Restricting Fermentative Potential by Proteome Remodeling
2012; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês
10.1074/mcp.m111.013102
ISSN1535-9484
AutoresGérémy Clair, Jean Armengaud, Catherine Duport,
Tópico(s)Probiotics and Fermented Foods
ResumoPathogenesis hinges on successful colonization of the gastrointestinal (GI) tract by pathogenic facultative anaerobes. The GI tract is a carbohydrate-limited environment with varying oxygen availability and oxidoreduction potential (ORP). How pathogenic bacteria are able to adapt and grow in these varying conditions remains a key fundamental question. Here, we designed a system biology-inspired approach to pinpoint the key regulators allowing Bacillus cereus to survive and grow efficiently under low ORP anoxic conditions mimicking those encountered in the intestinal lumen. We assessed the proteome components using high throughput nanoLC-MS/MS techniques, reconstituted the main metabolic circuits, constructed ΔohrA and ΔohrR mutants, and analyzed the impacts of ohrA and ohrR disruptions by a novel round of shotgun proteomics. Our study revealed that OhrR and OhrA are crucial to the successful adaptation of B. cereus to the GI tract environment. Specifically, we showed that B. cereus restricts its fermentative growth under low ORP anaerobiosis and sustains efficient aerobic respiratory metabolism, motility, and stress response via OhrRA-dependent proteome remodeling. Finally, our results introduced a new adaptive strategy where facultative anaerobes prefer to restrict their fermentative potential for a long term benefit. Pathogenesis hinges on successful colonization of the gastrointestinal (GI) tract by pathogenic facultative anaerobes. The GI tract is a carbohydrate-limited environment with varying oxygen availability and oxidoreduction potential (ORP). How pathogenic bacteria are able to adapt and grow in these varying conditions remains a key fundamental question. Here, we designed a system biology-inspired approach to pinpoint the key regulators allowing Bacillus cereus to survive and grow efficiently under low ORP anoxic conditions mimicking those encountered in the intestinal lumen. We assessed the proteome components using high throughput nanoLC-MS/MS techniques, reconstituted the main metabolic circuits, constructed ΔohrA and ΔohrR mutants, and analyzed the impacts of ohrA and ohrR disruptions by a novel round of shotgun proteomics. Our study revealed that OhrR and OhrA are crucial to the successful adaptation of B. cereus to the GI tract environment. Specifically, we showed that B. cereus restricts its fermentative growth under low ORP anaerobiosis and sustains efficient aerobic respiratory metabolism, motility, and stress response via OhrRA-dependent proteome remodeling. Finally, our results introduced a new adaptive strategy where facultative anaerobes prefer to restrict their fermentative potential for a long term benefit. Facultative anaerobes encompass all the major pathogens of the human gastrointestinal (GI) 1The abbreviations used are:GIgastrointestinalOhrorganic hydroperoxide resistanceOGDC2-oxo-acid dehydrogenase complexORPoxidoreduction potentialRACErapid amplification of cDNA endsROSreactive oxygen speciesOGDHC2-oxo acid dehydrogenase multienzyme complex. 1The abbreviations used are:GIgastrointestinalOhrorganic hydroperoxide resistanceOGDC2-oxo-acid dehydrogenase complexORPoxidoreduction potentialRACErapid amplification of cDNA endsROSreactive oxygen speciesOGDHC2-oxo acid dehydrogenase multienzyme complex. tract. The GI tract poses several challenges for pathogens because it is sliced into distinct niches with different oxygen concentrations and different oxidoreduction potentials (ORP) (1Marteyn B. Scorza F.B. Sansonetti P.J. Tang C. Breathing life into pathogens: The influence of oxygen on bacterial virulence and host responses in the gastrointestinal tract.Cell. Microbiol. 2011; 13: 171-176Crossref PubMed Scopus (72) Google Scholar, 2Marteyn B. West N.P. Browning D.F. Cole J.A. Shaw J.G. Palm F. Mounier J. Prévost M.C. Sansonetti P. Tang C.M. Modulation of Shigella virulence in response to available oxygen in vivo.Nature. 2010; 465: 355-358Crossref PubMed Scopus (236) Google Scholar, 3Moriarty-Craige S.E. Jones D.P. Extracellular thiols and thiol/disulfide redox in metabolism.Annu. Rev. Nutr. 2004; 24: 481-509Crossref PubMed Scopus (334) Google Scholar). Although much is known about gene expression and metabolism under fully aerobic and high ORP anaerobic condition (4Kramer G. Sprenger R.R. Nessen M.A. Roseboom W. Speijer D. de Jong L. de Mattos M.J. Back J. de Koster C.G. Proteome-wide alterations in Escherichia coli translation rates upon anaerobiosis.Mol. Cell. Proteomics. 2010; 9: 2508-2516Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 5Sawers G. The aerobic/anaerobic interface.Curr. Opin. Microbiol. 1999; 2: 181-187Crossref PubMed Scopus (82) Google Scholar), our knowledge about the physiological impact of low ORP anoxic conditions and the underlying molecular mechanisms is scarce (6Duport C. Zigha A. Rosenfeld E. Schmitt P. Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system.J. Bacteriol. 2006; 188: 6640-6651Crossref PubMed Scopus (67) Google Scholar). gastrointestinal organic hydroperoxide resistance 2-oxo-acid dehydrogenase complex oxidoreduction potential rapid amplification of cDNA ends reactive oxygen species 2-oxo acid dehydrogenase multienzyme complex. gastrointestinal organic hydroperoxide resistance 2-oxo-acid dehydrogenase complex oxidoreduction potential rapid amplification of cDNA ends reactive oxygen species 2-oxo acid dehydrogenase multienzyme complex. Bacillus cereus is a notorious food-borne pathogenic bacterium. Like the closely related Bacillus anthracis (7Klee S.R. Brzuszkiewicz E.B. Nattermann H. Bruggemann H. Dupke S. Wollherr A. Franz T. Pauli G. Appel B. Liebl W. Couacy-Hymann E. Boesch C. Meyer F.D. Leendertz F.H. Ellerbrok H. Gottschalk G. Grunow R. Liesegang H. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids.PLoS One. 2010; 5: e10986Crossref PubMed Scopus (115) Google Scholar, 8Kolstø A.B. Tourasse N.J. Økstad O.A. What sets Bacillus anthracis apart from other Bacillus species?.Annu. Rev. Microbiol. 2009; 63: 451-476Crossref PubMed Scopus (186) Google Scholar), it is a recognized agent of GI tract infections (9Bishop B.L. Lodolce J.P. Kolodziej L.E. Boone D.L. Tang W.J. The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection.Biochem. Biophys. Res. Commun. 2010; 394: 254-259Crossref PubMed Scopus (24) Google Scholar, 10Kim J.S. Bokoch G.M. 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Proteomics. 2010; 9: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Thus, how B. cereus adapts its catabolism and regulates its proteome across the range of physiologically relevant ORP and oxygen availabilities is important for its survival and growth. In B. cereus, anaerobic and aerobic catabolism work through different pathways. In the presence of oxygen, reducing equivalents generated by glycolysis and the TCA cycle (NADH and FADH) are reoxidized by the respiratory chain, resulting in the buildup of a proton motive force and the subsequent synthesis of ATP. Acetate excretion can occur aerobically when carbon flux into the cells exceeds TCA cycle capacity. In the absence of oxygen or other external electron acceptors (such as nitrate), NADH is reoxidized in terminal step fermentative reactions from pyruvate. When grown in pH-controlled anaerobic batch cultures (pH ∼7), the fermentative by-products of B. cereus are lactate, succinate, acetate, and ethanol. The relative rate of formation of these products is influenced by the ORP of the growth medium, which directly impacts the intracellular redox state (6Duport C. Zigha A. Rosenfeld E. Schmitt P. Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system.J. Bacteriol. 2006; 188: 6640-6651Crossref PubMed Scopus (67) Google Scholar, 13Duport C. Thomassin S. Bourel G. Schmitt P. Anaerobiosis and low specific growth rates enhance hemolysin BL production by Bacillus cereus F4430/73.Arch. Microbiol. 2004; 182: 90-95Crossref PubMed Scopus (50) Google Scholar, 14Laouami S. Messaoudi K. Alberto F. Clavel T. Duport C. Lactate dehydrogenase A promotes communication between carbohydrate catabolism and virulence in Bacillus cereus.J. Bacteriol. 2011; 193: 1757-1766Crossref PubMed Scopus (20) Google Scholar, 15Messaoudi K. Clavel T. Schmitt P. Duport C. Fnr mediates carbohydrate-dependent regulation of catabolic and enterotoxin genes in Bacillus cereus F4430/73.Res. Microbiol. 2010; 161: 30-39Crossref PubMed Scopus (18) Google Scholar, 16Rosenfeld E. Duport C. Zigha A. Schmitt P. Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain.Can. J. Microbiol. 2005; 51: 149-158Crossref PubMed Scopus (60) Google Scholar, 17Zigha A. Rosenfeld E. Schmitt P. Duport C. Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression.Arch. Microbiol. 2006; 185: 222-233Crossref PubMed Scopus (44) Google Scholar, 18Zigha A. Rosenfeld E. Schmitt P. Duport C. The redox regulator Fnr is required for fermentative growth and enterotoxin synthesis in Bacillus cereus F4430/73.J. Bacteriol. 2007; 189: 2813-2824Crossref PubMed Scopus (52) Google Scholar). The intracellular redox state is dependent on the degree of oxidation or reduction of various redox-active species. Among these species, NAD(P)H/NAD(P) and low molecular weight thiol/disulfide (SH/S-S) compounds are of special significance because they mediate redox regulation through direct effects on proteins. The activities of many metabolic enzymes depend on the steady-state NAD(P)H/NAD(P) ratio, whereas proteins with essential SH/S-S groups can be regulated by post-translational modification involving cellular thiols and disulfides. The main low molecular weight thiols in B. cereus ATCC 14579 cells are bacillithiol and cysteine (19Newton G.L. Rawat M. La Clair J.J. Jothivasan V.K. Budiarto T. Hamilton C.J. Claiborne A. Helmann J.D. Fahey R.C. Bacillithiol is an antioxidant thiol produced in Bacilli.Nat. Chem. Biol. 2009; 5: 625-627Crossref PubMed Scopus (213) Google Scholar). On the other hand, the NAD(P)H/NAD(P) and SH/S-S ratios are closely related to cellular levels of reactive oxygen species (ROS). NAD(P)H and thiols contribute to ROS formation via NAD(P)H oxidase activity and thiyl radicals, respectively, whereas thiol-dependent peroxidase-catalyzed reactions use NAD(P)H and thiols to scavenge hydrogen peroxide and limit ROS formation. At low concentrations, ROS play essential roles in redox homeostasis. Their high reactivity allows fast local oxidation of protein (i.e. protein disulfide bond formation) in the reducing cytoplasm. However, this high reactivity also leads to cell damage if cellular capacity to scavenge ROS is compromised (20Antelmann H. Helmann J.D. Thiol-based redox switches and gene regulation.Antioxid. Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar, 21Chi B.K. Gronau K. Maeder U. Hessling B. Becher D. Antelmann H. 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Duport C. ResDE-dependent regulation of enterotoxin gene expression in Bacillus cereus: Evidence for multiple modes of binding for ResD and interaction with Fnr.J. Bacteriol. 2009; 191: 4419-4426Crossref PubMed Scopus (28) Google Scholar) and Fnr (a one-component system) (18Zigha A. Rosenfeld E. Schmitt P. Duport C. The redox regulator Fnr is required for fermentative growth and enterotoxin synthesis in Bacillus cereus F4430/73.J. Bacteriol. 2007; 189: 2813-2824Crossref PubMed Scopus (52) Google Scholar, 25Esbelin J. Jouanneau Y. Armengaud J. Duport C. ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.J. Bacteriol. 2008; 190: 4242-4251Crossref PubMed Scopus (31) Google Scholar), and the catabolite control protein A (CcpA) (26van der Voort M. Kuipers O.P. Buist G. de Vos W.M. Abee T. Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereus ATCC 14579.BMC Microbiol. 2008; 8: 62Crossref PubMed Scopus (42) Google Scholar). Despite the knowledge currently gathered throughout these studies, we still lack a global quantitative understanding of the contribution of the multilevel regulatory events that govern metabolic modes (anaerobic fermentation versus aerobic respiration). One potential strategy for tackling this issue is to use a system biology approach (27Armengaud J. Proteogenomics and systems biology: Quest for the ultimate missing parts.Expert Rev Proteomics. 2010; 7: 65-77Crossref PubMed Scopus (50) Google Scholar), i.e. assessing the proteome components with high throughput techniques, rebuilding the main metabolic circuits, inactivating key regulators, and verifying their impacts. Here, we compare B. cereus ATCC 14579 proteomes established for cells grown under low ORP conditions (considered to mimic those encountered in the intestinal lumen (3Moriarty-Craige S.E. Jones D.P. Extracellular thiols and thiol/disulfide redox in metabolism.Annu. Rev. Nutr. 2004; 24: 481-509Crossref PubMed Scopus (334) Google Scholar)), oxic conditions (considered to mimic those encountered in zones adjacent to the mucosal surface (2Marteyn B. West N.P. Browning D.F. Cole J.A. Shaw J.G. Palm F. Mounier J. Prévost M.C. Sansonetti P. Tang C.M. Modulation of Shigella virulence in response to available oxygen in vivo.Nature. 2010; 465: 355-358Crossref PubMed Scopus (236) Google Scholar)), and intermediary high ORP anoxic conditions. This comparative analysis identified OhrA, a thiol-dependent peroxidase-like protein, as a putative low ORP sensor. OhrA is encoded by ohrA, which is the second cistron of the ohrR-ohrA operon in B. cereus ATCC 14579. The ohrR cistron is predicted to encode a MarR-like repressor of ohrA. The ΔohrR and ΔohrA mutants were constructed and analyzed for their effect on growth, glucose catabolism, and proteome composition under low and high ORP anaerobiosis and aerobiosis. Our results indicate that OhrA and OhrR are major factors controlling glucose catabolism and global proteome in B. cereus under both anaerobic fermentative and aerobic respiratory conditions. We discovered that the OhrRA system restricts B. cereus fermentative capacity under low ORP anoxic growth conditions while boosting motility, respiratory metabolism and resistance against external ROS. We conclude that the OhrRA system may function in B. cereus as a major redox signal transduction system to co-regulate catabolism, motility, and oxidoreductive stress resistance in an environment with varying oxygen and ORP conditions such as that encountered in the human intestine. Finally, our findings provide detailed insights into the metabolic events required to maximize survival in stressful environments characterized by strong temporal and/or spatial fluctuations in ORP and oxygen levels. Wild type, ΔohrA, and ΔohrR B. cereus ATCC 14579 (28Ivanova N. Sorokin A. Anderson I. Galleron N. Candelon B. Kapatral V. Bhattacharyya A. Reznik G. Mikhailova N. Lapidus A. Chu L. Mazur M. Goltsman E. Larsen N. D'Souza M. Walunas T. Grechkin Y. Pusch G. Haselkorn R. Fonstein M. Ehrlich S.D. Overbeek R. Kyrpides N. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.Nature. 2003; 423: 87-91Crossref PubMed Scopus (659) Google Scholar) strains were grown at 37 °C in synthetic MOD medium supplemented with 30 mm glucose as carbon source, as previously described (12Clair G. Roussi S. Armengaud J. Duport C. Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions.Mol. Cell. Proteomics. 2010; 9: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The pH of these cultures was maintained at 7.2 by automatic addition of 2 n KOH. The bioreactor was equipped with a Mettler Toledo polarographic oxygen electrode coupled with feedback regulation to maintain the set point dissolved oxygen tension (pO2) via air sparging and agitation speed. The bioreactor was sparged with air alone to set a pO2 value of 100%. A pO2 of 0% was obtained by continuously flushing the medium at 20 ml/h with either pure N2 or pure H2 gas previously filtered through a Hungate column. ORP was measured using a redox-combined electrode (AgCl, Mettler Toledo), and the values were corrected to the reference electrode value (Eref = +200 mV at 37 °C). Each bioreactor was inoculated with a culture grown overnight under aerobiosis or anaerobiosis in MOD medium supplemented with carbon source and the required electron acceptors. A sample of the overnight culture was diluted in fresh medium to obtain an initial optical density at 560 nm of 0.02. The cells were harvested by centrifugation at the early exponential growth phase, i.e. 1.5 h after reaching the maximal growth rate (when μ = 80 ± 10% of μmax) and immediately frozen at −80 °C until analysis. Supernatants were kept for metabolite and glucose assays. For motility and disk diffusion assays, B. cereus cells were grown in LB medium under aerobic or anaerobic conditions. For anaerobic growth, the cells were incubated in an Oxoid anaerobic jar. Growth at low temperature was performed at 15 °C. To investigate the transcriptional organization and expression of the ohrA-ohrR locus, RT-PCR was performed with total RNA isolated from B. cereus ATCC 14579 as previously described (12Clair G. Roussi S. Armengaud J. Duport C. Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions.Mol. Cell. Proteomics. 2010; 9: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Fragments corresponding to ohrA, ohrR, and the ohrA-ohrR intergenic regions were amplified by RT-PCR using the Titan one Tube RT-PCR system following the manufacturer's protocol (Roche Applied Science). The primer pairs used for ohrA, ohrR, and the ohrA-ohrR intergenic region are 5′-CAGGTGGAAGAAATGGGAAA-3′ plus 5′-GCCGAAACCACCATCTGTAT-3′, 5′-TGTTTTTCTATTTACGCCTGCTC-3′ plus 5′-TGTAGCCATCGTTGTCGGTA-3′, and 5′-CGAAACAAGCTGCATAACCA-3′ plus 5′-TACCGACAACGATGGCTACA-3′, respectively. To check whether contaminant genomic DNA was present, each sample was tested in a control reaction without reverse transcriptase. The 5′ end of ohrA-ohrR mRNA was mapped from a 5′-RACE PCR product obtained with the 3′/5′-RACE kit (Roche Applied Science). Briefly, the first strand cDNA was synthesized from total RNA with ohrA-ohrR-specific SP1 primer (5′-TGTAGCCATCGTTGTCGGTA-3′), avian myeloblastosis virus reverse transcriptase, and the deoxynucleotide mixture of the 3′/5′-RACE kit following the manufacturer's instructions. After purifying and dA-tailing the cDNA, PCR with the (dT)-anchor oligonucleotide primer and the ohrA-ohrR-specific SP2 primer (5′-TTTTCGCTCGTCTTCTTTGG-3′) followed by a nested PCR with SP3 primer (5′-CCATCTTGCTCCCATAGTACG-3′) led to a PCR product of ∼140 bp, as revealed by 2% agarose gel electrophoresis. This PCR product was purified and sequenced. Real time RT-PCR was performed using SYBR Green technology on a LightCycler instrument (Roche Applied Science), as described previously (6Duport C. Zigha A. Rosenfeld E. Schmitt P. Control of enterotoxin gene expression in Bacillus cereus F4430/73 involves the redox-sensitive ResDE signal transduction system.J. Bacteriol. 2006; 188: 6640-6651Crossref PubMed Scopus (67) Google Scholar). ΔohrA and ΔohrR cell mutants were constructed as follows. A BglII-SalI DNA fragment of 1,489 bp encompassing the ohrA and ohrR ORFs was amplified by PCR using chromosomal DNA as template and the primer pair 5′-AGATCTTCTTATGAATCTGACAATCGGG-3′ plus 5′-GTCGACCCGATAAAATTCCGAAAGGG-3′. The amplified DNA fragment was cloned into pCRXL-TOPO (Invitrogen). The resulting pCRXLohr plasmid was then digested with HpaI and BsgI, respectively. A 1.5-kb SmaI fragment containing the entire spectinomycin resistance gene spc (29Murphy E. Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3")(9).Mol. Gen. Genet. 1985; 200: 33-39Crossref PubMed Scopus (96) Google Scholar) was purified from pDIA(14). This purified DNA fragment was ligated into HpaI-digested pCRXLohr and BsgI-digested pCRXLohr, respectively. The resulting plasmids pCRXL-ohrRΔspc-ohrA and pCRXL-ohrR-ohrAΔspc were both digested with EcoRI plus BglII. The resulting ohrRΔspc-ohrA and ohrR-ohrAΔspc fragments were subsequently inserted between the corresponding pMAD sites. The resulting plasmids were introduced into B. cereus strains by electroporation. The ohrA and ohrR genes were deleted by a double-crossover event (30Arnaud M. Chastanet A. Débarbouillé M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria.Appl. Environ. Microbiol. 2004; 70: 6887-6891Crossref PubMed Scopus (704) Google Scholar). Chromosomal allele exchanges were confirmed by PCR with oligonucleotide primers located upstream and downstream of the DNA regions used for allelic exchange. To confirm the nonpolar effect of the spectinomycin resistance cassette insertion in ohrR (which precedes ohrA in the ohrR-ohrA operon), fragments encompassing the ohrA and ohrA-ohrR intergenic region mRNA were amplified from total RNA isolated from ΔohrR cells as described above. A low copy number plasmid, pHT304 (31Arantes O. Lereclus D. Construction of cloning vectors for Bacillus thuringiensis.Gene. 1991; 108: 115-119Crossref PubMed Scopus (352) Google Scholar), was used to complement the ohrR and ohrA genes in trans. The pCRXL-ohrRΔspc-ohrA and pCRXL-ohrR-ohrAΔspc plasmids were digested with EcoRI plus BamHI, and the ohrRΔspc-ohrA and ohrR-ohrAΔspc fragments were cloned into pHT304. The integrity of the inserts in the recombinant vectors was verified by sequencing, after which the vectors were used to transform B. cereus mutant strains. B. cereus growth was monitored spectrophotometrically at 560 nm and calibrated against cell dry weight measurements, as previously described (12Clair G. Roussi S. Armengaud J. Duport C. Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions.Mol. Cell. Proteomics. 2010; 9: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Specific growth rate (μ) was determined using the modified Gompertz equation (32Gompertz B. On the nature of the function expressive of the law of human mortality, and on a new mode of determining the value of life contingencies.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1925; 115: 513-585Google Scholar, 33Zwietering M.H. Jongenburger I. Rombouts F.M. van't Riet K. Modeling of the bacterial growth curve.Appl. Environ. Microbiol. 1990; 56: 1875-1881Crossref PubMed Scopus (0) Google Scholar). Glucose, lactate, ethanol, formate, acetate, and succinate concentrations were determined using Diffchamb, R-Biopharm, and Roche Applied Science kits. Specific glucose consumption rate, defined as the differential change in glucose concentration with time, was calculated from the equation qglucose = μ/Yx, where μ is specific growth rate (h−1), and Yx is biomass yield (g·mol carbon substrate−1). After thawing on ice, cell pellets were suspended in 1 ml of a lysis buffer consisting in 7.8 m urea (Sigma), 2.2 m thiourea (Sigma), 4.5% w/v CHAPS (Sigma), 44.5 mm DTT (Sigma), 2.2 mm Trizma base (Sigma)/HCl (Sigma) pH 7.5, and one tablet of antiprotease mini-mixture (Roche Applied Science). The suspension was transferred into a 2-ml sterilized tube containing 400 mg of 0.1-mm-diameter zirconium/silica beads (VWR). Each tube was placed in a Precellys 24 disruptor (Bertin Technologies) and shaken in three bursts at 6,500 rpm for 20 s with a pause of 5 s. After incubation for 45 min at room temperature, the samples were centrifuged at 3,500 × g for 4 min, and 600 μl of supernatant was recovered. The pellets were washed twice with 600 μl of lysis buffer and redisrupted. The supernatants were then pooled. Protein concentration of each lysate was determined using the Bio-Rad protein assay (Bio-Rad). A volume of 30 μl of lithium dodecyl sulfate 1× sample buffer (Invitrogen) was added to 40 μg of proteins. The samples were then sonicated for 10 min using a transonic 780H sonicator, boiled for 5 min at 95 °C, and loaded onto 4–12% gradient NuPAGE gels (Invitrogen). The gels were operated with MES buffer, run at 150 V (Invitrogen), and then stained with Coomassie Blue Safe stain (Invitrogen). After overnight destaining, the whole protein content from each well was excised as 10 polyacrylamide bands of equal volume from top to bottom. These bands were destained, and their protein contents were treated with iodoacetamide and then proteolyzed with proteasMAX (Promega). The resulting peptide mixtures were diluted 1:20 in 0.1% trifluoroacetic acid. NanoLC-MS/MS analyses were performed on an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex-LC Packings) operated as previously described (12Clair G. Roussi S. Armengaud J. Duport C. Expanding the known repertoire of virulence factors produced by Bacillus cereus through early secretome profiling in three redox conditions.Mol. Cell. Proteomics. 2010; 9: 1486-1498Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Peak lists were generated on Matrix Science MASCOT DAEMON software (version 2.2.2) using the extract_msn.exe data import filter (ThermoFisher) from the ThermoFisher Xcalibur FT software package (version 2.0.7). Data import filter options were set at: 400 (minimum mass), 5,000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1,000 (threshold). MS/MS spectra were searched using MASCOT 2.2.04 software (Matrix Science) against an in-house database containing all the annotated protein sequences of B. cereus ATCC 14579. This database comprises 5,255 polypeptide sequences specified by B. cereus ATCC 14579 chromosome (NC_004722) and plasmid pBClin15 (NC_004721), totaling 1,455,982 amino acids. Search parameters for tryptic peptides were as previously described (34Christie-Oleza J.A. Armengaud J. In-depth analysis of exoproteomes from marine bacteria by shotgun liquid chromatography-tandem mass spectrometry: The Ruegeria pomeroyi DSS-3 case-study.Mar. Drugs. 2010; 8: 2223-2239Crossref PubMed Scopus (42) Google Scholar): full trypsin specificity, a mass tolerance of 10 ppm on the parent ion, and 0.6 Da on the MS/MS spectra, static modifications of carboxyamidomethylated Cys (+57.0215), and dynamic modifications of oxidized Met (+15.9949). Maximum number of missed cleavages was set at 2. MASCOT results were parsed using IRMa 1.22.4 software (35Dupierris V. Masselon C. Court M. Kieffer-Jaquinod S. Bruley C. A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa.Bioinformatics. 2009; 25: 1980-1981Crossref PubMed Scopus (106) Google Scholar) filtering with a p value less than 0.05. A protein was considered valid when at least two different peptides were detected in the same experiment. The false-positive rate for protein identification was estimated using the appropriate decoy database as below 0.1% at these parameters. Mass spectrometry data were deposited in the PRIDE PRoteomics IDEntifications database (36Vizcaíno J.A. Côté R. Reisinger F. Barsnes H. Foster J.M. Rameseder J. Hermjakob H. Martens L. The Proteomics Identifications database: 2010 update.Nucleic Acids Res. 2010; 38: D736-D742Crossref PubMed Scopus (211) Google Scholar) under accession numbers 17312–17338. Peptide characteristics are reported in supplemental Table S4. The database comprises 14,679 unique peptide sequences. Supplemental Table S2 shows the 461,191 assigned spectra and the peptide coverage fo
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