Bacillus subtilis Spore Inner Membrane Proteome - Journal of

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

The Bacillus subtilis spore inner membrane proteome Linli Zheng, Wishwas Abhyankar, Natasja Ouwerling, Henk L. Dekker, Henk van Veen, Nicole N van der Wel, Winfried Roseboom, Leo J de Koning, Stanley Brul, and Chris G de Koster J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00976 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

The Bacillus subtilis spore inner membrane proteome Linli Zheng†‡, Wishwas Abhyankar†‡, Natasja Ouwerling†‡, Henk L. Dekker‡, Henk van Veen#, Nicole N van der Wel#, Winfried Roseboom‡, Leo J. de Koning‡, Stanley Brul†*§, Chris G. de Koster†*§ †Molecular Biology & Microbial Food Safety; ‡Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands; # Electron Microscopy Centre Amsterdam, Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, The Netherlands.

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

ABSTRACT

The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation these spore formers can survive very harsh physical and chemical conditions. Yet they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics based membrane protein predictions, ca. one-third could be predicted to be membrane localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were seen. In addition to the previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.

KEYWORDS: Bacillus subtilis, spore inner membrane, mass spectrometry, proteome


Page 3 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Spores of Bacillus and Clostridium species are dormant, metabolically inert structures formed particularly under nutrient starvation conditions. Spores are in their dormant form resistant to a variety of environmental insults including heat, desiccation, pressure, radiation and toxic chemicals.1, 2 Yet, the spores can detect nutrients in the surroundings and respond by triggering the resurgence of metabolism and growth via a process called germination. However, upon germination spores lose their stress resistant phenotype. Spore resistance and dormancy are attributed to the spore components and their structure (Figure 1). Exosporium is the outermost layer in spores of some species (but not B. subtilis), followed by a proteinaceous coat that can be subdivided into crust, outer coat, inner coat and basement.3 The outer membrane (OM) is beneath the coat layer, and is sometimes described as a permeability barrier4 but also often seen as only a vestigial structure5. The OM covers the cortex peptidoglycan layer and then the germ cell wall. The peptidoglycan of the germ cell wall and growing cell wall are identical however the cortex peptidoglycan has muramic acidδ-lactam residues which are unique to spores. Under the germ cell wall is the inner spore membrane (IM) and most of the proteins known to be involved in spore germination are present in this spore compartment.6 Finally, the core of the spore is where the spores’ DNA, ribosomes, and most enzymes are stored. The core possesses a low water content (25 to 50% of wet weight) and the spore-specific pyridine-2, 6-dicarboxylic acid i.e. dipicolinic acid (DPA; ~10% of total spore dry weight) chelated with divalent cations especially to Ca2+.7 The spore IM is considered to play an important role in the resistance properties of spores. This membrane is impermeable to small hydrophilic molecules unless these pass through appropriate membrane localized small molecule transporter proteins. The IM protects the spore core and becomes the plasma membrane of the outgrown cell after germination. The OM and IM differ from each other in their lipid compositions8 and the IM is the major barrier



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

to the passage of potentially harmful chemicals into the spore core2. Lipids in the IM are largely immobile, although the lipid mobility is regained upon germination.9 The protein and lipid components of the IM and the forces acting on this membrane due to the core or cortex could together play some role in determining the membrane properties. The IM of dormant spores exists in a compressed form, as in the absence of lipid synthesis its surface area is said to expand approximately 2-fold after the spore germination is completed.9 In previous studies it was observed that the damage to the IM plays an important role in spore killing by heat and oxidizing agents.10-12 High temperatures can cause disruption of the IM releasing DPA from the spore core.13 Oxidizing agents damage the IM and can cause spore killing without causing DPA release.14 Both the IM lipids and IM proteins can be targets for such spore killing.10 The inner membrane layer is also crucial for the process of spore germination. A number of proteins essential for spore germination, including the nutrient germinant receptors (GRs),15 the SpoVA proteins that facilitate DPA release during germination,16 and the GerD protein essential for GR-dependent germination, are generally considered to be present in or associated with the spore IM. The known and still unknown facts about spore germination have been extensively reviewed recently.6 The GRs, ie GerA, GerB, GerK have been identified as well as quantified. These GRs have also been shown to respond to specific nutrient molecules.17 Along with proteins such as CwlJ, SleB, YpeB, SpoVAD, the protease HtrC has been shown to play a pivotal role in spore germination.18 Yet questions about, for instance, the in vivo interaction between different GRs, regulated efflux of cations, regulated DPA efflux from spores as well as the coordination and the regulation of cortex lytic enzyme activity, remain unanswered.6 These unanswered questions, spur the need of detailed proteomics studies of the inner membrane to (a) obtain an exhaustive analysis of the minimal protein composition of an IM enriched fraction that is compatible with maintaining spore


Page 5 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


viability and (b) identify unknown proteins putatively linking the above mentioned key players of the spore germination process. In the current study we outline a robust protocol for spore IM isolation and coupled GeLCMS/MS based membrane proteomics. The most striking findings concerning the composition of the spore IM proteome are presented. The data obtained are discussed in the framework of open questions about spore germination and thus further the development of strategies for targeted inhibition of bacterial spore germination and subsequent outgrowth.

Materials and methods Culture and sporulation conditions B. subtilis strain 1A700 (trp2C) from a single colony was inoculated in tryptic soy broth and grown aerobically at 37°C and 200 rpm, until early exponential phase (OD600 0.3-0.4), and transferred to a defined minimal medium for sporulation buffered with 3-(Nmorpholino)propanesulfonic acid (MOPS), as described previously.19 Cells were then diluted into a fresh 20 ml of MOPS medium and grown until reaching early exponential phase, when they were transferred to larger volumes (250 ml) of MOPS medium. For vegetative cell preparation, cells were harvested upon reaching the early exponential phase. For sporulation, cells were incubated for 96 hours before the spore harvests. Spore inner membrane isolation Spores were harvested by centrifugation and washing with 0.1% Tween-80 and Milli-Q water.19 Purification of spores was done by centrifugation through a 20%–50% HistoDenz gradient and washing with water. The purity of the spore crops was examined by phase contrast microscopy. The spore IM isolation method that we adapted has been used in several studies aimed to determine the localization of IM proteins.20-22 We adapted this method to our proteomics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

studies with minor modifications. Approximately 100 to 150 mg (dry weight) of purified spores were divided in eight parts and treated in parallel by the subsequent steps. Spores were decoated by treatment with 0.15 M NaCl-0.1 M NaOH-0.5% sodium dodecyl sulfate (SDS) at 70°C for 1 h. After intensive washing with water, the decoated spores were incubated with TEP buffer (50 mM Tris-HCl [pH 7.4], 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) containing 2 mg/ml lysozyme and 40 µg/ml MgCl2 at 37°C for 30 min. The spores were intensively washed with TEP and then disrupted by bead-beating with 0.1 mm Zirconia-Silica beads (BioSpec Products, Bartlesville, OK) using a Precellys 24 homogenizer (Bertin Technologies, Aix en Provence, France) (6 rounds of 20 s at 6000 rpm with ice cooling between each round). The beads were rinsed with TEP buffer several times and the suspension was collected and centrifuged at 15,000 rpm for 5 min at 4°C to remove unbroken cells and integuments. NaCl was added at a final concentration of 1 M to the supernatant, which contained membrane and soluble fraction. The supernatant went through ultracentrifugation at 100,000 g for 1 h at 4°C. The pellet was washed with carbonate buffer (100 mM Na2CO3, 100 mM NaCl, 10 mM EDTA, pH 11) at 4°C on a shaker for 1 h and ultracentrifuged as mentioned earlier, producing the final pellet of IM. Peptidoglycan analysis The contents of peptidoglycan in different fractions were analyzed by colorimetric measurement of muramic acid following a reaction with p-hydroxydiphenyl.19,

23

N-

acetylmuramic acid was used to make a calibration curve. Spores at different treatment stages or isolated fractions of the same amount of starting material were measured. Vegetative cell membrane isolation Vegetative cells were harvested by centrifugation and washing with Tris-HCl buffer (pH 7.5). Cells were then disintegrated and the membrane fraction was pelleted by differential centrifugation using the same method as for spore IM isolation. To estimate the number of


Page 7 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spores in the vegetative cell crops, a germination assay was carried out. Briefly, cells were heat treated at 70°C for 30 min, cooled down on ice and plated on TSB agar at serial dilutions. Colonies were counted after overnight incubation at 37°C. Electron microscopy After each treatment, the spores or isolated membrane fraction were fixed in 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodiumcacodylate buffer ( McDowell fixative) and either directly negatively stained with Uranyl Acetate and imaged by TEM or epon embedded and sectioned. For sectioning, first postfixation with 1% osmiumtetroxide (OsO4, Electron microscopy sciences, Hatfield, PA, USA) in cacodylate buffer was performed. Subsequently, the spores were dehydrated in an alcohol series and embedded into Epon (LX‐112 resin Ladd research, Williston, VT, USA). Ultrathin Epon sections of 90 nm were collected on formvar‐coated grids, counterstained with uranyl acetate and lead citrate and analyzed with transmission electron microscopy (FEI Tecnai T12). Sample preparation for MS Membrane pellets from three replicate spores and three replicate vegetative cells were resuspended into 200 to 300 µl of water. Protein concentrations were evaluated by the Micro BCA Protein Assay (Pierce). The yield of spore IM proteins was 50-100 µg from 100-150 mg dry weight of spores, which is comparable to previously reported inner membrane isolations20-22. 50 to 100 µg of protein was loaded and separated by SDS-PAGE. SDS-PAGE was run on 10% Novex® Tris-Glycine gels (ThermoFisher Scientific) at 125V constant voltage with XCell SureLock® Mini-Cell (ThermoFisher Scientific). The gel was stained with Coomassie Blue. The lanes containing membrane proteins were fractionated into nine pieces. Gel pieces went through reduction by 10 mM dithiothreitol in 100 mM NH4HCO3 at 56°C for 1 h and alkylation by 55 mM iodoacetamide in100 mM NH4HCO3 in dark for 45 min. Proteins were digested by 12.5 µg/ml trypsin in 50 mM NH4HCO3 buffer overnight.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

Elutes of peptides were freeze-dried and finally reconstituted with 3% acetonitrile-0.1% trifluoroacetic acid for MS analysis. LC-MS/MS analysis Mass spectrometry analysis of the peptide samples was performed with an amaZon Speed Iontrap with a CaptiveSpray ion source (Bruker) coupled with an EASY-nLC II (Proxeon, Thermo Scientific) chromatographic system. 100 ng of peptides of each nine gel fractions from the three replicates of spore inner membrane and from the three replicates of the vegetative cell isolates were injected and separated with an eluent flow of 300 nL/min on an EASY-Column 10cm (SC 200 Thermo Scientific coupled with SC001 2 cm precolumn) using a 50 min gradient of 0–50% acetonitrile and 0.1% formic acid. Some selected MSMS instrument parameters were; MS mass range: m/z 400-1500; MSMS mass window from m/z 100: precursor mass window selection: m/z 4.0; number of precursor ion selections: 5; CID in SmartFrag mode with variable energy; Data dependent acquisition with active exclusion after 1 spectrum with release after 30 seconds. Raw MS/MS data of the gel peptide fractions were processed as multi-file (MudPIT) with the MASCOT DISTILLER program, version 2.4.3.1 (64 bits), MDRO 2.4.3.0 (MATRIX science, London, UK). Peak-picking for MS and MS/MS spectra was optimized for a mass resolution of up to 3500 (m/∆m) and 2500, respectively. Peaks were fitted to a simulated isotope distribution with a correlation threshold of 0.7, and with a minimum signal-to-noise ratio of 2. The processed data combined over the nine gel fractions were searched with the MASCOT server program 2.3.02 against B. subtilis Strain 168 protein database from the UniProt consortium (release: January, 2014; 4244 entries in total) with the redundancy removed with Dbtoolkit

24

, supplemented by a database of common contaminants

downloaded from Mascot website collected by Max Planck Institute of Biochemistry (Martinsried). The database was complemented with its corresponding decoy data base for


Page 9 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


statistical analyses of peptide false discovery rate (FDR). Trypsin was used as the enzyme and 1 missed cleavage was allowed. Carbamidomethylation of cysteine was used as a fixed modification and oxidation of methionine as a variable modification. Both peptide and MS/MS fragment mass tolerance was set to 0.3 Dalton. The MASCOT MudPIT peptide identification score was set to a cut-off of 18. 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 MASCOT protein identification reports were exported as XML and then imported in a custom made VBA software program running in Microsoft Excel. The program facilitates organization and data mining of large sets of proteomics data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium25 via the PRIDE partner repository with the dataset identifier PXD003268 and 10.6019/PXD003268. Details of the MS analyses and the protein identifications are listed in the Supporting Information.

Bioinformatics Membrane proteins were predicted using the following programs or databases: PSORTb 3.0

(http://www.psort.org/psortb/),

Phobius

(http://phobius.sbc.su.se/),

(http://services.cbu.uib.no/tools/bomp), (http://bioinformatics.biol.uoa.gr/PRED-LIPO/),

BOMP

PRED-LIPO LocateP

(http://www.cmbi.ru.nl/locatep-

db/cgi-bin/locatepdb.py), InterProScan 5 (http://www.ebi.ac.uk/interpro/). Identified proteins were categorized according to SubtiWiki (http://subtiwiki.uni-goettingen.de/).

Results and discussion Isolation of B. subtilis spore inner membrane



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

To investigate the Bacillus subtilis spore inner membrane proteome, we adapted the procedures that have been used for localization of several spore inner membrane proteins20, 22 to mass spectrometry based proteomics approaches (Figure 1). Dormant spores made from a defined minimal medium were purified using density gradient centrifugation and a purity of 99.5% of spores was obtained. Spores were first decoated using a harsh chemical treatment (see Materials and Methods). It was shown by Hudson et al that decoating could not completely remove the coat, and the remnant would eventually be in the integument fraction.15 Our earlier research also showed that extraction of the isolated coat still leaves a substantial amount of protein material (termed insoluble fraction).19 This is likely due to the high extent of cross-linking in coat proteins.26 Visualization of the spores by electron microscopy confirmed the presence of coat after the first extraction (Figure 1). The core is more intensely contrasted with the electron dense staining (osmium and uranyl acetate) as the staining can better penetrate the spores after the extraction. It was also reported that the outer membrane is absent after the decoating treatment.15 This is probably ensured by the extensive washing steps, although in our hands DacA (penicillin-binding protein 5*), which was reported to localize on the outer membrane27, was identified in the sample obtained. Thus we assume that by this procedure, the extractable fraction of coat proteins was removed, leaving the insoluble portion with fissures that allow lysozyme to enter and degrade the cortex and germ cell wall28. Electron micrographs of the lysozyme treated spores show a dramatic expansion of the spore core, suggesting the degradation of the cortex and germ cell wall. Breakage of the decoated spores followed by differential centrifugation gave a final pellet enriched for the IM. Electron micrographs show that the final pellet consist of IM (Figure 1 and Figure S3) while the integument pellet containing disrupted parts of the coat (Figure S4). To assess the efficiency of the lysozyme treatment, a muramic acid assay was deployed to probe for leftover


Page 11 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptidoglycan. The amount of muramic acid in the IM preparation measured below the detection limit, while all muramic acid in the original spore was recovered in the supernatant collected from lysozyme treatment.

Figure 1. (A) Schematic structures of B. subtilis spores. (B) The procedure of IM isolation with schematic illustration and electron micrographs of the spores and the membrane pellet (further information in supplementary figures). Dashed patterns show layers that are at least partially removed.

Identification of candidate IM proteins



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

Membrane proteins can be classified into different groups based on how they interact with the lipid bilayer29: proteins with transmembrane spanning regions (integral membrane proteins);

proteins

covalently

attached

via

fatty

acids

(lipoproteins)

or

glycosylphosphatidylinositol (GPI) anchors (in eukaryotes); and membrane associated proteins that are bound non-covalently or transiently to the membrane. Protocols to isolate integral membrane and covalently linked membrane proteins usually aim at removing membrane associated proteins and non-specifically adhering cytoplasmic proteins by washing with high-salt and high-pH buffers. Following a standard membrane purification method30, we were able to enrich our samples for spore IM integral membrane proteins and lipoproteins. It should be noted that it is virtually impossible to remove all cytoplasmic proteins and there is the distinct possibility that in vivo some of these proteins spend part of their life-time in the cell associated with the spore IM, hence classifying as membrane associated proteins (vide infra); instead, the goal is to enrich integral membrane proteins, membrane anchored and peripheral membrane proteins to boost the identification of lowabundant ones31. Next, SDS-PAGE was used to pre-fractionate the proteins to reduce the complexity of the samples and thus enhance the sensitivity of identification. Tryptic peptides were generated by in-gel digestion and analyzed by LC-MS/MS. As it is impractical to efficiently avoid co-isolation of cytoplasmic and core proteins in the membrane fraction, the identified proteins can be filtered further using bioinformatics tools which complement the biochemical protein subcellular localization.32 Results of six bioinformatic tools were included: Phobius is a combined transmembrane topology and signal peptide predictor.32 PRED-LIPO is a Hidden Markov Model method for the prediction of lipoprotein signal peptides of Gram-positive bacteria.33 PSORTb is a subcellular localization prediction program for bacterial and archaeal sequences using multiple analytical modules.34 LocateP combines many subcellular location predictors and distinguishes seven


Page 13 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different subcellular locations within Gram-positive bacteria.35 BOMP is a program to predict

β-barrel membrane proteins.36 Although β-barrel membrane proteins were thought to locate in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria, recently they were also reported in Gram-positive bacteria.37, 38 InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. It combines protein signatures from a number of member databases.39 InterProScan, the software package that allows sequences to be scanned against InterPro's signatures, was used. Each algorithm has its own trade-off between precision and recall. In order to reduce the omission rate, we assigned a protein as membrane protein if any of the tools indicated it to be membrane related. Out of the 929 proteins which were identified in at least two of the three replicates, 334 proteins were assigned as candidate membrane proteins (Table S3). Of these candidate membrane proteins 46 had not been reported by previous proteomics studies in B. subtilis. It is also worth noting that the uniqueness of the bacterial spore IM is not covered in the algorithms; hence in addition to intrinsic errors in the algorithm based identifications, extra caution needs to be taken when considering their sensitivity and specificity. The number of proteins identified is dramatically larger than that in the outer layers of the spore.19, 40 With several reported studies fewer proteins have been identified in the analyses of the whole spore proteome,41, 42 than in the present inner membrane isolate. This demonstrates the efficiency of the inner membrane isolation which overcomes the limitation of dynamic range of the MS analyses of the whole spore proteome. Vegetative cell membrane proteome There have been several studies on the proteome of B. subtilis vegetative cells.43 To validate that our methods are effective in identifying membrane proteins and to see whether in our culture conditions the vegetative cell proteome would turn out distinct from the spore



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

IM, we performed B. subtilis vegetative cell membrane protein identification as well. Microscopic examination of the cell crops could not find spores or spore-forming cells. Classical microbiological techniques used on the vegetative cell containing fraction identified less than 1 in 2000 cells as a spore. GeLC-MS/MS analysis of three biological replicates of vegetative cell membrane revealed 1316 proteins (Table S2). 880 of them were identified in at least two replicates, 721 of which were among the previously reported B. subtilis vegetative membrane subproteomes,44,

45

yet 42 of the rest were predicted as membrane

proteins (Table S4). The results illustrate that differences between growth conditions used in the various experiments reported in literature resulted in differential vegetative cell membrane proteomes. The identifications in the sample enriched for the vegetative cell membrane were compared with the data obtained for the spore IM proteins. There was a large portion of proteins, general or membrane predicted, overlapped in both cell types as well as unique to either (Figure 2).

Figure 2. Venn diagram of proteins identified from the B. subtilis spore IM isolation and vegetative cell membrane fraction. Membrane proteins were predicted by several bioinformatics tools (see Materials and methods). Functional categorization In order to have an overview of the functional distribution of identified proteins, protein identifications of the IM fraction as well as those of the vegetative cell membrane were 14 ACS Paragon Plus Environment

Page 15 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


categorized according to SubtiWiki46 (Figure 3; Figure S1 for subcategories). Proteins identified distributed throughout almost all of the groups. In many of the groups, spore IM proteins and vegetative cell membrane proteins represent an approximately similar number. The fact that each membrane has about half of its proteins unique, corroborates that the two cell types share many membrane localized processes, but also have unique membrane functions that are implemented by a differential set of proteins. As can be predicted, the spore IM contained more proteins in the category “Sporulation and Germination”, while vegetative cells have more in “Exponential and early post-exponential lifestyles”. In line with expectations, the membrane of motile vegetative cells showed a large number (35) of “Motility and chemotaxis” proteins. Only one protein belonging to this class was found in spores. Both cell types expressed a number of proteins that have a role in coping with environmental stress (also see below). It is also not surprising that spores had more proteins in the category of “Resistance against oxidative and electrophile stress”; however, proteins in the category “Cold stress proteins” “Coping with hyper-osmotic stress” were more enriched for vegetative cells than spores, reflecting that the spores did not meet cold or osmotic stress during sporulation;47, 48 Moreover, from an ecological point of view it is also less likely to encounter these protein classes in spores because cold and osmotic stresses are less damaging to them. The number of “DNA repair/ recombination” proteins in spores was twice that of vegetative cells. Interestingly, the category “Membrane proteins” included proteins that are not predicted as membrane proteins by us. These proteins are either peripheral membrane proteins (e.g. KtrC) or have been reported in other membrane proteome data but are in fact during most of their life cycle localized in the cytosol45. In the following sections we discuss a selection of proteins that has potentially significant roles in the spore physiology according to the categorization. Notice that the discussion in the following sections is not limited to the identifications predicted as membrane proteins.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

Figure 3. Categorization of proteins according to SubtiWiki. The six main categories are: 1. Cellular processes; 2. Metabolism; 3. Information processing; 4. Lifestyles; 5. Prophages and mobile genetic elements; 6. Groups of genes. SpoIM: identified in spore IM; VegM: identified in vegetative cell membrane; M+: predicted to be membrane proteins.

Expected IM proteins A few proteins have been reported to be localized in the spore’s IM by immunoblotting (Table 1 and references cited therein). The GRs of B. subtilis are heterocomplexes formed by three (A, B, and C) subunits encoded by tricistronic operons. The A-subunit consists of five or six predicted membrane-spanning domains next to N- and C-terminal hydrophilic 16 ACS Paragon Plus Environment

Page 17 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


domains; the B-subunit comprises 10 to 12 transmembrane helices; and the C-subunit is a lipid-anchored protein.49 We identified GerBA, GerKA, GerKB and GerKC with high confidence while one of the replicates allowed us to identify GerAB and GerBC. Although Stewart et al estimated that the number of GerAA and GerAC subunits per spore is higher than that of GerBA, GerBC and GerKA subunits, those two proteins were not identified in our isolates.20, 50 The proteins encoded by the spoVA operon are involved in DPA uptake during sporulation and DPA release during germination.51, 52 We identified five of the seven proteins, including the hydrophilic SpoVAEa, in line with the Western-blot analysis result of Perez-Valdespino et al.53 The missing of SpoVAB and SpoVAEb could be due to their low molecular weight (15.2 kDa and 12.3 kDa), as proteins of low molecular weight in the genome were less representative among the identifications (Figure S2). Western-blot analysis has high sensitivity, but its drawback is obvious as a specific antibody for each protein target is required. We used MS to analyze the IM fraction from spores in a high throughput way and identified most of the reported proteins as well as those predicted to be present in the spore IM based on their primary aminoacid sequence and reported functions. It is still possible to further optimize the methods to identify the missing ones, e.g. reducing complexity with more gel fractions, or using a complementary approach such as proteinase K pretreated membrane preparation45.

Table 1. Proteins reported or predicted to be present on IM

Protein GerAA GerAB

Reported IM localized Identified (References) this study

in

Function

15 54

Germinant receptor *

+ 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

GerAC

15

GerBA

20

Page 18 of 36

++

GerBB GerBC

+

GerKA

++

GerKB

++

GerKC

55

*

SpoVAA

++ ++

SpoVAB SpoVAC

++

SpoVAD

22

++

SpoVAEa

53

++

53

++

GerD

21, 56

++

Essential for GR assembly

SleB

57

++

Cortex-lytic enzyme

YpeB

57

++

Essential for SleB assembly

YhcN

58

++

Unknown

PrkC

59

++

Ser/Thr membrane kinase & GR to peptidoglycan fragments

DPA uptake and release

SpoVAEb SpoVAF

Proteins in this table are either experimentally shown to be localized on IM or predicted likely to be on IM based on their function. These reports used B. subtilis except that * GerAB was on C. botulinum and GerKC was on C. perfringens. +: identified in one replicate; ++: identified in at least two replicates.

Transporters A general notion is that the proteins encoded by the ger operon are involved in germinant binding but the further downstream signal-transduction mechanisms that mediate germination


Page 19 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


have not been unraveled yet. The release of cations followed by DPA release and divalent cations early in spore germination suggests that one or more channels for these ions must be opened in the inner spore membrane upon binding of a germinant to its receptor. It is possible that germination receptors activate certain antiporters in order to obtain a shift in the electrochemical gradient across the inner membrane.60 We identified several transporter proteins involved in different cellular processes. More than 50% of the identified transporter proteins belonged to the ABC transporters family. Of these, proteins YugO (potassium transporter), MrpA (sodium/proton antiporter), CorA (magnesium transporter), ZnuA (Zinc uptake protein), which were reported to be upregulated during sporulation61, were only identified from the spore inner membrane fraction and could play a major role in such efflux processes. In addition, BceB (Bacitracin exporter), MdxG & MdxF (Maltodextrin transporters permeases), RbsB (D-Ribose binding uptake protein), CydC (ATPbinding/permease) GlpT (glycerol-3-phosphate transporter), YcnJ (copper transporter), YflS (malate transporter) and YmfD (Bacillibactin transporter) were also identified from the spore inner membrane fraction. It is noteworthy that bacitracin has been seen to affect the size of parasporal crystals and the spores of B. thuringiensis62 and bacillibactin siderophore has been obtained when B. anthracis spores underwent germination and outgrowth in a low-iron medium63. Proteases Proteases play important roles during both sporulation and germination. Protease inhibitors have been shown to inhibit spore germination.64, 65 The membrane anchored protein YpeB is essential for proper cortex-lytic enzyme SleB assembly into spores. SleB is located interior to the cortex in the dormant spore, and our results corroborate its close association with the IM. Membrane protein YpeB has been suggested to maintain SleB inactive through YpeB's PepSY domains.66 A recent study showed in vivo and in vitro that B. anthracis HtrC cleaves



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

YpeB proteolytically to a stable fragment. We detect HtrC in Bacillus subtilis expressed by yyxA in our IM fraction. HtrC proteolytic action might disrupt YpeB interaction and permits SleB cortex-lytic activity. A B. subtilis htrC mutant was also shown to have an altered YpeB proteolysis; however, results suggested other proteases were also involved.18 Interestingly, in this context we identify the uncharacterized membrane protein TseB (YpmB) wich contains two PepSY domain and that might have an inhibitory function in cortex degradation.66 Further experimental evidence is needed to establish the function of inhibitory proteins and proteases in the activation of cortex-lytic proteins. Small, acid-soluble spore proteins (SASPs) are well known to be degraded by a germination protease Gpr during germination. A more recent study showed yet another protease, YmfB (or TepA), conserved among sporeforming species, was dedicated to the degradation of a specialized family of SASPs.67 These germination proteases were copurified in our spore IM fraction. Proteases that play regulatory functions during sporulation, such as SpoIVB and CtpB (for SigK), spoIIIAA (for SigG) and spoIIGA (for SigE) were not among the identifications, reflecting their spatially and temporally regulated expression. Functions of some proteases we identified, like CtpA and YmfH, remain to be elucidated. During germination, the spore coat composed of proteins needs to be breached and eventually shed. It is suggested that degradation is caused by specific hydrolytic enzymes located within the spore integument.68 But proteins responsible for this process have not been reported and a spore coat proteome analysis did not suggest a candidate.19 The protease YabG is shown to be involved in coat protein modification but not in degradation.69 It is possible that some uncharacterized proteins or the unidentified proteins in coat are the hydrolytic enzymes. Given that coat proteins are extensively cross-linked,70 unknown hydrolytic mechanisms might be needed. It might also be possible that the proteases locate in the inner


Page 21 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


layers (cortex, germ cell wall, IM) of dormant spores and their function is activated upon hydrolysis of the cortex. DNA repair, replication The spores’ DNA is well protected against damage from UV, heat, and oxidizing agents. The α/β-type SASPs bind to the negatively supercoiled spore DNA, reducing the chemical and enzymatic reactivity of the DNA. A certain number of DNA repair genes are expressed during sporulation and possibly exert their function upon spore germination. Involvement of nucleotide excision repair enzymes, the spore photoproduct lyase, base excision repair proteins in the protection of spores against UV radiation and wet heat has been shown previously.1 In our analysis of IM fraction, the nucleotide excision repair enzymes UvrA and UvrB, the base excision repair enzymes Nfo (YqfS) and Nth, the spore photoproduct lyase SplB, sporulation specific UV-damage-endonuclease YwjD were identified. In a similar manner, during outgrowth, the chromosomal DNA is required to relax rapidly to reactivate transcription. In addition to the degradation of the SASPs, an increased helicase activity in the initial stages of outgrowth may be necessary. In this view, we identified DNA 3'-5' helicase HelD (YvgS) and ATP dependent helicase DinG, YpvA and PcrA in our spore IM fraction. Concurrently, most of the above mentioned proteins have been reported to be overexpressed in the initial stages of germination in a previous gene expression study.71 Additionally, before onset of outgrowth, the DNA repair mechanisms scan the DNA for a possible damage. Such damage needs to be repaired prior to outgrowth. The identified DNA repair proteins RecA, RecF, RecO, RecN and the mismatch repair proteins MutS, MutSB may play a vital role in these processes. Other resistance proteins Besides the DNA recombination and replication machinery, many other proteins, enzymes and transporters are also responsible for the survival of spores and cells. In our analysis heat



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

shock proteins such as CtsR, GroES, GroEL and HtpG were identified in the spore IM fraction. Along with these and others, CssS, a two-component sensor kinase involved in the control of cellular responses to protein secretion stress was also identified. Proteins like MhqA, MhqD, AzoR1 etc. involved in resistance against oxidative and electrophilic stress were also identified from the IM fraction of spores. Interestingly proteins BshA, BshB2 and BshC involved in bacillithiol sysnthesis were also observed. Bacillithiol sysnthesis was reported to be upregulated in during spore outgrowth in B. anthracis.72 Speculatively, this may also be true in B. subtilis. In addition to protein LiaS, a two-component sensor kinase involved in response to bacitracin, protein BceB, a bacitracin ABC transporter (permease), was also identified in spores and high amounts of pre-synthesized bacitracin is known to inhibit germination in B. licheniformis.73 YxdK, a two-component sensor kinase, and putative multidrug resistance proteins YhcA and YitG, were identified in spores that may provide germinated spores with resistance agaist toxins. Additionally, SdpI, an immunity protein was identified in both spore and vegetative fractions being dominant in the spore fraction. This is a membrane bound protein required to protect the sporulating cells from self-killing.74 B. subtilis cells, after nutrient limitation, delay the commitment to spore formation by killing the other non-sporulating cells and feeding on them. This killing is mediated by the exported toxic protein SdpC.74 We identified also SdpC in the spore IM fraction. Interestingly SdpA, SdpB proteins involved in production and transport of SdpC were only identified in our vegetative cell fraction. Thus it is plausible that the SdpC and SdpI proteins are required for continuation of sporulation and are therefore conserved in the spore membrane. Others As reported previously in B. megaterium spores spermidine synthesis takes place during germination and outgrowth of spores. In our analysis of B. subtilis spores, we identified a couple of enzymes (SpeA, SpeE) which are part of the spermidine biosynthesis pathway


Page 23 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(KEGG pathway analysis). In addition, an arginase (ArgI) was also identified, which could be involved in conversion of arginine to ornithine. Ornithine can also be converted to putrescine (an intermediate in spermidine synthesis) by a predicted but not yet identified ornithine decarboxylase in B. subtilis. All the above mentioned enzymes have been identified to be present in germinating B. subtilis spores in a recent work by Sinai et al.75 In our analysis of the B. subtilis spore IM fraction, enzymes involved in co-enzyme A synthesis (according to KEGG pathway analysis) viz PanB, PanC, PanE, CoaBC, CoaE and IlvD, were identified. Incidentally, co-enzyme A has been previously reported to be in disulfide linkage to proteins in B. megaterium spores.76, 77 Thus CoA can play a role in the establishment of the structural integrity of spores as well as playing an important role as modulator of the metabolism that is operative in germinating and outgrowing spores as was already suggested in the past78. Conclusion Along with the extensive studies on the biochemistry and functions of individual proteins, the emergence of proteomics techniques, especially the mass spectrometry-driven proteomics, broadens our knowledge of spores in a more comprehensive way. Although the extensive efforts have been devoted to the spore coat proteome and have led to a detailed understanding of spore structure and functions as well as wide applications, our present study is the first to report a spore IM proteome. Cell membrane harbors proteins that play key roles in various physiological processes, and the spore IM is not an exception. Because of the low abundance of IM proteins and the limited dynamic range of mass spectrometers, a sweeping query to the whole spore is unable to give a satisfying number of IM proteins as many of them are masked by proteins of higher abundance from other layers. By fractionation and enrichment of the IM, we identified 336 membrane proteins along with 591 membrane associated proteins, a number that is far beyond that of the known spore proteins. By comparing with the vegetative cell membrane, the dynamics of the membrane proteome can



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

also be seen. Functional categorization gives hints on proteins involved in different processes that need further study. Future spore IM proteomics studies will be focused on quantitation and topology determination of the IM proteins, interaction between different IM proteins, between proteins on IM and other spore layers, and between IM proteins and small molecules such as germinants and inhibitory compounds. ASSOCIATED CONTENT Supporting Information Table S1a lists all identified proteins of the inner membrane isolate of the three Bacillus subtilis replicate spore cultures, with the mass, the mascot score and the number MSMS spectra per protein. Tables S1b, S1c and S1d list all the specifications of each Bacillus subtilis replicate spore culture, including the sequences, masses, number of peptide MSMS spectra, modifications and Mascot scores of all peptides assigned to an identified protein. Table S2a,S2b,S2c,S2d, list the same protein identification information for the three Bacillus subtilis vegetative cell replicate analyses. Table S3 lists the proteins identified in at least two Bacillus subtilis inner membrane isolate replicates, with the annotation of membrane protein candidates, and the membrane protein predictions for each bioinformatics algorithm used. Table S4 lists the proteins identified in at least two Bacillus subtilis vegetative replicate analyses for comparison with the inner membrane isolate analyses. Figure S1: Subcategorization of proteins according to SubtiWiki. Figure S2: Mass distribution of identified proteins in three replicates of the spore inner membrane fraction. Figure S3: Electron micrograph of the negatively stained Bacillus subtilis spore inner membrane fraction. Figure S4: Electron microscopic analysis of spores during the procedure of the Bacillus subtilis spore inner membrane isolation. This material is available free of charge via the Internet at http://pubs.acs.org


Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR INFORMATION Corresponding Author *C.G.d.K.: E-mail: [email protected]. Tel.: +31 (0)205255457. *S.B.: E-mail: [email protected]. Tel.: +31 (0)205257079 Author Contributions §S.B. and C.G.d.K. contributed equally to this work. S.B., C.G.d.K. and L.J.d.K. participated in the design, coordination of the study and supervision of the drafting of the manuscript. L.Z. performed the experiments and wrote the manuscript. W.A. participated in drafting the manuscript. N.O. and H.v.V. performed a part of the experiments. H.L.D., N.v.d.W. and W.R. provided expert technical assistance. Funding Sources L.Z. acknowledges the Erasmus Mundus program (EMEA3) and TNO (Healthy Living) for funding of his PhD project. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT Hereby we thank Chao Zhang and Xiyun Gan for their generous support on bioinformatics aspects, Hansuk Buncherd and Sacha Stelder for valuable advice, and Jolanda Verheul and Jos C. Arents for experimental assistance. ABBREVIATIONS MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid chromatography, GeLC, gel electrophoresis liquid chromatography; MudPIT, multidimensional protein



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

identification technique; IM, spore inner membrane; OM, spore outer membrane; DPA, dipicolinic acid; GR, germinant receptor. REFERENCES (1) Nicholson, W. L.; Munakata, N.; Horneck, G.; Melosh, H. J.; Setlow, P., Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 2000, 64, (3), 548-572. (2) Setlow, P., Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 2006, 101, (3), 514-525. (3) McKenney, P. T.; Driks, A.; Eichenberger, P., The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat. Rev. Microbiol. 2013, 11, (1), 33-44. (4) Black, S. H.; Gerhardt, P., Permeability of bacterial spores IV. Water Content, uptake, and distribution. J. Bacteriol. 1962, 83, (5), 960-967. (5) Henriques, A. O.; Moran, J. C. P., Structure, assembly, and function of the spore surface layers. Annu. Rev. Microbiol. 2007, 61, (1), 555-588. (6) Setlow, P., The Germination of spores of Bacillus species: what we know and don't know. J. Bacteriol. 2014, JB.01455-13. (7) Paredes-Sabja, D.; Setlow, P.; Sarker, M. R., Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. Trends Microbiol. 2011, 19, (2), 8594. (8) Setlow, B.; Setlow, P., Measurements of the pH within dormant and germinated bacterial spores. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, (5), 2474-6.


Page 27 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Cowan, A. E.; Olivastro, E. M.; Koppel, D. E.; Loshon, C. A.; Setlow, B.; Setlow, P., Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, (20), 7733-7738. (10) Young, S. B.; Setlow, P., Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents. J. Appl. Microbiol. 2004, 96, (2), 289-301. (11) Young, S. B.; Setlow, P., Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone. J. Appl. Microbiol. 2004, 96, (5), 1133-42. (12) Young, S. B.; Setlow, P., Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide. J. Appl. Microbiol. 2003, 95, (1), 54-67. (13) Coleman, W. H.; Chen, D.; Li, Y.-q.; Cowan, A. E.; Setlow, P., How moist heat kills spores of Bacillus subtilis. J. Bacteriol. 2007, 189, (23), 8458-8466. (14) Melly, E.; Cowan, A. E.; Setlow, P., Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide. J. Appl. Microbiol. 2002, 93, (2), 316-25. (15) Hudson, K. D.; Corfe, B. M.; Kemp, E. H.; Feavers, I. M.; Coote, P. J.; Moir, A., Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J. Bacteriol. 2001, 183, (14), 4317-4322. (16) Vepachedu, V. R.; Setlow, P., Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores. FEMS Microbiol. Lett. 2007, 274, (1), 42-47.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

(17) Atluri, S.; Ragkousi, K.; Cortezzo, D. E.; Setlow, P., Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor. J. Bacteriol. 2006, 188, (1), 28-36. (18) Bernhards, C. B.; Chen, Y.; Toutkoushian, H.; Popham, D. L., HtrC isinvolved in proteolysis of YpeB during germination of Bacillus anthracis and Bacillus subtilis spores. J. Bacteriol. 2015, 197, (2), 326-336. (19) Abhyankar, W.; Beek, A. T.; Dekker, H.; Kort, R.; Brul, S.; de Koster, C. G., Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics 2011, 11, (23), 4541-4550. (20) Paidhungat, M.; Setlow, P., Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J. Bacteriol. 2001, 183, (13), 3982-3990. (21) Pelczar, P. L.; Setlow, P., Localization of the germination protein GerD to the inner membrane in Bacillus subtilis spores. J. Bacteriol. 2008, 190, (16), 5635-5641. (22) Vepachedu, V. R.; Setlow, P., Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis. J. Bacteriol. 2005, 187, (16), 5677-5682. (23) Hadžija, O., A simple method for the quantitative determination of muramic acid. Anal. Biochem. 1974, 60, (2), 512-517. (24) Martens, L.; Vandekerckhove, J.; Gevaert, K., DBToolkit: processing protein databases for peptide-centric proteomics. Bioinformatics 2005, 21, (17), 3584-3585. (25) Vizcaíno, J. A.; Deutsch, E. W.; Wang, R.; Csordas, A.; Reisinger, F.; Ríos, D.; Dianes, J. A.; Sun, Z.; Farrah, T.; Bandeira, N., ProteomeXchange provides globally


Page 29 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coordinated proteomics data submission and dissemination. Nat. Biotechnol. 2014, 32, (3), 223-226. (26) Abhyankar, W.; Pandey, R.; Ter Beek, A.; Brul, S.; de Koning, L. J.; de Koster, C. G., Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation. Food Microbiol. 2015, 45, Part A, 54-62. (27) Buchanan, C. E.; Neyman, S. L., Correlation of penicillin-binding protein composition with different functions of two membranes in Bacillus subtilis forespores. J. Bacteriol. 1986, 165, (2), 498-503. (28) Setlow, P., Summer meeting 2013 - when the sleepers wake: the germination of spores of Bacillus species. J. Appl. Microbiol. 2013, 115, (6), 1251-1268.

(29) Bendz, M.; Möller, M. C.; Arrigoni, G.; Wåhlander, Å.; Stella, R.; Cappadona, S.; Levander, F.; Hederstedt, L.; James, P., Quantification of membrane proteins using nonspecific proteasedigestions. J. Proteome Res. 2009, 8, (12), 5666-5673. (30) Molloy, M. P., Isolation of bacterial cell membranes proteins using carbonate extraction. In 2D PAGE: Sample Preparation and Fractionation, Posch, A., Ed. Humana Press: 2008; pp 397-401. (31) Cordwell, S. J.; Thingholm, T. E., Technologies for plasma membrane proteomics. Proteomics 2010, 10, (4), 611-627. (32) Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H., Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2007, 2, (4), 953-971.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

(33) Bagos, P. G.; Tsirigos, K. D.; Liakopoulos, T. D.; Hamodrakas, S. J., Prediction of lipoprotein signal peptides in Gram-positive bacteria with a Hidden Markov Model. J. Proteome Res. 2008, 7, (12), 5082-5093. (34) Yu, N. Y.; Wagner, J. R.; Laird, M. R.; Melli, G.; Rey, S.; Lo, R.; Dao, P.; Sahinalp, S. C.; Ester, M.; Foster, L. J.; Brinkman, F. S. L., PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010, 26, (13), 1608-1615. (35) Zhou, M.; Boekhorst, J.; Francke, C.; Siezen, R. J., LocateP: Genome-scale subcellular-location predictor for bacterial proteins. BMC Bioinformatics 2008, 9, (1). (36) Berven, F. S.; Flikka, K.; Jensen, H. B.; Eidhammer, I., BOMP: a program to predict integral β-barrel outer membrane proteins encoded within genomes of Gram-negative bacteria. Nucleic Acids Res. 2004, 32, (suppl 2), W394-W399. (37) Heuck, A. P.; Moe, P. C.; Johnson, B. B., Thecholesterol-dependent cytolysin family of Gram-positive bacterial toxins. In Cholesterol Binding and Cholesterol Transport Proteins:, Harris, J. R., Ed. Springer Netherlands: 2010; pp 551-577. (38) Yamashita, D.; Sugawara, T.; Takeshita, M.; Kaneko, J.; Kamio, Y.; Tanaka, I.; Tanaka, Y.; Yao, M., Molecular basis of transmembrane beta-barrel formation of staphylococcal pore-forming toxins. Nat. Commun. 2014, 5, 4897. (39) Mitchell, A.; Chang, H.-Y.; Daugherty, L.; Fraser, M.; Hunter, S.; Lopez, R.; McAnulla, C.; McMenamin, C.; Nuka, G.; Pesseat, S.; Sangrador-Vegas, A.; Scheremetjew, M.; Rato, C.; Yong, S.-Y.; Bateman, A.; Punta, M.; Attwood, T. K.; Sigrist, C. J. A.; Redaschi, N.; Rivoire, C.; Xenarios, I.; Kahn, D.; Guyot, D.; Bork, P.; Letunic, I.; Gough, J.; Oates, M.; Haft, D.; Huang, H.; Natale, D. A.; Wu, C. H.; Orengo, C.; Sillitoe, I.; Mi, H.;


Page 31 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thomas, P. D.; Finn, R. D., The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res. 2015, 43, (D1), D213-D221. (40) Lai, E.-M.; Phadke, N. D.; Kachman, M. T.; Giorno, R.; Vazquez, S.; Vazquez, J. A.; Maddock, J. R.; Driks, A., Proteomic analysis of the sporecoats of Bacillus subtilis and Bacillus anthracis. J. Bacteriol. 2003, 185, (4), 1443-1454. (41) Kuwana, R.; Kasahara, Y.; Fujibayashi, M.; Takamatsu, H.; Ogasawara, N.; Watabe, K., Proteomics characterization of novel spore proteins of Bacillus subtilis. Microbiology 2002, 148, (12), 3971-3982. (42) Mao, L.; Jiang, S.; Wang, B.; Chen, L.; Yao, Q.; Chen, K., Protein profile of Bacillus subtilis spore. Curr. Microbiol. 2011, 63, (2), 198-205. (43) Becher, D.; Büttner, K.; Moche, M.; Heßling, B.; Hecker, M., From the genome sequence to the protein inventory of Bacillus subtilis. Proteomics 2011, 11, (15), 2971-2980. (44) Dreisbach, A.; Otto, A.; Becher, D.; Hammer, E.; Teumer, A.; Gouw, J. W.; Hecker, M.; Völker, U., Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques. Proteomics 2008, 8, (10), 2062-2076. (45) Hahne, H.; Wolff, S.; Hecker, M.; Becher, D., From complementarity to comprehensiveness – targeting the membrane proteome of growing Bacillus subtilis by divergent approaches. Proteomics 2008, 8, (19), 4123-4136. (46) Michna, R. H.; Commichau, F. M.; Todter, D.; Zschiedrich, C. P.; Stulke, J., SubtiWiki-a database for the model organism Bacillus subtilis that links pathway, interaction and expression information. Nucleic Acids Res. 2013, 42, (D1), D692-D698.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

(47) Méndez, M. B.; Orsaria, L. M.; Philippe, V.; Pedrido, M. E.; Grau, R. R., Novel roles of the master transcription factors Spo0A and σB for survival and sporulation of Bacillus subtilis at low growth temperature. J. Bacteriol. 2004, 186, (4), 989-1000. (48) Hahne, H.; Mader, U.; Otto, A.; Bonn, F.; Steil, L.; Bremer, E.; Hecker, M.; Becher, D., A comprehensive proteomics and transcriptomics analysis of Bacillus subtilis salt stress adaptation. J. Bacteriol. 2009, 192, (3), 870-882. (49) Ross, C.; Abel-Santos, E., The Ger receptor family from sporulating bacteria. Curr. Issues Mol. Biol. 2010, 12, (3), 147-158. (50) Stewart, K. A. V.; Setlow, P., Numbers of inividual ntrient grminant rceptors and oher grmination poteins in sores of Bacillus subtilis. J. Bacteriol. 2013, 195, (16), 3575-3582. (51) Vepachedu, V. R.; Setlow, P., Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme. J. Bacteriol. 2006, 189, (5), 1565-1572. (52) Li, Y.; Davis, A.; Korza, G.; Zhang, P.; Li, Y. q.; Setlow, B.; Setlow, P.; Hao, B., Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis. J. Bacteriol. 2012, 194, (8), 1875-1884. (53) Perez-Valdespino, A.; Li, Y.; Setlow, B.; Ghosh, S.; Pan, D.; Korza, G.; Feeherry, F. E.; Doona, C. J.; Li, Y.-Q.; Hao, B.; Setlow, P., Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores. J. Bacteriol. 2014, 196, (11), 2077-2088. (54) Alberto, F. o.; Botella, L.; Carlin, F. d.; Nguyen-the, C.; Broussolle, V. r., The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores. FEMS Microbiol. Lett. 2005, 253, (2), 231-235.


Page 33 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(55) Banawas, S.; Paredes-Sabja, D.; Korza, G.; Li, Y.; Hao, B.; Setlow, P.; Sarker, M. R., The Clostridium perfringens germinant receptor protein GerKC is located in the spore inner membrane and is crucial for spore germination. J. Bacteriol. 2013, 195, (22), 5084-5091. (56) Mongkolthanaruk, W.; Robinson, C.; Moir, A., Localization of the GerD spore germination protein in the Bacillus subtilis spore. Microbiology (Reading, England) 2009, 155, (Pt 4), 1146-1151. (57) Korza, G.; Setlow, P., Topology and accessibility of germination proteins in the Bacillus subtilis spore inner membrane. J. Bacteriol. 2013, 195, (7), 1484-1491. (58) Bagyan, I.; Noback, M.; Bron, S.; Paidhungat, M.; Setlow, P., Characterization of yhcN, a new forespore-specific gene of Bacillus subtilis. Gene 1998, 212, (2), 179-188. (59) Shah, I. M.; Laaberki, M.-H.; Popham, D. L.; Dworkin, J., A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell 2008, 135, (3), 486-496. (60) Xiao, Y.; Francke, C.; Abee, T.; Wells-Bennik, M. H. J., Clostridial spore germination versus bacilli: Genome mining and current insights. Food Microbiol. 2011, 28, (2), 266-274. (61) Nicolas, P.; Mäder, U.; Dervyn, E.; Rochat, T.; Leduc, A.; Pigeonneau, N.; Bidnenko, E.; Marchadier, E.; Hoebeke, M.; Aymerich, S.; Becher, D.; Bisicchia, P.; Botella, E.; Delumeau, O.; Doherty, G.; Denham, E. L.; Fogg, M. J.; Fromion, V.; Goelzer, A.; Hansen, A.; Härtig, E.; Harwood, C. R.; Homuth, G.; Jarmer, H.; Jules, M.; Klipp, E.; Chat, L. L.; Lecointe, F.; Lewis, P.; Liebermeister, W.; March, A.; Mars, R. A. T.; Nannapaneni, P.; Noone, D.; Pohl, S.; Rinn, B.; Rügheimer, F.; Sappa, P. K.; Samson, F.; Schaffer, M.; Schwikowski, B.; Steil, L.; Stülke, J.; Wiegert, T.; Devine, K. M.; Wilkinson, A. J.; Dijl, J. M. v.; Hecker, M.; Völker, U.; Bessières, P.; Noirot, P., Condition-dependent transcriptome



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

reveals high-level regulatory architecture in Bacillus subtilis. Science 2012, 335, (6072), 1103-1106. (62) Garcia-Patrone, M., Bacitracin increases size of parasporal crystals and spores in Bacillus thuringiensis. Mol. Cell. Biochem. 1985, 68, (2), 131-7. (63) Wilson, M. K.; Abergel, R. J.; Arceneaux, J. E.; Raymond, K. N.; Byers, B. R., Temporal production of the two Bacillus anthracis siderophores, petrobactin and bacillibactin. Biometals 2010, 23, (1), 129-34. (64) Cortezzo, D. e.; Setlow, B.; Setlow, P., Analysis of the action of compounds that inhibit the germination of spores of Bacillus species. J. Appl. Microbiol. 2004, 96, (4), 725741. (65) Boschwitz, H.; Milner, Y.; Keynan, A.; Halvorson, H. O.; Troll, W., Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination. J. Bacteriol. 1983, 153, (2), 700-708. (66) Üstok, F. I.; Chirgadze, D. Y.; Christie, G., Crystal structure of the PepSY‐containing domain of the YpeB protein involved in germination of bacillus spores. Proteins: Structure, Function, and Bioinformatics 2015, 83, (10), 1914-1921. (67) Traag, B. A.; Pugliese, A.; Setlow, B.; Setlow, P.; Losick, R., A conserved ClpP-like protease involved in spore outgrowth in Bacillus subtilis. Mol. Microbiol. 2013, 90, (1), 160166. (68) Plomp, M.; Leighton, T. J.; Wheeler, K. E.; Hill, H. D.; Malkin, A. J., In vitro highresolution structural dynamics of single germinating bacterial spores. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, (23), 9644-9649.


Page 35 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(69) Kuwana, R.; Okuda, N.; Takamatsu, H.; Watabe, K., Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores. J. Biochem. 2006, 139, (5), 887-901. (70) Abhyankar, W.; de Koning, L. J.; Brul, S.; de Koster, C. G., Spore proteomics: the past, present and the future. FEMS Microbiol. Lett. 2014, 358, (2), 137-144. (71) Keijser, B. J. F.; Beek, A. T.; Rauwerda, H.; Schuren, F.; Montijn, R.; Spek, H. v. d.; Brul, S., Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth. J. Bacteriol. 2007, 189, (9), 3624-3634. (72) Bergman, N. H.; Anderson, E. C.; Swenson, E. E.; Niemeyer, M. M.; Miyoshi, A. D.; Hanna, P. C., Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J. Bacteriol. 2006, 188, (17), 6092-6100. (73) Egorov, N.; Loriia, Z.; Vybornykh, S.; Khamrun, R. In [Effect of bacitracin on the sporulation of Bacillus licheniformis 28 KA], Nauchnye Doki. Vyss. Shkoly Biol. Nauki, 1984; 1984; pp 89-91. (74) Ellermeier, C. D.; Hobbs, E. C.; Gonzalez-Pastor, J. E.; Losick, R., A three-protein signaling pathway governing immunity to a bacterial cannibalism toxin. Cell 2006, 124, (3), 549-59. (75) Sinai, L.; Rosenberg, A.; Smith, Y.; Segev, E.; Ben-Yehuda, S., The molecular timeline of a reviving bacterial spore. Mol. Cell 2015, 57, (4), 695-707. (76) Setlow, B.; Setlow, P., Most of the coenzyme A in dormant spores of Bacillus megaterium is in disulfide linkage to protein. Biochem. Biophys. Res. Commun. 1977, 75, (1), 45-50.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

(77) Setlow, B.; Setlow, P., Levels of acetyl coenzyme A, reduced and oxidized coenzyme A, and coenzyme A in disulfide linkage to protein in dormant and germinated spores and growing and sporulating cells of Bacillus megaterium. J. Bacteriol. 1977, 132, (2), 444-52. (78) Setlow, P.; Kornberg, A., Biochemical studies of bacterial sporulation and germination. XXII. Energy metabolism in early stages of germination of Bacillus megaterium spores. J. Biol. Chem. 1970, 245, (14), 3637-44.

For TOC only


Bacillus subtilis Spore Inner Membrane Proteome - Journal of

Recommend Documents