Abstract
The l-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of d-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting l-alanine to the germination inhibitor d-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of l- to d-alanine. Unlike ATCC 14579 spores, l-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.
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References
Abee T, Groot MN, Tempelaars M, Zwietering M, Moezelaar R, van der Voort M (2011) Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors. Food Microbiol 28:199–208
Abhyankar W, Ter Beek A, Dekker H, Kort R, Brul S, de Koster CG (2011) Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics 11:4541–4550
Bagyan I, Noback M, Bron S, Paidhungat M, Setlow P (1998) Characterization of yhcN, a new forespore-specific gene of Bacillus subtilis. Gene 212:179–188
Barlass PJ, Houston CW, Clements MO, Moir A (2002) Germination of Bacillus cereus spores in response to l-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089–2095
Boydston JA, Yue L, Kearney JF, Turnbough CL (2006) The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. J Bacteriol 188:7440–7448
Chesnokova O, McPherson SA, Steichen CT, Turnbough CL (2009) The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development. J Bacteriol 191:1303–1310
Couñago RM, Davlieva M, Strych U, Hill RE, Krause KL (2009) Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames). BMC Struct Biol 9:53
Del Torre M, Della Corte M, Stecchini ML (2001) Prevalence and behaviour of Bacillus cereus in a REPFED of Italian origin. Int J Food Microbiol 63:199–207
Del Vecchio VG, Connolly JP, Alefantis TG, Walz A, Quan MA, Patra G, Ashton JM, Whittington JT, Chafin RD, Liang X, Grewal P, Khan AS, Mujer CV (2006) Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl Environ Microbiol 72:6355–6363
Dodatko T, Akoachere M, Muehlbauer SM, Helfrich F, Howerton A, Ross C, Wysocki V, Brojatsch J, Abel-Santos E (2009) Bacillus cereus spores release alanine that synergizes with inosine to promote germination. PLoS ONE 4:e6398
Driks A (2009) The Bacillus anthracis spore. Mol Aspects Med 30:368–373
Faille C, Tauveron G, Le Gentil-Lelièvre C, Slomianny C (2007) Occurrence of Bacillus cereus spores with a damaged exosporium: consequences on the spore adhesion on surfaces of food processing lines. J Food Protect 70:2346–2353
Faille C, Lequette Y, Ronse A, Slomianny C, Garénaux E, Guerardel Y (2010a) Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium. Consequences on their ability to adhere to stainless steel. Int J Food Microbiol 143:125–135
Faille C, Sylla Y, Le Gentil C, Bénézech T, Slomianny C, Lequette Y (2010b) Viability and surface properties of spores subjected to a cleaning-in-place procedure: consequences on their ability to contaminate surfaces of equipment. Food Microbiol 27:769–776
Fazzini M, Schuch R, Fischetti VA (2010) A novel spore protein, ExsM, regulates formation of the exosporium in Bacillus cereus and Bacillus anthracis and affects spore size and shape. J Bacteriol 192:4012–4021
Fey G, Gould GW, Hitchins AD (1964) Identification of d-alanine as the auto inhibitor of germination of Bacillus globigii spores. J Gen Microbiol 35:229–236
Gould GW (1966) Stimulation of l-alanine-induced germination of Bacillus cereus spores by d-cycloserine and O-carbamyl-d-serine. J Bacteriol 92:1261–1262
Grossman AD (1995) Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet 29:477–508
Henriques AO, Moran CP (2000) Structure and assembly of the bacterial endospore coat. Methods 20:95–110
Henriques AO, Moran CP (2007) Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61:555–588
Henriques AO, Melsen LR, Moran CP (1998) Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J Bacteriol 180:2285–2291
Hornstra LM, de Vries YP, de Vos WM, Abee T, Wells-Bennik MHJ (2005) gerR, a novel ger operon involved in l-alanine and inosine-initiated germination of Bacillus cereus ATCC 14579. Appl Environ Microbiol 71:774–781
Hornstra LM, de Vries YP, Wells-Bennik MHJ, de Vos WM, Abee T (2006) Characterization of germination receptors of Bacillus cereus ATCC 14579. Appl Environ Microbiol 72:44–53
Kanda-Nambu K, Yasuda Y, Tochikubo K (2000) Isozymic nature of spore coat-associated alanine racemase of Bacillus subtilis. Amino Acids 18:375–387
Kanodia S, Agarwal S, Singh P, Agarwal S, Singh P, Bhatnagar R (2009) Biochemical characterization of Alanine racemase—a spore protein produced by Bacillus anthracis. BMB Reports 42:47–52
Kim H, Hahn M, Grabowski P, McPherson DC, Otte MM, Wang R, Ferguson CC, Eichenberger P, Driks A (2006) The Bacillus subtilis spore coat protein interaction network. Mol Microbiol 59:487–502
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Liu H, Bergman NH, Thomason B, Shallom S, Hazen A, Crossno J, Rasko DA, Ravel J, Read T, Peterson SN, Yates J, Hanna PC (2004) Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186:164–178
McPherson S, Li M, Kearney JF, Turnbough CL (2010) ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis. Mol Microbiol 76:1527–1538
Moir A (2003) Bacterial spore germination and protein mobility. Trends Microbiol 11:452–454
Moir A (2006) How do spore germinate? J Appl Microbiol 101:526–530
Moir A, Corfe BM, Behravan J (2002) Spore germination. Cell Mol Life Sci 59:403–409
Nicholson WL, Setlow P (1990) Sporulation, germination, and outgrowth. In: Harwood CR, Cutting SM (eds) Molecular biological methods for Bacillus. Wiley and sons, Chichester UK, pp 391–450
Pandey NK, Aronson AI (1979) Properties of the Bacillus subtilis spore coat. J Bacteriol 137:1208–1218
Preston RA, Douthit HA (1984) Germination of Bacillus cereus spores: critical control by dl-alanine racemase. J Gen Microbiol 130:3123–3133
Redmond C, Baillie LW, Hibbs S, Moir AJ, Moir A (2004) Identification of proteins in the exosporium of Bacillus anthracis. Microbiology 150:355–363
Setlow P (2003) Spore germination. Curr Opin Microbiol 6:550–556
Severson KM, Mallozzi M, Bozue J, Welkos SL, Cote CK, Knight KL, Driks A (2009) Roles of the Bacillus anthracis spore protein ExsK in exosporium maturation and germination. J Bacteriol 191:7587–7596
Steichen C, Chen P, Kearney JF, Turnbough CL (2003) Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol 185:1903–1910
Steichen C, Kearney JF, Turnbough CL (2005) Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis. J Bacteriol 187:5868–5876
Steichen CT, Kearney JF, Turnbough CL (2007) Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth. Mol Microbiol 64:359–367
Thompson BM, Waller LN, Fox KF, Fox A, Stewart GC (2007) The BclB glycoprotein of Bacillus anthracis is involved in exosporium integrity. J Bacteriol 189:6704–6713
Todd SJ, Moir AJG, Jonhson MJ, Moir A (2003) Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J Bacteriol 185:3373–3378
Waller L, Fox KF, Fox A, Price RL (2004) Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and B. subtilis. J Microbiol Meth 58:23–30
Yasuda Y, Kanda K, Nishioka S, Tanimoto Y, Kato C, Saito A, Fukuchi S, Nakanishi Y, Tochikubo K (1993) Regulation of l-alanine initiated germination of Bacillus subtilis spores by alanine racemase. Amino Acids 4:89–99
Zilhão R, Serrano M, Isticato R, Ricca E, Moran CP Jr, Henriques AO (2004) Interactions among CotB, CotG, and CotH during assembly of the spore coat in Bacillus subtilis. J Bacteriol 186:1110–1119
Acknowledgments
This research was supported by the MiPAAF (Ministero delle Politiche Agricole, Alimentari e Forestali) MIERI project. Thanks are due to Giovanna Lippe for advice and assistance with gel electrophoresis and to Massimiliano Spaziani for his technical contribution.
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Communicated by Erko Stackebrandt.
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Venir, E., Del Torre, M., Cunsolo, V. et al. Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium. Arch Microbiol 196, 79–85 (2014). https://doi.org/10.1007/s00203-013-0946-y
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DOI: https://doi.org/10.1007/s00203-013-0946-y