Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Anne Moir. Tel. +44 114 2224418. Fax: +44 114 2728697. e-mail: a.moir{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: Bacillus anthracis, gerA, germinant, receptor, endospore
c The GenBank accession numbers for the gerL and gerQ operons reported in this paper are AF387344 and AY037930, respectively.
a Present address: BIOS Scientific Publishers, 9 Newtec Place, Magdalen Road, Oxford OX4 1RE, UK.
b Present address: Wolfson Institute of Biomolecular Research, University College London, Gower Street, London WC1E 6BT, UK.
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INTRODUCTION |
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Bacillus cereus is widely distributed in nature, and can be found in milk and cereals, on plant surfaces and in a variety of other foods. It causes two types of food poisoning: the emetic type resulting from the growth of vegetative cells spoiling food and creating toxins; and the diarrhoeal type caused by an enterotoxin (Kramer & Gilbert, 1989 ; Granum & Lund, 1997
). There is a close genetic relationship between B. cereus, Bacillus thuringiensis and Bacillus anthracis (Helgason et al., 2000
), and much of the information on B. cereus germination systems is likely also to be of relevance to these others. B. cereus spores germinate in response to L-alanine and to ribosides, such as inosine (Warren & Gould, 1968
), as do also B. thuringiensis and B. anthracis. Molecular genetic techniques, including transposon mutagenesis and insertional inactivation, have recently been applied to the study of spore germination in B. cereus. Already identified are the gerI operon, a member of the gerA operon family, required for the response to inosine as sole germinant member and contributing to alanine-stimulated germination (Clements & Moir, 1998
), gerN, a Na+/K+-H+ ion antiporter required for inosine germination (Thackray et al., 2001
; Southworth et al., 2001
) and gerP, a locus affecting the permeability of spore coats to germinants (Behravan et al., 2000
).
This work describes the characterization of two additional operons, both gerA operon homologues, identified by transposon mutagenesis. The gerL operon, a second homologue in B. cereus of the gerA receptor-encoding family, is responsible for the rapid response of spores to L-alanine as sole germinant; the relative contributions to alanine germination from proteins encoded in gerL and gerI loci are assessed under optimized conditions, using the appropriate mutants. A third homologue, gerQ, is required for germination in inosine as sole germinant, but has no role in the alanine response.
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METHODS |
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Spore-germination assays.
Spores were heat-activated in distilled water at a concentration of 810 mg dry weight ml-1 in a water bath at 70 °C for 30 min and then placed on ice. Spores were used within 810 h of activation.
Spores were germinated under two sets of conditions. For inosine germination, spores were suspended in 10 mM Tris/HCl, pH 8·0, 10 mM NaCl, incubated for 15 min at 37 °C, and germination initiated by the addition of inosine to 5 mM. For L-alanine germination, spores were suspended in 50 mM Tris/HCl, 50 mM NH4Cl, pH 8·9, at 30 °C, and germination was initiated by the addition of L-alanine (to 50 mM). For L-alanine germination, O-carbamyl-D-serine (5 µg ml-1) was added to inhibit the alanine racemase activity of the spores. For the estimation of the effects of pH, 50 mM Tris/HCl50 mM NH4Cl or 50 mM CHES/NaOH50 mM NH4Cl buffers were used.
The OD580 of the spore suspensions was monitored continuously on a chart recorder linked to a Unicam SP1800 ultraviolet spectrophotometer fitted with a constant-temperature cell holder.
Transposon mutagenesis, enrichment and screening.
Transposon mutagenesis was carried out with B. cereus 569 UM20.1(pLTV1) as previously described (Clements & Moir, 1998 ). Libraries of transposon-insertion mutants were enriched for mutants and screened by using a tetrazolium overlay as described previously (Clements & Moir, 1998
).
Phage transduction.
Phage transduction was performed using phage CP51ts, a heat-sensitive derivative of generalized transducing phage CP51 (Thorne, 1968 ), as described previously (Clements & Moir, 1998
).
Screening of a B. cereus genomic library.
B. cereus 569 UM20.1 genomic DNA was prepared according to the protocol supplied with the Puregene kit (Flowgen Instruments). The DNA was partially digested with Sau3A, separated by gel electrophoresis, and fragments of 46 kb were excised, purified with GeneClean II (Bio 101), then ligated into BamHI-pre-digested ZAP Express vector as described by the manufacturer (Stratagene). The ligation products were packaged using Stratagenes Gigapack II according to the manufacturers instructions. Approximately 10000 plaques were screened by plaque blotting, probing and hybridization detection using the Digoxigenin DNA labelling and detection kit according to the instructions of the manufacturer (Boehringer Mannheim).
Sequencing and analysis.
DNA sequencing was performed with the Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) and an Applied Biosystems DNA sequencer. Plasmid DNA for sequencing was prepared using the PEG method according to the manufacturers instructions. The 3' ends of the operons were completed using PCR; for each operon, two independent PCR products were sequenced The DNA sequence, complete on both strands and fully overlapped, was assembled and analysed using the Staden suite of programs (Staden, 1990 ). Preliminary sequence data from the B. anthracis genome was obtained from The Institute for Genomic Research website at http://www.tigr.org.
The sequences of the gerL (accession no. AF387344) and gerQ (accession no. AY037930) operons have been submitted to GenBank.
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RESULTS |
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Cloning and sequencing of the gerL region
The gerL region is shown in Fig. 1. Chromosomal DNA from the gerLp1 and gerLA4 mutants was digested with EcoRI, then diluted and ligated as described previously (Clements & Moir, 1998
). Transformation of Escherichia coli DH5
allowed recovery of a plasmid carrying the chromosomal region flanking the end of the transposon, yielding pALA1 and pALA4, respectively, from the two mutants. SalI cuts at 276 bp into the transposon sequence; a double digest identified the SalIEcoRI fragments containing chromosomal DNA as approximately 2·2 and 3·6 kb for the two plasmids, respectively.
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Three ORFs representing the gerLA, gerLB and gerLC genes are separated from the upstream ORF1, which is a close homologue of YhjB of B. subtilis (69% amino acid identity), by an intergenic region of just over 200 bp. This intergenic region contains an extended inverted repeat, possibly representing a termination site for the upstream yhjB gene, and also a potential forespore-specific G-dependent promoter region for the gerL genes (Fig. 2A
). In database searches against finished and unfinished microbial genomes at NCBI, the GerL proteins have close homologues in B. anthracis (94, 93 and 87 % identity, respectively, for the GerLA, GerLB and GerLC proteins) and lower similarity to other homologues in the GerA family (up to 41% identity for GerLA, up to 27% for GerLB, and up to 28% for GerLC). The site of transposon insertion in gerLp1 is between the predicted -35 and -10 sequences, 26 bases upstream of the ribosome-binding site for the gerLA gene. The insertion in the gerLA4 mutant removes the last 54 codons of GerLA.
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A more extensive clone of this region was recovered from a lambda library, by probing with labelled cloned DNA from pINO2. The excised phagemid from this clone, pBKGQ100, contains the complete gerQ operon, except for the 3' end of the gerQC ORF. The N-terminal part of the gerQA gene corresponds closely to gerX, described as a partial sequence of a germination gene homologue located downstream of the hblCDAB gene cluster in the B. cereus type strain ATCC14579 (Økstad et al., 1999 ). We have identified the rest of the equivalent gerQ operon in B. cereus strain ATTC 14579, by searching the preliminary and partial genome sequences made available by Integrated Genomics (http://ergo.integratedgenomics.com/B_cereus.html). This allowed the amplification, from DNA of B. cereus ATTC 10876, of a PCR fragment extending into the end of gerQC from the next downstream ORF. A 4·36 kb sequence containing the entire gerQ operon of B. cereus ATCC 10876 has been submitted to GenBank (accession no. AY037930).
The intergenic region upstream of gerQA in the ATCC 10876 sequence contains several regions also conserved in the ATCC 14579 intergenic sequence. This includes an inverted repeat thought to represent the rho-independent terminator of an upstream KinA-like gene (Økstad et al., 1999 ) and a region that contains a potential sigG-dependent promoter sequence (Fig. 2B
), although it is not very close to the consensus. The gerQhbl region is not represented in the unfinished B. anthracis genome sequence; B. cereus ATCC 10987 and B. anthracis (A.-B. Kolstø, personal communication) both appear to lack the hbl gene cluster. The Ger homologues with the greatest similarity to the GerQ proteins are the GerI proteins of B. anthracis (45, 37 and 33% for the QA, QB and QC proteins, respectively) and homologues in Bacillus halodurans (49, 39 and 33%, respectively). The identities with respect to B. subtilis homologues are up to 34, 23 and 22%, respectively.
Germination behaviour of mutants
Differences were noted in optimal pH, temperature and monovalent-cation dependence for inosine and L-alanine germination by B. cereus (Clements & Moir, 1998 ) Optimized conditions, based on their data, have been used in this work. Measurement of the loss of OD580 of a spore suspension essentially demonstrates the time to germination summed over the population of spores. Fig. 3
shows the germination behaviour in alanine of spores with defects in one of the three recognized loci gerL, gerI and gerQ. Alanine germination is unaffected by the gerQA2 mutation, but the rate of germination is a little slower in a gerIA5 mutant, and the gerLp1 and gerLA4 mutants respond much more slowly to L-alanine. Fig. 4
shows that the gerQA2 mutant, like a gerIA5 mutant, is very slow to germinate in inosine. The gerLp1 and gerLA4 mutant spores showed the same germination kinetics in inosine as the wild-type parent.
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Washed spore suspensions of strains UM20.1, AM1314 (gerIA5) and AM1401 (gerLA4) were tested for germination. Rates of germination at different pH values are shown in Table 2. In Tris/HCl buffer, the gerL-dependent response (in a gerIA5 mutant) increased over a wide pH range above 7, but still showed significant activity at neutral pH. In contrast, little activity of the gerI-dependent response was seen below pH 8·9. To obtain buffering at a more alkaline pH, germination in a CHES/NaOH buffer was examined. The gerL-dependent response was again strongest at 8·58·9; germination dependent on gerI, maximal at pH 8·9 in Tris/HCl, was very low in CHES. The summed rates of germination in the individual mutants do not equal the overall rate of germination in the wild-type, especially at pH values around 78. This suggests that the products of both the gerL and gerI operons may be required to act in concert (and possibly with additional receptors) to cause efficient germination at pH values close to neutrality, or that additional receptors contribute at this pH.
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DISCUSSION |
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This work brings to six the total number of Bacillus germinant receptors of the GerA family that meet both the criterion of proven function and that of defined germinant specificity (the gerA, gerB and gerK operons in B. subtilis, and the gerI, gerQ and gerL operons in B. cereus), although there are numerous additional members of this family encoded in the available genomic DNA sequences from different spore-formers. The gerL operon of B. cereus and the gerA operon of B. subtilis are the closest in terms of germinant specificity, but they are widely divergent (e.g. GerLB and GerAB share 23% identical amino acids). We do not yet have enough information to identify the residues conserved in these proteins that contribute directly to L-alanine binding.
Three operons of the gerA family, all with the same gene order as gerA, have now been defined in B. cereus as encoding receptors for alanine or inosine germination. Additional operon homologues are present in the sequences in the incomplete B. cereus and B. anthracis databases and are presumably involved in the response to other individual germinants or to combinations of germinants. In earlier work (Clements & Moir, 1998 ), we measured much more rapid germination responses to inosine than to alanine in wild-type spores, but the temperature and pH used for germination were not optimized for the latter. It is also important to include an alanine racemase inhibitor, as D-alanine is commonly a competitive inhibitor of L-alanine germination (Foerster & Foster, 1966
).
Germination in L-alanine as sole germinant is mediated by at least two separate receptors; GerL, reported here, is the major contributor under our optimized conditions. In contrast, germination in inosine as sole germinant strictly requires two receptors, i.e. the protein products of both the gerI and gerQ operons.
GerI and GerQ receptors in B. cereus are both part of the nucleoside response. We do not understand why both receptors are required for a response to a single germinant; perhaps they are recognizing different parts of the germinant molecule, or, simply, perhaps neither provides sufficient activation individually. As yet there is no experimental evidence illuminating, even for multiple germinants, how different receptors act in concert.
We have identified mutations (all affecting inosine germination; Barlass, 1998 ) in each of the gerI genes, but the mutations described here for gerL and gerQ all lie in the first gene in the operons. Past precedent from B. subtilis indicates that the genes in an individual operon have co-evolved and do not substitute for each other, so we tentatively assume the same scenario for these operons, but this should be tested experimentally.
Germination in a combination of inosine and L-alanine, a common combination of germinants in experiments, can involve multiple germinant receptors, and detailed physiological analysis addressing germination mechanisms will require dissection of the responses of different receptors, as they clearly vary: inosine germination, but not alanine germination, for example, is dependent on GerN, a Na+/K+-H+ antiporter. Germination rates in inosine and a very low concentration of alanine are significant in a gerQ mutant but not in a gerI mutant (Clements, 1996 ), suggesting that the role of gerQ can be bypassed if some alanine is present. Double mutants have not yet been constructed to determine whether gerL and gerI are both required for this response to combinations of germinants.
B. cereus, B. thuringiensis and B. anthracis represent a closely related group of organisms that could be considered as a single species (Helgason et al., 2000 ). The germination responses are related (all are reported to germinate in alanine plus inosine) but are not identical. For example, in B. anthracis, the gerX operon, encoded in the pathogenicity gene cluster of pXO1, contributes to rapid spore germination in the macrophage (Guidi-Rontani et al., 1999
). Differences in the germination properties of different strains in the B. cereus continuum may well reflect the activity, and even the presence, of different germinant receptor operons, and these may contribute to the exploitation of particular biological niches.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Battisti, L., Green, B. D. & Thorne, C. B. (1985). Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. J Bacteriol 162, 543-550.[Medline]
Behravan, J., Chirakkal, H., Masson, A. & Moir, A. (2000). Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect the access of germinants to their target in the spore. J Bacteriol 182, 1987-1994.
Camilli, A., Portnoy, D. A. & Youngman, P. (1990). Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions. J Bacteriol 172, 3738-3744.[Medline]
Clements, M. O. (1996). Molecular and biochemical analysis of Bacillus cereus 569 spore germination. PhD thesis, University of Sheffield.
Clements, M. O. & Moir, A. (1998). Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180, 6729-6735.
Errington, J. (1993). Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57, 1-33.[Abstract]
Foerster, H. F. & Foster, J. W. (1966). Response of Bacillus spores to combinations of germinative compounds. J Bacteriol 91, 1168-1177.[Medline]
Gould, G. W. & Dring, G. J. (1972). Biochemical mechanisms of spore germination. In Spores V , pp. 401-408. Edited by H. O. Halvorson, R. Hanson & L. L. Campbell. Washington, DC: American Society for Microbiology.
Granum, P. E. & Lund, T. (1997). Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett 157, 223-228.[Medline]
Guidi-Rontani, C., Pereira, Y., Ruffie, S., Sirard, J.-C., Weber-Levy, M. & Mock, M. (1999). Identification and characterization of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis. Mol Microbiol 33, 407-414.[Medline]
Helgason, E., Økstad, O. A., Caugant, D. A., Johansen, H. A., Fouet, A., Mock, M., Hegna, I. & Kolstø, A.-B. (2000). Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis one species on the basis of genetic evidence. Appl Environ Microbiol 66, 2627-2630.
Hudson, K. D., Corfe, B. M., Kemp, E. H., Feavers, I. M., Coote, P. J. & Moir, A. (2001). Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol 183, 4317-4322.
Kramer, J. M. & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In Foodborne Bacterial Pathogens , pp. 21-70. Edited by P. M. Doyle. New York: Marcel Dekker.
Moir, A. & Smith, D. A. (1990). The genetics of bacterial spore germination. Annu Rev Microbiol 44, 531-553.[Medline]
Moir, A., Kemp, E. H., Robinson, C. & Corfe, B. M. (1994). The genetic analysis of bacterial spore germination. J Appl Bacteriol 76, 9S-16S.
Moir, A., Corfe, B. M. & Behravan, J. (2002). Spore germination. Cell Mol Life Sci 59, 403-409.[Medline]
Økstad, O. A., Gominet, M., Purnelle, B., Rose, M., Lereclus, D. & Kolstø, A.-B. (1999). Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin. Microbiology 145, 3129-3138.
Paidhungat, M. & Setlow, P. (1999). Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine. J Bacteriol 181, 3341-3350.
Paidhungat, M. & Setlow, P. (2000). Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182, 2513-2519.
Paidhungat, M. & Setlow, P. (2001). Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores. J Bacteriol 183, 3982-3990.
Paidhungat, M., Ragkousi, K. & Setlow, P. (2001). Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J Bacteriol 183, 4886-4893.
Southworth, T. W., Guffanti, A. A., Moir, A. & Krulwich, T. A. (2001). GerN: an endospore germination protein of Bacillus cereus is a Na+/H+-K+ antiporter. J Bacteriol 183, 5896-5903.
Staden, R. (1990). Finding protein coding regions in genomic sequences. Methods Enzymol 83, 163-180.
Stewart, G. S. A. B., Johnstone, K., Hagelberg, E. & Ellar, D. J. (1981). Commitment of bacterial spores to germinate: a measure of the trigger reaction. Biochem J 198, 101-106.[Medline]
Thackray, P. D., Behravan, J., Southworth, T. W. & Moir, A. (2001). GerN, an antiporter homologue important in the germination of Bacillus cereus endospores. J Bacteriol 183, 476-482.
Thorne, C. B. (1968). Transducing bacteriophage for Bacillus cereus. J Virol 2, 657-662.[Medline]
Warren, S. C. & Gould, G. W. (1968). Bacillus cereus spore germination: absolute requirement for an amino acid. Biochim Biophys Acta 170, 341-350.[Medline]
Wuytack, E. Y., Soons, J., Poschet, F. & Michiels, C. W. (2000). Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores. Appl Environ Microbiol 66, 257-261.
Received 26 November 2001;
revised 11 March 2002;
accepted 21 March 2002.