of the light chain of BoNT/A. Thus protease CD3652 is an interesting target to study for its role in spore-mediated infections. As mentioned above, for orthologue evaluation using PSI-BLAST and to study the conservation of coat and exosporium proteins we used the identified proteins from B. cereus ATCC 14579 (this study), C. difficile 630 (this study) and B. subtilis 168[15, 22] as the initial dataset. Figure 2 shows the distribution of orthologues respective to the three species mentioned. As seen, identified B. subtilis coat proteins are very unique to the organism with 25 being only present in B. subtilis. Although B. cereus and B. subtilis are evolutionary distant, 53 B. subtilis coat proteins had orthologues present in B. cereus ATCC 14579 with > 30% sequence identity. From the identified B. subtilis 168 spore coat protein dataset, 9 proteins - CotJB, CotJC, DacF, SpoIVA, SleB, YabG, YhxC, and YtfJ were found to be conserved in selected Bacilli and Clostridia (Figure 2, Supplementary Table 4). Focusing on the identified proteins from B. cereus ATCC 14579, it was seen that 77 proteins are conserved in the B. cereus group while 44 had orthologues in B. subtilis 168 and 11 in C. difficile 630. Eleven of the identified proteins emerged to be conserved in all of the spore forming species considered for comparative analysis. Amongst the known exosporium proteins, 27 proteins have conserved orthologues (Supplementary Table 5) in the B. cereus group including the Bcl-family of proteins. Alanine racemase is conserved throughout the spore formers while in addition to BclA, only SodF has an orthologue present in C. difficile and few strains of C. botulinum. From the C. difficile 630 identified proteins, 13 are unique to this strain, 12 have orthologues in B. subtilis 168, 13 have orthologues in B. cereus and 8 are conserved


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across the spore formers considered for comparison. Our orthologue predictions for certain B. subtilis and C. difficile proteins were also supported by previous transcriptomic [73] and the phylogenetic studies [74] making our orthologue identification method confident. Figure 3 summarizes the orthologue conservation results highlighting potential protein targets for easy detection and removal of spores. There are 13 coat proteins conserved in all spore formers considered in this study. From the exosporium proteins, 11 are conserved in the B. cereus group and only Alr has orthologues in all Clostridia. The exact localization of three proteins BC_0987, BC_0996 both conserved in the B. cereus group and CD3613 conserved in Clostridia is not known. Lastly, despite severe and harsh treatments to the sample pellets, we identified a number of cytosolic proteins including highly abundant ribosomal proteins and elongation factors (Supplementary Table 8). We think that since in both species the exosporium was not completely removed, these proteins might have been trapped in the interspace region between the coat and exosporium layers during sporulation. The fact that Liu et al.[52] also identified orthologues of many of these proteins from the exosporium fraction of B. anthracis indicates that the process of exosporium attachment to the coat occurs in a highly regulated and precise manner inside the mother cell cytoplasm. It is suggested that efficient removal of the exosporium is a must to diminish the contribution of cytosolic proteins. Recently an effort has been made to remove exosporium from C. difficile 630 spores [75] but in Bacillus spp. efficient removal of exosporium has not been achieved [76]. For the same reason, it is also difficult to classify the identified proteins as solely coat or exosporium proteins. Concluding Remarks

In conclusion, our gel-free method performs comprehensively by identifying coat and exosporium proteins from different aerobic and anaerobic spore formers. We provide potential protein targets emerging from this study, for use in food and health sectors for selective removal of spores from the samples, and these targets need to be studied in detail. Also our study outlines the general classes of proteins that are necessary for the spore integrity namely - the structural proteins (eg. SpoIVA, SafA, CotE), proteins involved in resistance mechanisms (eg. oxidoreductases, catalases, ferritin like proteins) and proteins that facilitate germination (eg. hydrolases, peptidases). Additionally, we provide a wide view of protein conservation in the structure of spores from different aerobic and anaerobic pathogenic spore formers in an attempt to derive a universal set of spore surface proteins. We propose that proteins CotJB, CotJC, DacF, Gpr, Eno, SleB, SpoIVA, RnjA, YabG, YabP, YhxC, YloB, YtfJ make up the universally conserved set of proteins in bacterial spore formers. Nevertheless, functional annotation of the genomes, localization studies for the identified putative spore coat and exosporium proteins as well as quantitative studies are necessary for gaining more detailed insights of the spore structure.

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Figure 2. Orthologue distribution for identified spore coat & exosporium proteins from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630. Query protein sequences used for orthologue identification comprised of proteins identified from B. subtilis 168 (92 proteins) [15],[22], B. cereus ATCC 14579 (100 proteins; this study) and C. difficile 630 (54 proteins; this study). The bars indicate the number of proteins from the respective query datasets that have orthologues present in the species or groups indicated below. *Number of proteins exclusive for indicated organisms studied in our work (See Supplementary Tables 2, 3 & 4 for details).


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Figure 3. Potential target proteins from spore coat and/or exosporium layers emerging from this study. Spore coat proteins presented are conserved in the spore formers considered for the comparison. a Exosporium proteins conserved in the B. cereus group (Alr is conserved in Bacilli as well as in Clostridia). b Localization of the proteins is unknown. BC_0987 & BC_0996 are conserved in the B. cereus group and CD3613 is conserved in Clostridia. Materials and Methods

Media and bacterial strains used

The strains used in this study were B. cereus ATCC 14579 and C. difficile 630 (tcdA+ tcdB+). For B. cereus ATCC 14579, growth and sporulation were carried out in Tryptic Soy Broth (TSB) medium as well as a chemically defined growth and sporulation (CDGS) medium, as described previously[77]. The CDGS medium consisted of the following components (final concentrations): 10 mM D-glucose, 20 mM L-glutamic acid, 6 mM leucine, 2.6 mM L-valine, 1.4 mM L-threonine, 0.47 mM L-methionine, 0.32 mM L-histidine, 5 mM D/L-lactic acid, 1 mM acetic acid, 50μM FeCl3, 2.5 μM CuCl2, 12.5 mM ZnCl2, 66 μM MnSO4, 1 mM MgCl2, 5 mM (NH4)2SO4, 2.5 μM Na2MoO4, 2.5 μM CoCl2, 1 mM Ca(NO3)2, buffered to pH 7.2 using 100 mM potassium phosphate buffer. C. difficile 630 was routinely grown in vegetative state on Brain-Heart Infusion Supplemented (BHIS) agar plates and in Tryptone Glucose Yeast extract (TGY)-vegetative medium (3% TSB, 2% glucose, 1% yeast extract, 0.1% L-cysteine). Pre-culturing was done in SMC broth (90 g peptone, 5 g proteose peptone, 1 g (NH4)2SO4, 1.5 g Tris) containing 0.1% L-cysteine, while sporulation was initiated on SMC agar plates [24]. Conditions for growth and sporulation

For B. cereus ATCC 14579 and C. difficile 630 respectively four and three independent biological spore crops were made. A B. cereus culture was grown overnight in 50 mL TSB medium (pH 7.5) at 30⁰C in a shaker-incubator at 200 rpm. Cells were harvested by centrifugation at 10000 rpm for 30 min at 4⁰C, resuspended in 500 mL CDGS medium, and incubated at 30⁰C for 96 h at 200 rpm. After 4 days, the spores were harvested by centrifugation. To lyse the remaining vegetative cells, the spore pellet was treated twice with 0.1% (final concentration) Tween-20 and subsequently washed five times with sterile Milli-Q water. From each B. cereus spore crop, three sample fractions were prepared. The first fraction contained spores with intact exosporium and coat (Fraction 1), the second fraction contained spores with exosporium removed (spore coat sample; Fraction 2) and the third fraction contained the concentrated exosporium fragments (Fraction 3). For C. difficile, a colony from a BHIS agar plate was inoculated into 10 ml TGY medium and incubated at 37°C overnight. This TGY culture was then subcultured into SMC broth and incubated until the cells achieved the logarithmic phase, and then plated onto SMC agar plates. The plates were further

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incubated for 7 days at 37°C. After 7 days, the spores were harvested by washing twice in water and followed by suspension in phosphate-buffered saline (PBS) containing 125 mM Tris, 200 mM EDTA, 0.3 mg/ml proteinase K (Fermentas), and 1% sarcosyl with further incubation and gentle shaking at 37°C for 2 h. Finally, spores were centrifuged; the pellets were resuspended in water, and washed a further 10 times [24]. Only the coat fraction was analyzed from C. difficile in this study. Removal of exosporium

To separate the exosporium layer from B. cereus spores, spores (1010 spores / mL) were resuspended in 50 mM Tris-HCl, 0.5 mM Na-EDTA (pH 7.5) and passaged 4 times through a French pressure cell (Thermo Fisher Scientific, MA, USA) at 20000 psi. Spores were separated from exosporium fragments by centrifugation at 10000 rpm for 15 min at 4⁰C. The supernatant (containing loose exosporium fragments) was centrifuged one more time, at 10000 rpm for 15 min at 4oC and pressed through a 0.22 μm PES filter (VWR International, PA, USA) to remove any remaining spores and stored at 4oC. The pelleted spore fraction was resuspended in sterile Milli-Q water and stored at 4oC until further use. The protocol was adapted from the work of Todd and co-workers [78]. Isolation of spore coat

The spore fraction of B. cereus ATCC 14579 (obtained after French press treatment) and C. difficile 630 was centrifuged and the spore pellets were resuspended in 10 mM Tris-HCl (pH 7.5). The spores were disintegrated by bead-beating with 0.1 mm Zirconia-Silica beads (BioSpec Products, USA) using a Precellys 24 homogenizer (Bertin Technologies, Aix en Provence, France). Nine rounds of bead-beating were performed, with each round consisting of a 40 s cycle at 6000 rpm and a 1 min interval between each round for cooling. After every three rounds of beating, 10 min cooling periods (on ice) were allowed to prevent protein degradation by overheating. The disintegrated spore pellets were then washed 5-6 times with 1 M NaCl to remove non-covalently bound proteins and intracellular contaminants. Spore coat protein extraction was done by thermal heating for 10 min using a water bath, starting at ambient temperature and reaching a final temperature of 80⁰C in SDS extraction buffer consisting of (in final concentrations): 50 mM Tris-HCl (pH 7.8), 100 mM Na-EDTA, 150 mM NaCl, 0.2% SDS and 100 mM β-mercaptoethanol. After protein extraction, the remaining SDS insoluble spore coat protein pellets were washed four times with sterile Milli-Q water to remove SDS. The pellets were then frozen in liquid nitrogen and freeze-dried overnight and stored at -80⁰C. Concentration and analysis of exosporium

For B. cereus spores, the suspension (obtained after French press) containing the exosporium fragments, was concentrated by ultracentrifugation at 40000 rpm for 1 h at 4⁰C. The pellet containing exosporium fragments was then washed in 1 M NaCl and subsequently twice in TEP buffer, consisting of 50 mM Tris-HCl (pH 7.5), 10 mM Na-EDTA, 0.2% SDS and 2 mM β-mercaptoethanol. After washing in TEP buffer, the pellet was washed once in TEP buffer without SDS. Exosporium protein extraction was done as mentioned above for spore coat protein extraction. After protein extraction, the remaining SDS insoluble exosporium pellet was washed four times with sterile Milli-Q water to remove residual SDS. The obtained pellet was then frozen in liquid nitrogen, freeze-dried overnight and stored at -80 ⁰C. Though C. difficile spores have exosporium, its presence in our sample was variable. This is likely because of its loss during spore harvest. Therefore we did not proceed to isolate exosporium from C. difficile spores. The method was adapted from the work of Redmond et al [54]. Analysis of spore cortex peptidoglycan

The muramic acid from the spore cortex peptidoglycan is an efficient marker to estimate the purity (i.e. the absence of cortex peptidoglycan) of the spore coat sample. Therefore muramic acid assays were performed where the muramic acid is estimated based on the absorbance measurements as described


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previously[79, 80]. To eliminate any possible interference in the assay from the exosporial sugars (indicating inefficient exosporium removal as reported previously by Thompson & co-workers[76]) deglycosylation of the sample pellets was done by β-elimination according to the manufacturer’s instructions (GlycoProfile™ IV Chemical Deglycosylation Kit, Sigma-Aldrich Co. LLC). Lysozyme is known to act on the 1,4-β-linkage between N-acetylmuramic acid and N-acetyl-D-glucosamine from the vegetative bacterial cell wall thus we also subjected the pellets to lysozyme (Sigma-Aldrich Chemie B.V.; final concentration 250 μg/ml) treatment[81] to remove the remaining muramic acid residues, if any. Intact spores, disintegrated spores before and after NaCl wash, and the final reduced and alkylated spore extracts were used for the assay. Preparation for MS analysis

Preparation of samples from spore coat & exosporium fraction, spore coat fraction and exosporium fraction was done separately in the following manner. For reduction of disulfide bridges, the freeze-dried samples were incubated with 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 for 1 h at 55⁰C. The reducing reaction was followed by an alkylation treatment with 55 mM iodacetamide (IAA) in 100 mM NH4HCO3 for 45 min. at room temperature in the dark. The samples were immediately digested for 18 h at 37⁰C with trypsin (Trypsin Sequencing Grade Promega, Madison, WI, USA) using 1:60 w/w protease: protein ratio. The tryptic digests were desalted using Omix μC18 pipette tips (80 μg capacity, Varian, Palo Alto, CA, USA) according to the manufacturer’s instructions and the peptides were collected in 25μL 50% acetonitrile (ACN), 0.1 % trifluoroacetic acid (TFA) and stored at -80⁰C. Before analysis a fraction of eluted peptide material was freeze-dried and concentrated in 10 μL of 0.1% TFA and peptide concentration was measured at 205 nm [82] with a Nanodrop ND1000 spectrophotometer (Isogen Life Sciences, De Meern, The Netherlands).

LC-FT-ICR MS/MS analysis

LC-MS/MS data were acquired with an Bruker ApexUltra Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 7 T magnet and a nano-electrospray Apollo II DualSource™ coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) HPLC system. Samples containing up to 60 ng of the tryptic peptide mixtures were injected as a 10 μl 0.1% TFA, 3% ACN aqueous solution and loaded onto a PepMap100 C18 (5-μm particle size, 100-Å pore size, 300-μm inner diameter x 5 mm length) precolumn. Following injection, the peptides were eluted via an Acclaim PepMap 100 C18 (3-μm particle size, 100-Å pore size, 75-μm inner diameter x 250 mm length) analytical column (Thermo Scientific, Etten-Leur, The Netherlands) to the nano-electrospray source. Gradient profiles of up to 120 minutes were used from 0.1% formic acid / 3% CH3CN / 97% H2O to 0.1% formic acid / 50% CH3CN / 50% H2O at a flow rate of 300 nL/min. Data dependent Q-selected peptide ions were fragmented in the hexapole collision cell at an Argon pressure of 6x10-6 mbar (measured at the ion gauge) and the fragment ions were detected in the ICR cell at a resolution of up to 60000. In the MS/MS duty cycle, 3 different precursor peptide ions were selected from each survey MS. The MS/MS duty cycle time for 1 survey MS and 3 MS/MS acquisitions was about 2 s. Instrument mass calibration was better than 1 ppm over a m/z range of 250 to 1500. Raw FT-MS/MS data were processed with the MASCOT DISTILLER program, version 2.4.3.1 (64bits), MDRO 2.4.3.0 (MATRIX science, London, UK), including the Search toolbox and the Quantification toolbox. Peak-picking for both MS and MS/MS spectra were optimized for the mass resolution of up to 60000. Peaks were fitted to a simulated isotope distribution with a correlation threshold of 0.7, with minimum signal to noise of 2. The processed data, combined from the three independent biological replicates, were searched in a MudPIT approach with the MASCOT server program 2.3.02 (MATRIX science, London, UK) against a complete B. cereus ATCC 14579 and C. difficile 630 ORF translation database (Uniprot 2011 update, downloaded from http://www.uniprot.org/uniprot), respectively. The databases were complemented with their corresponding decoy data bases for statistical analyses of peptide false discovery rate (FDR). Trypsin was used as enzyme and 2 missed cleavages were allowed. Carbamidomethylation of cysteine was used as a fixed modification and oxidation of methionine as a

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variable modification. The peptide mass tolerance was set to 30 ppm and the peptide fragment mass tolerance was set to 0.03 Dalton. If needed the search was repeated with the same parameters but with semitrypsin as the enzyme. Error-tolerant MASCOT search was also done to identify possible modification in the peptides. In addition, MS/MS data were matched, allowing semi-tryptic peptides. MASCOT MudPIT peptide identification score was set to a cut-off of 20. At this cut-off and based on the number of assigned decoy peptide sequences, a peptide false discovery rate (FDR) of ~2% for all analyses was obtained. The identified coat and/or exosporium proteins of Bacillus cereus ATCC 14579 and Clostridium difficile 630 are listed in Table 1 and 2 respectively. The identified cytosolic proteins are listed in Supplementary Table 6.

Tertiary structure prediction and Sequence alignment

For tertiary structure prediction Phyre2 tool (www.sbg.bio.ic.ac.uk/phyre2/) was used in the default mode. Sequence alignment was done using the Clustal Omega tool (http://www.ebi.ac.uk/Tools/msa/clustalo/) with the default parameters. Conserved Domain Search

The annotation of protein sequences for the identification of domains is important in the protein sequence analysis. The identification of a conserved domain may aid in understanding the cellular and molecular function of a protein, as well as the evolutionary history of proteins. Identification of conserved domains from the identified proteins was performed using the automatic mode of the Conserved Domain (CD)-search tool from NCBI database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The default parameters were used. Orthologue identification and PSI-BLAST analysis

For orthologue identification, PSI-BLAST (Position-Specific Iterated Basic Local Alignment Search Tool) option from BLASTP tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) version 2.2.27+ was used. The thresholds for orthologue identification: maximum identity ≥30%, E-value ≤0.005. Only single iteration for PSI-BLAST was performed. Proteins were said to be conserved if orthologues were found in at least 80% of the strains selected for comparison.

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