In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

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Article

In Pursuit of Protein Targets: Proteomiccharacterization of Bacterial Spore Outer Layers.

Wishwas Abhyankar, Abeer H. Hossain, André Djajasaputra, Patima Permpoonpattana, AlexanderS. Ter Beek, Henk L. Dekker, Simon Cutting, Stanley Brul, Leo J de Koning, and Chris G de Koster

J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4005629 • Publication Date (Web): 02 Sep 2013

Downloaded from http://pubs.acs.org on September 9, 2013

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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*,†

†Mass Spectrometry of Bio-macromolecules and ×Molecular Biology & Microbial Food safety

Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands;

‡School of Biological Sciences, Royal Holloway, University of London, UK.

¤ Current address : Faculty of Science and Industrial Technology, Prince of Songkla University, Surat

Thani Campus, Surat Thani 84000, Thailand.

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

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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.

Keywords: B. cereus ATCC 14579, C. difficile 630, spore coat, exosporium, insoluble protein

fraction

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 cereus1. B. cereus is mainly known as a food-

borne pathogen causing two types of gastrointestinal diseases: emesis and diarrhea2. 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 infections4. Spore forming Clostridium

spp. (e.g. Clostridium botulinum, Clostridium tetani), like Bacilli, are also known for the

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infections and diseases caused by them. Pathogenic C. difficile can colonize the intestinal tracts

of humans and other mammals5, 6. Routine treatments containing use of antibiotics such as

Metronidazole, Vancomycine have led to emergence of resistant strains7, 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 people9, 10. C. difficile is reported to be a continuously evolving species11,

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 growth14. In B. subtilis

the outermost thick concentric proteinaceous layers - the inner and outer spore coat15 - 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 surfaces16.

Though the cortex layer from spores might have evolved from the cell wall 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 germination

receptor assembly is a relatively later event in the evolution of the spore structure as the ancestral

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Bacilli and Clostridia moved to different niches after their divergence about 2.3 billion years

ago17. 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 spore structure18. 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” method22 allowed us for the first

time to focus on the insoluble protein fraction from the spore coat, which makes up to 30% of the

proteins15, 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

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proteins we also assess different spore formers for the conservation of the spore surface proteins

that were identified from B. subtilis22, B. cereus and C. difficile in our studies.

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 previously24. 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 stage 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 plates25.

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

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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 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 times25. 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

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resuspended in sterile Milli-Q water and stored at 4oC until further use. The protocol was adapted

from the work of Todd and co-workers26.

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

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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 al27.

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 previously28, 29. To eliminate any

possible interference in the assay from the exosporial sugars (indicating inefficient exosporium

removal as reported previously by Thompson & co-workers30) 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) treatment31 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

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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 nm32 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

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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 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

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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.

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

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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 previously25.

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

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 S1). 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 16822, B. cereus ATCC 14579 and C. difficile 630 (Figure 1).

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

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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 been considered the best33. 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

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(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, CdeC34 (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 previously35 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).

We divided the identified proteins from both species into six different categories based

on their possible function 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

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coat proteins possibly involved in spore germination and (6) other putative spore coat and/or

exosporium proteins.

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 development15, 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 identified36 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 CotE25, 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 they are mostly identified in the soluble fraction of spores from B.

subtilis37, 38. 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 role39-42. Genes for

CotC, CotG, CotS, CotT and CotW are not present in the B. cereus genome15. 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. anthracis43.

Identical to B. anthracis ExsB43, we identified Threonine residues at positions 76, 79, 82, 105,

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114, 117, 129 from BC_2030 as phosphorylated in our error-tolerant MASCOT search.

Identified protein CD2399 is a paralogue of CD0596 (renamed as CotF44) 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 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. anthracis45.

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 spore46. Although the expression of YhcN in

B. subtilis is controlled by the transcription factor σG (expressed late in the forespore during

sporulation47) 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

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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 peptidoglycan48. 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. subtilis49. 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 assemblies50. 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 spores44 was identified. The newly identified protein BC_4047 is an

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orthologue of the outer coat protein Cotα (BAS3957) in B. anthracis Sterne and contains a

DUF3915 domain of unknown function. There are three Cys residues at the amino terminus (first

20 residues) and similar to the case in B. anthracis spores51, 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

hypochlorite51. 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 (CD167)),

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 stress52, 53 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 attachment to 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 exosporium54, 55. We could not identify this protein (accession no.

HM071986.116) 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

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regions that possess the (Gly-X-Y)n motifs, which is characteristic for a triple helix, rod-like

structure formation56. A recent study57 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, BclB19, 58, 59 have also been identified

in B. anthracis. Protein BxpA (BC_2149) has an amino acid sequence unusually rich in

glutamine and proline60. 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 subunits61. BxpB

is an exosporium basal layer protein in B. anthracis Sterne58 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. 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 spores58. 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

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exosporium protein ExsK identified from the exosporium fraction in B. anthracis (BA_2554) in

a study of Redmond et al.27

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 group62. These genes are

expressed in the last developmental time waves during sporulation63. 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

spores62. 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 Clostridia64 and may

facilitate spore dissemination, assist in spore nutrition during formation, or have no function and

result from a deranged metabolism65.

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 domain55, 66. Though not

required for pathogenesis67 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

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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 manner68. 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.

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 germination69 and the inosine-uridine preferring nucleoside

hydrolase (IunH; BC_2889) by converting the germinant inosine to D-ribose could play a role in

preventing germination70. 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.

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subtilis spore coat71. However, in other studies GerQ/YwdL and CwlJ were identified in the

exosporium fraction of B. cereus ATCC 10876 and B. anthracis spores, respectively60, 72.

Another enzyme identified from the insoluble fraction in this study and reported to be involved

in spore cortex degradation73 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 colleagues74 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 germination75 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 important target for the B. cereus group of spore formers. It is an orthologue

of BAS5303 in B. anthracis Sterne76 and BA5699 in B. anthracis Ames, identified previously in

the exosporium fraction in the Ames strain60 and also shown to be immunogenic in animal

models76, 77. BC_0996 could therefore also be located in the exosporium of B. cereus ATCC

14579, although its function remains unclear. Protein BC_0987 is much less conserved in the B.

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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 abundant35 and cysteine-rich (~9%) protein CD1067, recently shown to be

important for exosporium assembly34, 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 genomes78. 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 and 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 domain79. 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

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ATCC 14579 (this study), C. difficile 630 (this study) and B. subtilis 16815, 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 (Fig. 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 across the spore formers considered for

comparison. Our orthologue predictions for certain B. subtilis and C. difficile proteins were also

supported by previous transcriptomic80 and the phylogenetic studies81 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

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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.60 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 spores82 but in Bacillus spp. efficient removal of exosporium

has not been achieved30. 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.

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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.

Associated content

Supplementary Table 1. Conserved superfamily domains from the identified spore coat and

exosporium proteins from Bacillus cereus ATCC 14579 and Clostridium difficile 630.

Supplementary Table 2. Orthologue identification for the identified spore coat and exosporium

proteins from Bacillus cereus ATCC 14579.

Supplementary Table 3. Orthologue identification for the identified spore coat and exosporium

proteins from Clostridium difficile 630.

Supplementary Table 4. Orthologue identification for the previously assigned spore coat

proteins in Bacillus subtilis 168.

Supplementary Table 5. Orthologue identification for the previously assigned exosporium

proteins.

Supplementary Table 6. Cytosolic proteins (possibly from the interspace) identified in this

study.

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Author information

Corresponding author

*E-mail: [email protected]

Notes

The authors declare no competing financial interest.

Acknowledgements

Rokus van den Dool and Jolanda Verheul are thanked for the experimental help in this study.

W.A. acknowledges the Erasmus Mundus program (EMECW 15) for funding of his PhD project.

A.T.B. is supported by a grant from the Dutch Foundation for Applied Sciences (STW 10431).

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Figures

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.

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).

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.

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

aNo. 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

1 BC_4420 (safA) d Q812R2 SpoVID-dependent spore coat assembly factor SafA

65354 1286 46 2357 70

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

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

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

5 BC_3770 (cotE) d Q81A24 Spore coat protein E 20615 971 37 122 5

6 BC_0390 (cotB) Q81IJ6 Spore coat protein B 17339 744 29 1563 39 328 11

7 BC_4419 (yhcN) d Q817V9 Putative uncharacterized protein 24526 672 21 628 21

8 BC_1222 Q81GH8 Spore coat protein Y 17596 486 19 729 21 5668 188

9 BC_5056 d Q815S6 Collagen adhesion protein 34657 383 19 1427 37

10 BC_2874 (cotX) Q81CA1 Spore coat protein X 21850 306 12 293 12 36 1

11 BC_1509 (spoIVA) Q81FR0 Stage IV sporulation protein A 55636 242 11

12 BC_4640 (ytfJ) d Q817B7 Putative uncharacterized protein 13968 237 10 81 3

13 BC_1560 (cotD) d Q81FM0 Spore coat protein D 15393 214 10 565 16

14 BC_0212 (yusW) d Q81IY1 Putative uncharacterized protein 17725 122 3 204 6

15 BC_4075 (dacF) d Q819B1 D-alanyl-D-alanine carboxypeptidase

44032 85 3 150 5

16 BC_1279 (cotN) Q81GC8 Spore coat-associated protein N 21848 79 3

17 BC_1245 Q81GF8 Putative uncharacterized protein 15287 39 2

18 BC_0822 (cotJB) Q81HI6 CotJB protein 10523 45 1

19 BC_0821 (cotJC) Q81HI7 CotJC protein 21694 37 1

20 BC_0823 (cotJA) Q81HI5 CotJA protein 8475 30 1

21 BC_3534 (yxeE) d Q81AM8 IG hypothetical 17193 16298 21 1 33 1

22 BC_2095 (ytfJ) d Q81E93 Putative uncharacterized protein 15087 120 5 32 1

23 BC_0063 (yabP) Q81J89 Putative uncharacterized protein 11627

45 1

24 BC_2677 Q81CR9 L-alanyl-D-glutamate peptidase 31439

41 1

25 BC_0047 (yabG) Q81JA4 Sporulation-specific protease YabG

33219

23 1

26 BC_1559(yppG)f Q81FM1 Spore coat protein 22228

23 1

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

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

28 BC_4639 d Q817B8 Thiol peroxidase 18055 158 8 48 2

29 BC_1391 (yqfX) d Q81G20 Putative uncharacterized protein 13457 187 5 69 1

30 BC_2099 (yqfX) d Q81E89 Putative uncharacterized protein 12239 26 1 67 1

31 BC_4774 (yisY) d Q816P9 Non-heme chloroperoxidase 30166 54 1 62 1

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

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

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

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pept Score no.

pept Score no.

pept

34 BC_3712 (bclC) Q812Y5 Hypothetical Membrane Spanning Protein

75838 673 37 483 21 820 30

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

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

37 BC_2374 (exsFB) Q813L4 Hypothetical Membrane Spanning Protein

17496 141 6 88 3 164 5

38 BC_2382 (bclB)f Q81DI4 Putative uncharacterized protein 39119

50 1

39 BC_2639 d Q81CV2 Cell surface protein 521640 80 1 29 1

40 BC_3345 Q81B46 Collagen-like triple helix repeat protein

77508

116 2

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

42 BC_2149 (bxpA) d Q81E43 Putative uncharacterized protein 32484 47 1 412 8

(IV) Exosporium proteins possibly involved in pathogenicity

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

137 5

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

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

46 BC_1281 (calY) e Q81GC6 Cell envelope-bound metalloprotease (Camelysin)

21801 409 18

21 1

(V) Spore coat proteins involved in spore germination

47 BC_3607 (yaaH) d Q81AG3 Spore peptidoglycan hydrolase (N-acetylglucosaminidase)

48167 1185 54 1235 41

48 BC_5391 (gerQ) d Q814N4 Putative uncharacterized protein 16090 587 21 227 8

49 BC_0264 (alr1) Q81IT5 Alanine racemase 1 43789 382 22 678 18 1393 47

50 BC_2889 (iunH) Q81C90 Inosine-uridine preferring nucleoside hydrolase

36552 306 11 580 20 1797 61

51 BC_2207 Q81DY9 Sporulation-specific N-acetylmuramoyl-L-alanine amidase

35924 129 8

52 BC_5390 (cwlJ) d Q814N5 Cell wall hydrolase cwlJ 16467 93 5 45 3

53 BC_4319 (gpr) Q818E2 Germination protease 40450 90 3

54 BC_1591 e Q81FJ1 Putative uncharacterized protein 58965 87 2

5360 170

55 BC_3552 (iunH) Q81AL1 Inosine-uridine preferring nucleoside hydrolase

34507

27 1

56 BC_2752 (ypeB) Q813I5 Spore germination protein 50109 81 2

57 BC_2753 (sleB) d P0A3V0 Spore cortex-lytic enzyme 28297 66 2 32 1

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

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

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

60 BC_5135 (eno) d Q815K8 Enolase 46400 516 16 95 4

61 BC_p0002 d Q814F0 Putative uncharacterized protein 17989 333 11 97 4

62 BC_2858 Q81CB5 Putative uncharacterized protein 9626 266 12 232 7 52 1

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

64 BC_2026 Q81EF3 Oligopeptide-binding protein oppA 63075 237 11

65 BC_1613 d Q81FH4 Zn-dependent hydrolase 62004 193 8 187 5

66 BC_4410 (yajC) d Q817W7 Protein translocase subunit YajC 9486 132 6 72 2

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pept Score no.

pept Score no.

pept

67 BC_0337 d Q814A8 Hypothetical Membrane Spanning Protein

14189 126 8 151 9

68 BC_3195 Q813E6 Hypothetical Cytosolic Protein 15025 113 7 74 3 109 5

69 BC_3787 Q81A08 Zinc protease 48972 112 3

70 BC_3786 Q81A09 Zinc protease 49288 112 2

71 BC_3586 Q81AI2 Oligopeptide-binding protein oppA 63759 104 3

72 BC_3133 Q81BM0 Putative hydrolase 42323 98 3

73 BC_1424 Q81FY8 Ferredoxin--nitrite reductase 60770 97 3

74 BC_3986 d Q812V3 Hypothetical Cytosolic Protein 9170 95 3 61 2

75 BC_4480 (tig) d Q812Q9 Trigger factor 47316 88 4 35 1

76 BC_0344 (rocA) d Q81IP0 1-pyrroline-5-carboxylate dehydrogenase

56418 84 4 23 1

77 BC_2375 Q81DJ0 Putative uncharacterized protein 9489 70 2

78 BC_0825 Q81HI3 Putative uncharacterized protein 30284 65 1

79 BC_2745 Q81CL1 Putative uncharacterized protein 43460 62 3

80 BC_1029 d Q81GZ8 IG hypothetical 18063 33466 57 4 25 1

81 BC_3090 d Q81BR0 Putative uncharacterized protein 18565 44 4 48 2

82 BC_5181 d Q815H3 UPF0145 protein BC_5181 11143 42 1 51 1

83 BC_3992 Q819I6 Putative uncharacterized protein 12700 30 1 44 2 22 1

84 BC_2969 Q81C15 Putative uncharacterized protein 12769 30 1

85 BC_2481 Q81D93 Putative uncharacterized protein 12069 28 1

86 BC_1456 Q81FV9 Putative uncharacterized protein 16272 27 2

87 BC_1708 Q81F89 Putative uncharacterized protein 25183 27 1

88 BC_3977 Q819J9 Zn-dependent hydrolase 62055 27 1

89 BC_3515 d Q813A3 Hypothetical Glycosyltransferase 82417 24 1 38 1

90 BC_0395 Q81IJ1 Metal-dependent hydrolase 23560 23 1

91 BC_3784 Q81A11 IG hypothetical 16623 9209

195 5

92 BC_4387 Q817Y7 Putative uncharacterized protein 4832

58 1

93 BC_3582 (yodI) Q81AI6 Putative uncharacterized protein 14601

213 4 25 1

94 BC_1334 Q813U4 Hypothetical Exported Protein 28367

106 1

95 BC_2878 Q81C97 Putative uncharacterized protein 29564

42 2 66 4

96 BC_0263 Q81IT6 Putative uncharacterized protein 38513

42 1

97 BC_1468 (sodF)f Q81FV0 Superoxide dismutase 26798

29 1

98 BC_0944 f Q81H77 Putative uncharacterized protein 61227

28 1

99 BC_2237f Q81DW2 Putative uncharacterized protein 74111

26 1

100 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.

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Table 2. Identified spore proteins from Clostridium difficile 630.



Spore coat

bFDR= 1.72%

cScore cno. pept

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

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

2 CD1581 e Q186D6 Putative uncharacterized protein 20043 2572 103

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

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

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

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

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

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

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

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

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

12 CD2399 e Q181Y3 Putative uncharacterized protein 8247 169 5

13 CD2598 Q182T7 Putative oligosaccharide deacetylase 29780 70 2

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

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

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

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

17 CD2845 (rbr) e Q183T4 Rubrerythrin 22603 373 12

18 CD2864 (yisY) e Q183V0 Putative hydrolase 30520 243 13

19 CD1524 Q18C45 Putative rubrerythrin 20319 126 9

20 CD1623 Q186H7 Putative oxidoreductase 94987 110 5

21 CD0825 (rbr) e Q18A24 Rubrerythrin 20829 64 3

22 CD0116 Q18CK8 Putative ferredoxin/flavodoxin oxidoreductase,alpha subunit

39290 106 4

23 CD0117 Q18CK7 Putative ferredoxin/flavodoxin oxidoreductase,beta subunit

27031 61 2

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

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

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

26 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

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

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

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

29 CD3032 Q184M0 Putative pyridoxal phosphate-dependent transferase 47977 332 18

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38



Spore coat

bFDR= 1.72%

cScore cno. pept

30 CD3620 Q181M3 Putative uncharacterized protein 14256 232 11

31 CD1133 Q18AZ2 Putative uncharacterized protein 23857 217 10

32 CD3580 Q181I4 Putative uncharacterized protein 29071 157 5

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

58649 126 9

34 CD3664 Q181R8 Putative aminotransferase 44997 119 7

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

36 CD3522 Q181C2 Putative uncharacterized protein 50689 91 4

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

38 CD2808 Q183P2 Putative uncharacterized protein 23705 87 1

39 CD1463 Q18BY4 Putative uncharacterized protein 17988 68 5

40 CD3652 Q181Q6 Putative peptidase, M1 family 54828 62 5

41 CD3170 (eno) e Q181T5 Enolase 46289 67 3


43 CD2865 Q183U9 Putative bacterioferritin 20747 54 3

44 CD3613 Q181L4 Putative uncharacterized protein 17425 42 3

45 CD2431 Q182B6 Putative nitrite/sulphite reductase 58684 40 1

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

8140 40 2

47 CD1622 Q186H8 Putative uncharacterized protein 23461 39 1

48 CD3232 Q17ZX7 UPF0597 protein CD630_32320 45787 39 1

49 CD3457 Q180V4 Putative uncharacterized protein 15859 37 2

50 CD1063.1 e Q18AR2 Putative uncharacterized protein 8221 35 2

51 CD0894 Q18A86 Putative iron-dependent hydrogenase 56384 32 1


53 CD0279 Q18D22 Putative uncharacterized protein 14922 24 1

54 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 studies25, 34, 44. eProteins identified previously from the whole spore protein extract35.

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39

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 tick mark (��) indicates experimental identification of the proteins. Proteins CotJC, DacF, SpoIVA and

YisY were identified experimentally from all the three organisms.

Protein Bacillus cereus

ATCC 14579

Clostridium difficile

630

Bacillus subtilis

168

YabG ��

YabP ��

YdbD ��

CotJC ��

CotJB ��

SpoIVA ��

SleB ��

��

RnjA ��

YmfH ��

YmfF ��

DacF ��

Gpr ��

Tig ��

YtfJ ��

��

YtjP

��

YisY ��

Eno ��

CwlJ ��

��

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40

Graphic for abstract

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Graphical Abstract

43x22mm (300 x 300 DPI)

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Fig.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. Total 148 superfamily domains were identified in the proteins from three species indicated. The numbers correspond to the number of

superfamily domains identified. The eleven superfamily domains common to the three organisms are listed in the box.

122x42mm (300 x 300 DPI)

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Fig.2 Orthologue distribution for identified spore coat & exosporium proteins from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630. Initial datasets used for orthologue identification include proteins identified

from B. subtilis 16816, 21, B. cereus ATCC 14579 (this study) and C. difficile 630 (this study). The bars

indicate the number of proteins from the respective initial datasets that have orthologues present in the species or groups indicated below. * Number of proteins exclusive for indicated organisms studied in our

works (See Supplementary Tables 2,3 & 4 for details). 18x20mm (600 x 600 DPI)

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69x37mm (300 x 300 DPI)

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In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

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