Protection of Bacillus pumilus Spores by Catalases

Aleksandra Checinska,a Malcolm Burbank,b and Andrzej J. Paszczynskia,b

Environmental Science Program, Food Research Center, University of Idaho, Moscow, Idaho, USA,a and University of Idaho and Washington State University School ofFood Science, Food Research Center, Moscow, Idaho, USAb

Bacillus pumilus SAFR-032, isolated at spacecraft assembly facilities of the National Aeronautics and Space Administration JetPropulsion Laboratory, is difficult to kill by the sterilization method of choice, which uses liquid or vapor hydrogen peroxide.We identified two manganese catalases, YjqC and BPUM_1305, in spore protein extracts of several B. pumilus strains by usingPAGE and mass spectrometric analyses. While the BPUM_1305 catalase was present in six of the B. pumilus strains tested, YjqCwas not detected in ATCC 7061 and BG-B79. Furthermore, both catalases were localized in the spore coat layer along with lac-case and superoxide dismutase. Although the initial catalase activity in ATCC 7061 spores was higher, it was less stable over timethan the SAFR-032 enzyme. We propose that synergistic activity of YjqC and BPUM_1305, along with other coat oxidoreducta-ses, contributes to the enhanced resistance of B. pumilus spores to hydrogen peroxide. We observed that the product of the cata-lase reaction, gaseous oxygen, forms expanding vesicles on the spore surface, affecting the mechanical integrity of the coat layer,resulting in aggregation of the spores. The accumulation of oxygen gas and aggregations may play a crucial role in limiting fur-ther exposure of Bacilli spore surfaces to hydrogen peroxide or other toxic chemicals when water is present.

The genus Bacillus is among a few bacterial genera that formendospores to survive adverse conditions. A bacterial endo-

spore is a metabolically dormant form of life that is much moreresistant to environmental challenges than a vegetative cell. Theseinclude heat, desiccation, lack of nutrients, and exposure to UVand gamma radiation, as well as organic chemicals and oxidizingagents (43). This exceptional resistance is attributed to the spore’sstructure and the biochemical properties of its components. Thisfact has been supported primarily by the investigation of Bacillussubtilis and Bacillus anthracis spores (19, 22, 24, 35). However,more information on the spore’s physiology and biochemicalcomposition is essential to understand their contribution to thesurvival strategies of different members of the Bacilli class.

Three compartments of spores are identified on the basis oftheir morphology, i.e., the core, cortex, and coat. The latter is theoutermost layer, which directly interacts with the surroundingenvironment (18, 47). The coat actively limits the passage ofharmful chemicals and is a gate that recognizes germinants (e.g.,water and nutrients) (6, 17). The coat layer is composed largely ofspecific proteins that are not present in vegetative cells (23, 27, 35).For example, the coat protein laccase (CotA) is responsible for theformation and deposition of the brown protective pigment of B.subtilis spores that may protect them from hydrogen peroxide (25,37), while CotE is involved in coat assembly (49). The CotJC pro-tein has a manganese catalase domain, but catalase activity has notbeen reported in B. subtilis spores (44).

Bacillus pumilus SAFR-032 was repeatedly isolated from thespacecraft assembly facilities at the Jet Propulsion Laboratory(JPL), Pasadena, CA (36). Endospores of B. pumilus SAFR-032 areresistant to high concentrations of H2O2 (5% for 60 min) andsimulated Martian UV irradiation (29, 38). The SAFR-032 ge-nome was sequenced, and numerous protein-encoding geneswere identified as putative candidates responsible for the strain’senhanced resistance to the various sterilization treatments tested(21). Understanding its hydrogen peroxide resistance is particu-larly important since H2O2 is used in current and newly developedsterilization methods (29, 46). So far, the major role in peroxideresistance has been attributed to small acid-soluble proteins asso-

ciated with DNA (42). Recently, Bosak et al. (12) described a cyclic“sporulene” compound that is located in the inner membranesurrounding the core and limits hydrogen peroxide diffusion intothe endospore. The coat was also found to protect the spore, asdecoated spores have a lower ability to survive hydrogen peroxidetreatment (40). From the genome data, Gioia et al. (21) reportedthat SAFR-032 has two germination catalase genes, katX1 andkatX2, that are homologs of the B. subtilis germination catalase,the KatX protein. In addition, the SAFR-032 genome has two cat-alase genes, yjqC and bpum_1305, with two manganese ions re-placing heme in the enzyme catalytic centers; hence, the proteinsthey encode are called pseudocatalases or manganese catalases(15). Manganese catalase (YjqC) was found in B. subtilis sporeextract, but its biochemical function was not elucidated (33). B.subtilis also has the ydbD gene, which codes for a putative proteinwith a manganese domain, but there is no counterpart in the B.pumilus SAFR-032 genome (21, 32).

Although multiple manganese catalase genes are present in Ba-cillus species, manganese catalases are not widespread in natureand all of them have a lower affinity for hydrogen peroxide thanheme catalases do (15). The presence of manganese catalases hasbeen reported in Thermoleophilum album (2), Pyrobaculum calidi-fontis (3), Lactobacillus plantarum (31), and Thermus thermophilus(48). Manganese catalases from L. plantarum (9) and T. thermo-philus (4) have been crystallized, and their molecular structuresrevealed that the active site contains dimanganese cations linkedby two oxygen atoms. Both catalases are hexamers of about 30-kDa monomers with a four-helix bundle motif containing con-served glutamate, aspartate, and histidine residues, some of whichare involved in manganese chelation (4, 9).

Received 16 April 2012 Accepted 25 June 2012

Published ahead of print 29 June 2012

Address correspondence to Andrzej J. Paszczynski, andrzej@uidaho.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01211-12

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In this study, we identified two manganese catalase proteins inthe SAFR-032 spore coat that were annotated earlier as YjqC andBPUM_1305 and we detected catalase activity in spores of all ofthe B. pumilus strains investigated. The research presented heresuggests that both proteins may contribute to the spores’ H2O2

resistance. We observed that catalase activity and the product ofthe catalase reaction, gaseous oxygen, both contribute to hydro-gen peroxide resistance. We hypothesize that the oxygen vesiclesform a gaseous barrier on the spore surface that limits hydrogenperoxide diffusion into the spores’ interior, further enhancingtheir resistance to hydrogen peroxide. As vesicles expand andbreak, they cause mechanical disruption of the coat layer, result-ing in adjacent spore aggregation. The aggregate traps oxygen gasand allows spores to float on the liquid surface, further limitingtheir exposure to hydrogen peroxide.

MATERIALS AND METHODSBacterial strains and spore preparation. The B. pumilus strains used inthis work were SAFR-032 (JPL, Pasadena, CA), ATCC 7061 (AmericanType Culture Collection, Manassas, VA), BG-B79 (Department of Bacte-riology, National Veterinary Institute, Uppsala, Sweden), 8A4, 8A6, and14A1 (Bacillus Genetic Stock Center, The Ohio State University, Colum-bus, OH). Vegetative cells were maintained on tryptic soy agar (TSA; BD,Franklin Lakes, NJ) at 37°C.

Spores were prepared by inoculating Difco sporulation medium(DSM) plates (39) with overnight DSM broth cultures. The plates wereallowed to grow for 48 h at 37°C and then placed at a suboptimal growthtemperature of 25°C until �99% of the cells had sporulated. Sporulationand endospore purity were monitored with a phase-contrast microscope(Nikon Eclipse 55i, NIS-147 Elements Br. 3.0). Spores were purified toremove the remaining vegetative cells and cellular debris by using a mod-ified method described earlier (39). Briefly, cells from agar plates werescraped into sterile deionized water and pelleted again by centrifugationafter each washing step (30,000 � g, 10 min, 4°C). The pellets were washedwith a solution containing 1 M KCl and 0.5 M NaCl, with 1 M NaCl, andthen three times with deionized water. Finally, the endospore suspensionwas heat shocked at 80°C for 15 min and stored at 4°C while protectedfrom light. Spore viability was estimated by serial dilutions, plating onTSA, and enumeration of CFU.

Preparation of spore protein extracts. Total spore protein extracts ofintact or decoated spores were prepared by the method described previ-ously (10), with modification as follows. A spore suspension (450 �l,optical density at 580 nm [OD580] of 20) in water was centrifuged(16,000 � g, 10 min, 25°C), and the pellet was suspended in 100 �l of 1�PAGE sample loading buffer (Bio-Rad, Hercules, CA). The suspensionwas boiled for 5 min, homogenized in a bead mill beater (Biospec Prod-ucts Inc., Bartlesville, OK) with 0.1 g of 0.1 mm zirconia/silica (BiospecProducts, Inc., Bartlesville, OK) for a total of 5.5 min (settings: 1 minfollowed by 9 � 30 s at 30-s intervals). The homogenate was boiled for 5min and centrifuged at 16,000 � g for 1 min. Thirty microliters of super-natant was analyzed by 12% SDS-PAGE and visualized by Coomassiebrilliant blue R-250 staining (41).

Decoating of spores and preparation of coat protein extracts. Sporesuspension (450 �l, OD580 of 20) was centrifuged at 16,000 � g for 10 minto remove water. The spore coat was removed using detergent in thepresence of salt and a reducing agent under alkaline conditions as de-scribed by Bagyan and Setlow (6), with the following modifications. Thesupernatant obtained from decoated spores was desalted using SephadexG-25 PD-10 disposable columns (Amersham Biosciences, Uppsala, Swe-den). All fractions containing proteins were combined and freeze-dried(Freeze Zone 6; Labconco Corporation, Kansas City, MO). The lyophi-lized material was suspended in 100 �l of 1� SDS sample loading buffer(Bio-Rad, Hercules, CA). A 30-�l sample of each extract was analyzed by

12% SDS-PAGE and visualized by Coomassie brilliant blue R-250 stain-ing (41).

In-gel digestion. For functional distribution of proteins and single-band identification, PAGE lanes were cut horizontally into slices 1.5 mmwide and then cut into cubes of �1 mm3. In-gel digestion was performedby a modified method described earlier (45). Briefly, each slice wasdestained overnight in 500 �l water containing 50% acetonitrile and 25mM ammonium bicarbonate. The extract was discarded, and gel pieceswere suspended in 200 �l of 100% acetonitrile to dehydrate until thepieces turned white. Gel pieces were rehydrated in a 25 mM solution ofammonium bicarbonate containing 10 mM dithiothreitol and incubatedfor 45 min at 50°C, followed by 30 min of incubation in a 25 mM ammo-nium bicarbonate solution containing 55 mM iodoacetamide (both fromSigma-Aldrich, St. Louis, MO) at room temperature in the dark. Gelpieces were washed with 25 mM ammonium bicarbonate and dehydratedby adding 200 �l of acetonitrile until the pieces turned white. Finally, thegel pieces were rehydrated in trypsin solution (12.5 ng/�l in 25 mM am-monium bicarbonate) and incubated at 37°C for 16 h. The peptides wereeluted from the gel by 50% acetonitrile-water containing 0.1% formicacid, and the extract was concentrated with a SpeedVac. Just before liquidchromatography-tandem mass spectrometry (LC-MS/MS) analysis, drysamples were dissolved in 5% acetonitrile– 0.1% formic acid and clearedby centrifugation at 16,000 � g for 10 min.

Peptide sequencing by LC-MS/MS. Peptides were separated usingreverse-phase LC on a nano-ACQUITY Ultra Performance Liquid Chro-matograph (Waters Corporation, Milford, MA) and analyzed using a Q-TOF Premier tandem mass spectrometry system equipped with a nano-electrospray ionization (nano-ESI) source (7, 8). A 2-�l sample wasloaded onto a Symmetry C18 trap column (0.18 by 20 mm) and thenseparated on a BEH 130 C18 analytical column (0.075 by 200 mm; WatersCorporation, Milford, MA). A 0.1% formic acid solution in H2O was usedas solvent A, and 0.1% formic acid in acetonitrile was used as solvent B.Peptides were trapped by 100% solvent A for 3 min at a 10-�l/min flowrate. Separation was performed at 35°C at 0.4 �l/min under the followingconditions: (i) isocratic for 1 min, 95% solvent A and 5% solvent B; (ii)gradient for the next 44 min of separation, solvent A concentration grad-ually decreased from 95% to 50% and solvent B concentration increasedfrom 5% to 50%; (iii) gradient for the next 5 min, solvent A concentrationdecreased to 10% and solvent B concentration increased to 90%; (iv)isocratic for 5 min, 10% solvent A and 90% solvent B; and (v) gradient for5 min, solvent A concentration increased to 80% and solvent B concen-tration decreased to 20%. The nano-ESI source was set as follows: capil-lary voltage, 3.7 kV; cone voltage, 30 V; source temperature, 120°C; neb-ulizing gas pressure, 0.45 � 105 Pa; collision energy, 5.0 V; detectorvoltage, 1,825 V. The data were acquired in MS survey mode. The follow-ing settings were used for the MS survey: a 300- to 2,000-Da mass range, a1-s scan time, and a 0.1-s interscan delay. The threshold of MS/MS acqui-sition was set to 20 counts per second. The MS/MS acquisition settingswere as followed: a mass range 50 to 2,000 Da, 3 ions selected from a singleMS survey scan, a 2-s scan time, and a 0.05-s interscan delay. [Glu1]-fibrinopeptide B (Sigma-Aldrich, St. Louis, MO) was used as a lockmasswith a 30-s frequency.

Proteomic data analysis. The raw data files were generated to peaklists and saved as pkl files by ProteinLynx Global Server (PLGS) 2.3 soft-ware (Waters, Milford, MA). For protein identification, Mascot software(Matrix Science, London, United Kingdom) was used and files were searchedagainst the B. pumilus SAFR-032 protein sequence database. The database wascreated from the genome sequence information available at the National Cen-ter for Biotechnology Information website (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Bacillus_pumilus_SAFR_032_uid59017/NC_009848.faa). The pa-rameters used by Mascot were as follows. (i) Trypsin was the specificenzyme, (ii) the peptide window tolerance was �0.2, (iii) the fragmentmass tolerance was �0.1, (iv) the number of missed cleavage sites was 1,(v) an individual ion score of �18 was significant for peptide identifica-tion [P � 0.05; the individual ion score is �10 � log(P), where P is the

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probability that the observed match is the random match], and (vi) carb-amidomethyl C was the only fixed amino acid modification set.

Enzyme assays. Catalase activity was determined spectrophotometri-cally (Agilent/Hewlett Packard 8453 UV-Vis spectrophotometer; AgilentTechnologies, Santa Clara, CA) by measuring the decomposition of H2O2

at 240 nm (ε240 � 43.6 M�1 cm�1) (11). The spore suspension (OD580 of�20) was centrifuged at 16,000 � g for 10 min, and water was discarded.The spores were suspended in 1 ml of 50 mM potassium phosphate buffer(pH 7.0) containing 15 mM (0.05%) H2O2 (EMD Chemicals, MerckKGaA, Darmstadt, Germany) so that the OD580 was 1.0. Spore suspensionaliquots of 1 ml were removed at T0 and Tn at n time points and centri-fuged at 16,000 � g for 5 min, and the concentration of hydrogen peroxidein the supernatant was measured.

Optimization of catalase activity. The pH optimum for the catalaseactivity was determined at 25°C by measuring hydrogen peroxide decom-position by spores suspended in the following buffers: 50 mM glycine-HCl at pH 3.6, 50 mM potassium phosphate buffer at pH 5.8, 50 mMpotassium phosphate buffer at pH 7.0, 50 mM potassium phosphate buf-fer at pH 8.0, and 50 mM glycine-NaOH at pH 10.0.

The optimum temperature for the catalase activity in spores was de-termined by using 15 mM (0.05%) H2O2 potassium phosphate buffer atpH 7.0 in a temperature range of 25 to 80°C.

Catalase inhibition assay. One milliliter of spore suspension(OD580 � 1.0) was centrifuged at 16,000 � g for 10 min, and the super-natant was discarded. Spores were suspended in 1 ml of prewarmed 15mM (0.05%) H2O2 in 50 mM potassium phosphate buffer at pH 7.0. Theinhibitors were added to final concentrations of 0.1 mM NH2OH (freshlyprepared before the measurement) and 5 mM NaN3 (both from Sigma, St.Louis, MO) (2). After incubation at 37°C for 60 min with shaking (200rpm), the spore suspension was centrifuged at 16,000 � g for 10 min. TheH2O2 concentration in the supernatant was determined by measuringabsorbance at 240 nm.

Hydrogen peroxide resistance. The assay of resistance to 5% hydro-gen peroxide was performed for all Bacillus strain spores as describedpreviously by using �108 spores ml�1 (29). Also, the same experimentwas performed with the SAFR-032 strain with hydroxylamine as a man-ganese catalase inhibitor (30). To assess the contribution of manganesecatalase to spore survival, 10 �l of 0.1 M NH2OH at pH 6.0 was added tothe 823-�l spore suspension, the suspension was incubated at room tem-perature (25°C) for 10 min, and 167 �l of 30% H2O2 was added. Thesuspension was incubated at 25°C (with shaking at 150 rpm) for 60 min.At the end of the incubation period, a 100-�l sample was diluted 1:10 withbovine catalase (100 �g/ml in phosphate-buffered saline [PBS; Sigma-Aldrich, St. Louis, MO], sterilized with a 0.2-�m-pore-size polyethersul-fone filter; VWR) and then further diluted. The bovine catalase was usedto decompose any remaining hydrogen peroxide that would potentiallyinhibit spore germination. Dilutions of 1:100 and 1:1,000 contained 100�l of bovine catalase solution (100 �g/ml in PBS) because hydroxylaminealso inhibited bovine catalase activity.

Scanning electron microscopy. A 50-�l volume of B. pumilus SAFR-032 spore suspension (OD580 � 20) was added to 450 �l of 2% albumincontaining 15 mM (0.05%) H2O2. After 5 min of incubation on ice, thealiquots were subjected to flash freezing in liquid nitrogen, followed by asolvent replacement procedure (26). The samples frozen in 2-ml micro-tubes were overlaid with 1.5 ml of a �80°C chilled solution of acetonecontaining 5% glutaraldehyde (Electron Microscopy Sciences, Hatfield,PA). After 3 days, the solution was decanted and fresh chilled 5% glutar-aldehyde in acetone was added to the samples. The next day, the pro-cedure was repeated and samples were moved from �80°C to �20°C.In the final step, the glutaraldehyde solution was replaced with anhy-drous acetone and the samples were kept for 2 days at 5°C. The sampleswere dried at room temperature for 2 days. Both samples were carboncoated before examination with an Amray 1830 scanning electron mi-croscope (SEMTech Solutions, Inc., North Billerica, MA).

RESULTSProteomic analysis of total spore protein extracts. B. pumilusSAFR-032 and ATCC 7061 spore protein extracts were analyzedby SDS-PAGE. The protein profiles obtained from the gel sliceextracts of both strains’ spores were similar, with few significantdifferences in the intensities of protein bands. We identified 374and 350 proteins for SAFR-032 and ATCC 7061 (data not shown).The most numerous group in both strains were proteins dedicatedto translation, including proteins of both ribosomal subunits(19%), but there were only a few proteins dedicated to amino acidmetabolism, suggesting that germinating spores use proteinstored in spores as an amino acid source. We found several oxida-tive stress response proteins that may contribute to the higherreactive oxygen species (ROS) resistance of SAFR-032 spores (Ta-ble 1). However, manganese catalase (BPUM_1305), manganesesuperoxide dismutase (SodA), laccase (CotA), catalase KatX1,peroxiredoxin (YkuU), and thiol peroxidase (Tpx) were detectedin both strains. We did not detected manganese catalase (YjqC),superoxide dismutase (SodF), glutathione peroxidase (Bsa), orthioredoxin (TrxA) in our proteomic analysis of ATCC 7061 gelslices. YjqC was very abundant in SAFR-032 spore protein extractbut was not present in that of ATCC 7061 (Fig. 1). The PAGE-predicted molecular mass of YjqC corresponded to the calculatedmass (�31 kDa). The BPUM_1305 protein, with a predicted mo-lecular mass of 33 kDa, was detected in both strains, although aband corresponding to BPUM_1305 was difficult to distinguishbecause of the similarity of the molecular masses of other proteins.However, the band of this protein was possible to visualize whenthe coat protein fractions were extracted and analyzed by PAGEseparately. The two manganese catalases showed 27.6% identity(data not shown), and the peptide amino acid sequences detectedby LC-MS/MS were unique to each of the proteins. In addition tothe main band, YjqC was detected in multiple PAGE slices in theSAFR-032 extract, which did not corresponded to its molecularmass, so the PAGE and mass spectrometry results confirmed theexistence of cross-linking between coat proteins.

We also screened other B. pumilus strains for the presence ofthe manganese catalases. BPUM_1305 was detected in all of thestrains analyzed, and ATCC 7061 had the highest number of pep-tides detected, Mascot score, and sequence coverage (Fig. 2 andTable 2). YjqC was absent from ATCC 7061 and BG-B79 spores.

Localization of oxidative stress response proteins. We per-formed spore decoating by using detergent in the presence of saltand a reducing agent under alkaline conditions to extract the coatproteins of SAFR-032 and ATCC 7061 spores. SDS-PAGE analysisof decoated spores and coat protein extracts revealed that some ofthe proteins are present only in the coat (Fig. 1). Bands from thecoat fraction were excised from the PAGE gel for ESI-MS/MSsequencing and Mascot identification. The distinctive abundantband in native spores of SAFR-032 was identified as a sporulation-related manganese catalase (YjqC). YjqC was not detected in de-coated spore protein extracts, but it was abundant in the coatfraction, confirming the localization of this protein in the sporecoat. BPUM_1305 was identified in the coat fractions of bothstrains. Additional oxidoreductases were identified in the coatbands as well. Table 3 summarizes the results for the oxidativestress response enzymes detected in the SAFR-032 coat fraction.

Catalase enzymatic properties. Catalase activity optimizationwas performed at various pHs and temperatures for SAFR-032

Catalases of Bacillus pumilus Spores

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and ATCC 7061 spore suspensions (data not shown). Catalaseactivity was measured over a pH range of 3.6 to 10.0, and thehighest activity was detected in 50 mM potassium phosphate buf-fer at pH 7.0. Catalase activity was detected from 25 to 80°C, with

optimal temperatures of 70 and 37°C for spores of SAFR-032 andATCC 7061, respectively (Table 4). Moreover, catalase activitywas detected in the spores of all of the strains at all of the temper-atures tested. The ATCC 7061 strain had the highest activity at

TABLE 1 Oxidative stress response proteins identified in B. pumilus SAFR-032 spore extract

Identified proteinaNCBI accessionno.

Expected/calculatedmolecular masses (Da)

% sequencecoveragea

Mascotscorea,b Peptide(s)a

Catalase KatX1 YP_001488916.1 60,947/67,350 4 55 K.FYTEEGNWDLVGNNLK.I, R.TFSYSDTQR.Y

5 53 R.DALKFPDLVHAFKPDPVTNR.Q, K.NLVNTLATCQK.D

Glutathione peroxidase (BsaA) YP_001487155.1 18,227/17,470 7 19 K.ELESSIIALLDK

Iron superoxide dismutase (SodF) YP_001487090.1 32,422/27,390 6 55 R.YDALEPFISK.E, R.LEILQAER.H

Manganese catalase YP_001486548.1 33,407/37,770 21 60 R.LLIDLPRPDHPDANGAAAVQELLGGK.F, K.ALEVATGVDVGK.M

12 93 K.GSHPIDGKPLTVIEGTPQGAPVPDYR.E

Manganese superoxide dismutase (SodA) YP_001487459.1 22,397/22,590 14 117 K.LEITSTPNQDSPLTEGK.T, K.FKSDFAAAAAGR.F,K.HHNTYVTNLNK.A,

12 80 R.FGSGWAWLVVNNGK.L

Multicopper oxidase/laccase (CotA) YP_001485796.1 58,882/40,280 4 117 K.VWPYLEVEPR.K

2 65 R.LGSVEVWSIVNPTR.G

Peroxiredoxin (YkuU) YP_001486562.1 20,578/22,590 26 112 K.YPLAADTNHTVSR.E, R.EYGVLIEEEGIALR.G

31 179 R.VLQALQTGGLCPANWKPGQK.T,R.GLFIINPEGELQYQTVFHNNIGR.D

Sporulation-related manganese catalase (YjqC) YP_001487572.1 31,115/31,150 38 438 K.ELQYEAKPSKPDPLYAK.K, K.YIIASGNLMADFR.A,R.ANLNAESQGR.L, R.GVKDMLSFLIAR.D, K.DMLSFLIAR.D,R.DTYHQNMWIAAIK.E, R.DTYHQNMWIAAIKELEER.E,K.ELEEREGDVVVPTTFPR.S

Thiol peroxidase (Tpx) YP_001487800.1 17,954/17,470 21 60 R.WCGANGIENVETLSDHR.D, K.VVYTEYVSEATNHPNYEK.A

43 199 K.VGEQAPDFTVLTNSLGEVSLSDLTGK.V,K.VTIISVIPSIDTGVCDAQTR.R,R.FNEEAAGLGPVNIYTISADLPFAQAR.W

Thioredoxin (TrxA) YP_001487729.1 11,445/8,610 32 72 K.IDVDDNQETAGK.Y, K.YGVMSIPTLLVLK.D, K.EALAELVNK.H

a Bold font indicates results common to both SAFR-032 and ATCC 7061, while lightface font and italics indicate results for SAFR-032 and ATCC 7061, respectively.b Mascot search probability-based MOWSE score. The ion score is �10 � log(P), where P is the probability that the observed match is a random event. Individual ion scores of�18 indicate identity (P � 0.05). Protein scores are derived from ion scores as a nonprobabilistic basis for ranking of proteins hits.

FIG 1 SDS-PAGE analysis of proteins extracted from intact and decoated B. pumilus spores. Lanes: ST, molecular mass standards (sizes in kDa are on the left);1, SAFR-032; 2, ATCC 7061; 1A and 2A, intact spore protein extracts; 1B and 2B, decoated spore protein extracts; 1C and 2C, coat protein extracts. Lanes 1A and1B were cut into �1.5-mm slices from the bottom to the top for protein identification by LC-MS/MS. A similar amount of protein (�10 �g) was loaded into eachwell. The black arrowheads indicate a sporulation-related manganese catalase (YjqC). The white arrowheads indicate the second manganese catalase(BPUM_1305). The numbers indicate the excised PAGE bands.

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37°C, while the spores of the other strains showed similar activityat 70°C, which suggests that this enzyme is thermostable.

Hydroxylamine is a good inhibitor of manganese catalase at aconcentration of 0.1 mM (30). The catalase activities were inhib-ited by hydroxylamine in all spores of B. pumilus strains. Sodiumazide was used as an inhibitor of heme catalase at a concentrationof 5 mM (28). SAFR-032 catalase activity was inhibited by 75%and that of ATCC 7061 was inhibited by 83% by incubation withhydroxylamine, while the activities of both strains decreased byonly 12% in the presence of sodium azide (Table 4).

Spore resistance to hydrogen peroxide. A small percentage ofthe spores of all of the B. pumilus strains (�108 ml�1), exceptATCC 7061, survived 1 h of incubation in 5% H2O2. We then used�5.63 � 108 ml�1 SAFR-032 spores for the enumeration test withhydroxylamine. The survival of spores treated with 5% H2O2 was

0.12%; however, in the presence of 0.1 mM hydroxylamine, sporesurvival decreased to 0.0046%. In the control containing 0.1 mMhydroxylamine, approximately 62% of the SAFR-032 spores sur-vived, suggesting some toxicity of hydroxylamine for B. pumilusspores (Fig. 3).

We also found that the catalase activity of SAFR-032 decreasedby 53% and that of ATCC 7061 decreased by 89% after a 60-minexposure to 5% hydrogen peroxide (Table 4). We know fromprevious studies (29) and work performed in our laboratory thatATCC 7061 spores do not survive a 60-min exposure to 5% H2O2.

The addition of albumin, followed by rapid freezing and sol-vent replacement, allowed the visualization and preservation ofoxygen vesicles deforming the spore coat as they emerge from thespore surface in the presence of hydrogen peroxide. During thegradual temperature increase, the acetone solubilized the ice inthe sample and glutaraldehyde-albumin copolymer fixed frozenoxygen vesicles on the B. pumilus spore surface (Fig. 4B and D).Control spore samples (Fig. 4A and C) without hydrogen peroxideshowed some deformations of the spore surface, but the differencefrom H2O2-treated samples is profound.

DISCUSSION

The research presented here suggests that catalase activity detectedin the B. pumilus spore coat contributes to the spore’s resistance tohydrogen peroxide exposure. Genes for sporulation-related man-ganese catalase (YjqC) and other oxidative stress response pro-teins were previously identified in the genome of B. pumilusSAFR-032 (21).

To verify the genomic information, we investigated SAFR-032and ATCC 7061 spore protein extracts. The latter strain served asa control since it is more sensitive to the 5% hydrogen peroxidetreatment (29). Proteomic analysis of these two strains revealedthat manganese catalase (BPUM_1305) was present in both

FIG 2 SDS-PAGE analysis of B. pumilus spore protein extracts. Lanes: ST, molecular mass standards (sizes in kDa are on the left); 1, SAFR-032; 2, ATCC 7061;3, BG-B79; 4, 8A4; 5, 8A6; 6, 14A1. A similar amount of protein was loaded into each 12% SDS-PAGE well. The rectangles (�1 cm) were excised, digested in thegel with trypsin, and sequenced. The arrowheads indicate the sporulation-related manganese catalase (YjqC). BPUM_1305 was identified in all of the gel extractstested.

TABLE 2 Manganese catalases identified by SDS-PAGE and LC-MS/MSin B. pumilus spores

Strain

YjqC BPUM_1305

Mascotscorea

%coverage

No. ofpeptides

Mascotscorea

%coverage

No. ofpeptides

SAFR-032 461 34 9 119 16 3ATCC 7061 —b — — 199 23 5BG-B79 — — — 154 15 48A4 142 20 5 43 4 18A6 248 25 6 28 4 114A1 269 20 5 15 8 1a Mascot search probability-based MOWSE score. The ion score is �10 � log(P),where P is the probability that the observed match is a random event. Individual ionscores of �18 indicate identity (P � 0.05). Protein scores are derived from ion scores asa nonprobabilistic basis for ranking of proteins hits.b —, not detected.

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strains, while the sporulation-related manganese catalase protein(YjqC) was found only in SAFR-032. Generally, our goal was tocompare the model organism (SAFR-032) with the control(ATCC 7061), as another research group did (29). We includedother B. pumilus strains for comparison of hydrogen peroxideresistance. Catalase activity was detected in other strains, andthose strains survived treatment with 5% hydrogen peroxide. Fur-ther investigation revealed the presence of BPUM_1305 inother B. pumilus strains tested. In addition to ATCC 7061spores, YjqC was also absent from B. pumilus BG-B79, a strainisolated from a biogas plant in Sweden (5). Interestingly, theBG-B79 strain has 100% 16S rRNA sequence similarity toSAFR-032 on the basis of the sequences deposited in the NCBIdatabase. This result indicates that proteomic analysis can dis-tinguish differences between strains that genomic analysis can-not. The two manganese catalases have features in commonwith enzymes characterized earlier. The YjqC catalase has ex-tensive homology to crystallized L. plantarum catalase (46.8%)(9), while the BPUM_1305 amino acid sequence is more simi-lar to that of T. thermophilus catalase (54.5%) (4). The highoptimum temperature of the enzyme may suggest a horizontal

gene transfer from a thermophile. The molecular masses ofYjqC and BPUM_1305 are 31 and 33 kDa, respectively. Ourresults indicated that BPUM_1305 may be common in B. pumi-lus spores, while YjqC may be more strain specific.

Though it is accepted that the coat protects spores fromhydrogen peroxide, the exact mechanism has not been deter-mined (40). CotA present in B. subtilis 168 spores was identi-fied as a laccase (multicopper oxidase) and found to protectspores from hydrogen peroxide (25, 37). We detected CotA inthe whole spore extracts and the coat fractions of SAFR-032and ATCC 7061. However, B. pumilus spores are white and lackthe brown pigment produced by laccase, which is a character-istic trait of B. subtilis spores. The proteomic analyses of SAFR-032 coat proteins confirmed both catalase proteins’ localiza-tion in the coat. Furthermore, the genomic analysis (21) andour proteomic data revealed other oxidoreductase proteins incoat fraction as potential contributors to resistance to otherROS (Table 3). Our results strongly indicate that the SAFR-032spore coat is filled with oxidative stress response proteins. TheYjqC protein was detected previously in B. subtilis spore pro-tein extracts by Kuwana et al. (33), and Abhyankar et al. (1)found that YjqC is located in the outer spore coat of this spe-cies. In B. anthracis, superoxide dismutases are located in thespore exosporium, which is a characteristic spore structurepresent in most pathogenic members of the class Bacilli (16).The presence of superoxide dismutases in B. anthracis sporesfacilitates their virulence-enhancing survival in the macro-phage vacuole. Analogously, the presence of large amounts ofmanganese catalase enzymes in the outer layers in SAFR-032spores might help this strain to survive the multiple hydrogenperoxide treatments used in clean rooms at the National Aero-nautics and Space Administration (NASA) JPL. Our observa-tions suggest that although BPUM_1305 has higher activity, itis less stable over reaction time than YjqC. Therefore, both

TABLE 3 Oxidative stress response proteins identified in B. pumilus SAFR-032 coat fraction

Identified protein/band no.aNCBI accessionno.

Expectedmolecularmass (Da)

Sequencecoverage(%)c

Mascotscoreb,c Peptidesa

Iron superoxide dismutase/4, 9 YP_001487090.1 32,422 9 195 R.YDALEPFISK.E, K.HHQSYVDGLNQAELALK.R,R.QIEQSFESYDAFK.A, R.LEILQAER.H, K.AAYVDR.W

Outer spore coat protein YP_001485796.1 58,882 7 138 K.FVDELPIPEVAEPVK.K, R.DFEATGPFFER.E, K.VWPYLEVEPR.K(CotA)/multicopper oxidase(laccase)/3, 5, 8; 3, 7, 9

6 65 R.ILNASNTR.T, R.TLTLTGTQDK.Y, R.LGSVEVWSIVNPTR.G

Manganese catalase/6, 8; 5, 6 YP_001486548 0.1 33,407 36 329 R.VYEMTDHPTAR.E, R.EMIGYLLVR.G, K.ALEVATGVDVGK.M,K.LYTFSDTDYKDINK.I, R.GGVHVVAYAK.A

24 215 K.YFDSAR.K, R.KFEDQNIHTK.L, K.LYTFSDTDYK.D,K.GSHPIDGKPLTVIEGTPQGAPVPDYR.E, R.ELPEEFAPGISK.E,R.LLIDLPRPDHPDANGAAAVQELLGGK.F

Sporulation-related manganesecatalase (YjqC)/1, 2, 3, 4, 5,6, 7

YP_001487572.1 31,115 49 611 MFYHIK.E, K.ELQYEAKPSKPDPLYAK.K R.LLDKAPVK.E,K.YIIASGNLMADFR.A, R.ANLNAESQGR.L, R.LYEMTDDR.GK.DMLSFLIAR.D, R.DTYHQNMWIAAIK.E,R.EGDVVVPTTFPR.S, K.QQVSYDLFNFSR.G R.SMDGKGEFR.Y,R.YISAPVVFGSAPK.L, K.ELFNTPK.K

a Results common to both SAFR-032 and ATCC 7061 are in bold, while results for SAFR-032 and ATCC 7061 are in lightface and italics, respectively.b Mascot search probability-based MOWSE score. The ion score is �10 � log(P), where P is the probability that the observed match is a random event. Individual ions score �18indicate identity (P � 0.05). Protein scores are derived from ions scores as a nonprobabilistic basis for ranking proteins hits.c Normal font and italics indicate results for SAFR-032 and ATCC 7061, respectively.

TABLE 4 Catalase specific activities detected in B. pumilus sporesuspensions

Conditionsa

Mean catalase activity (nmolmin�1 ml�1 OD580

�1) � SD

SAFR-032 ATCC 7061

37°C 112 � 1 224 � 370°C 180 � 3 208 � 837°C, 60-min H2O2 treatment 53 � 1 25 � 537°C, 0.1 mM NH2OH 24 � 3 35 � 437°C, 5 mM NaN3 87 � 7 186 � 10a Each in 15 mM H2O2.

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catalases’ activities may contribute to the higher resistance ofSAFR-032 spores to hydrogen peroxide. Similar conclusionsabout the additive effect of enzyme activities were reachedwhen superoxide dismutases of B. anthracis spores were inves-tigated (16). Knocking out two out of four superoxide dismu-tases surprisingly led to an increase in superoxide dismutaseenzyme activity, presumably to compensate for the loss of thedeleted dismutases. There was a total loss of activity when allfour superoxide dismutases were deleted. Therefore, the in-creased catalase activity of ATCC 7061 spores that we observedmay compensate for the absence of YjqC, although spores ofthis strain are not as resistant because of the enzymatic insta-bility of BPUM_1305.

We were not able to obtain yjqC or bpum_1305 knockoutmutants by using a method described previously for B. pumilusstrains that involves the use of high-voltage electroporationtransformation (13). As B. pumilus SAFR-032 lacks comS, acompetence gene that is essential for competence regulation inB. subtilis (20), it was not possible to transform the bacteriumthrough natural competence. Therefore, we took a biochemicalapproach and used hydroxylamine, a manganese catalase-spe-cific inhibitor. Hydrogen peroxide disproportionation by B.pumilus spore catalase was strongly inhibited by hydroxyl-amine, which was selected as the strongest manganese catalaseinhibitor in a previous study (30). Sodium azide weakly inhib-ited the activity further, confirming that the heme catalasesmay contribute to spore resistance as well. Also, the ability of B.pumilus SAFR-032 spores to survive was weaker in the presenceof hydroxylamine, indicating the protective role of manganesecatalases against hydrogen peroxide.

We conclude that YjqC and BPUM_1305 take part in the de-fense of B. pumilus against hydrogen peroxide. The localization ofthese two enzymes in the spore coat contributes to the dynamiccontrol of hydrogen peroxide influx into the spore’s interior. Inaddition, evolving oxygen as a final product of the catalase reac-tion may further protect spores. Our hypothesis is supported by

the scanning electron microscope observation of SAFR-032 sporestreated with hydrogen peroxide (Fig. 4). The solvent replacementmethod allowed us to preserve the ice-trapped oxygen vesicles thatemerge from the spore’s surface in a solution containing hydrogenperoxide. We conclude that oxygen-filled vesicles accumulatingwithin the spore coat form a gas barrier that limits the diffusion ofperoxide to the spores’ interior so that not only do catalases enzy-matically destroy hydrogen peroxide but the accumulated reac-tion product, oxygen, is retained on the spore surface, providingsome additional protection. Our findings also suggest that trappedoxygen provides buoyancy that helps the spores to escape full sub-mersion in H2O2. This is especially evident when pelleted sporesfloat to the surface of water containing more than 1% hydrogenperoxide. Previously, other researchers observed a “doughnut-like” deformation of spores of Bacillus sp. strain 34hs1 (34) and anundulated surface of spores of B. pumilus FO-036b after a 60-minexposure to 5% hydrogen peroxide (29), confirming our observa-tions that exposure to H2O2 mechanically disturbs the spore coat(Fig. 4E). The sterilization method developed recently by ourgroup uses supercritical fluid carbon dioxide containing 3.3% wa-ter and 0.1% hydrogen peroxide (vol/vol/vol) to achieve a 4- to8-log reduction of the viability of various microbial species, in-cluding SAFR-032 spores, and kills dry spores of SAFR-032 withgreater efficiency (14, 46) than wet spores. We believe that dryspores lack the gaseous oxygen barrier formation that protects wetspores in an aqueous environment. Also, the sterilization withhydrogen peroxide vapor used by the NASA JPL kills sporesthrough the formation of a gaseous H2O2 plasma cloud in anelectromagnetic field (29), thus preventing spores from formingprotective oxygen vesicles.

We observed that SAFR-032 is one of the most efficient sporeformers we have used in our laboratory and it is able to form a veryrigid biofilm that floats on the surface of liquid medium (14).There are still some questions to be answered. What is the physi-ological role of oxygen retention on the spore surface? Are cata-lases involved in the change in buoyancy of SAFR-032 vegetative

FIG 3 Viability of SAFR-032 spores (5.63 � 108) after 1 h of exposure to 5% hydrogen peroxide. The bars represent means of three replicates, and the errorbars represent the standard deviations. Bars: 1, untreated control (standard deviation [SD], �1.2 � 108); 2, treatment with 5% H2O2 (SD, �5.6 � 105);3, control treated with 0.1 mM NH2OH (SD, �1.3 � 108); 4, treatment with 0.1 mM NH2OH for 10 min before exposure to 5% H2O2 (SD, �7 � 103).

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FIG 4 Spores of B. pumilus SAFR-032 visualized by scanning electron microscopy after treatment with H2O2. In order to preserve oxygen vesicles, the reactionwas performed in 2% albumin and a freezer substitution solvent replacement procedure (acetone containing 5% glutaraldehyde) was used after the rapid freezingof samples in liquid nitrogen. Shown are control spores (magnifications: A, �20,000; C, �50,000) and spores treated with H2O2 (magnifications: B, �20,000; D,�50,000; E, �60,000; F, �110,000). The white arrows indicate oxygen gas vesicles fixed on the spore surface during hydrogen peroxide treatment. The spores insample E were suspended in water, treated with H2O2, and freeze-dried; no albumin or glutaraldehyde was used. Panel E provides evidence that treatment withH2O2 causes part of the spore surface to be dislodged and that the resulting membrane fragments bind spores together.

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cells before they float, form a biofilm, and sporulate? We believethat SAFR-032 has emerged as an important microbial model forfurther studies of how members of the class Bacilli respond totoxic environmental conditions.

ACKNOWLEDGMENTS

This work was supported by NASA EPSCoR cooperative agreementsNX08AT68A and NN11AQ30A. Any opinions, findings, and conclusionsor recommendations expressed in this report are ours and do not neces-sarily reflect the views of NASA.

We thank Kasthuri Venkateswaran for providing the B. pumilusstrains SAFR-032 and ATCC 7061, and we thank Elizabeth Bagge forproviding B. pumilus BG-B79. We thank Thomas Williams for takingelectron microscopic pictures at the University of Idaho electron micros-copy analytical center.

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