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Journal of Bacteriology, February 2003, p. 1462-1464, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1462-1464.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of the gerH Operon of Bacillus anthracis Endospores: a Differential Role for Purine Nucleosides in Germination

Matthew A. Weiner,¹ Timothy D. Read,² and Philip C. Hanna^1*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan,¹ The Institute for Genomic Research, Rockville, Maryland²

Received 19 August 2002/ Accepted 18 November 2002

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We identified a tri-cistronic operon, gerH, in Bacillus anthracis that is important for endospore germination triggered by two distinct germination response pathways termed inosine-His and purine-Ala. Together, the two pathways allow B. anthracis endospores a broader recognition of purines and amino acids that may be important for host-mediated germination.

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Bacterial endospores are metabolically inactive and are capable of surviving extended periods of time under harsh environmental conditions but germinate rapidly in the presence of small molecules termed germinants (2). Work by Hachisuka demonstrated that the addition of exogenous adenosine and L-alanine was required for in vivo germination of Bacillus anthracis in the rat peritoneal cavity, thus establishing nucleosides as potentially contributing to in vivo germination (5). Germination is characterized by the hydration of the core and the breakup of the endospore cortex, though the molecular mechanisms underlying these activities remain undetermined (2, 9). Endospore germination results in the expulsion of Ca²⁺ and dipicolinic acid and the initiation of metabolic activity (11). Nutrient-triggered endospore germination is facilitated by ger operons, which are believed to encode germinant sensor proteins (9).

Identification and characterization of gerH in B. anthracis. In Bacillus cereus, gerI (gerIABC) is necessary for the triggering of germination by inosine and the disruption of gerIA or gerIB abolishes inosine-triggered germination (1). A BLAST search of the B. anthracis genome (http://www.tigr.org) with gerIA, gerIB, and gerIC from B. cereus (GenBank accession number AF067645) identified homologs in B. anthracis referred to here as gerHA, -B, and -C (open reading frames 02625, 02624, and 02623, respectively). The putative proteins encoded by gerHA, gerHB, and gerHC have 78, 92, and 89% amino acid identity, respectively, to their B. cereus GerI homologs. Unlike with B. cereus, inosine alone does not trigger B. anthracis endospore germination but acts as a potent cogerminant with several amino acids (Table 1) (1, 6).

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TABLE 1. Germination of B. anthracis Sterne 34F2 endospores in a subgerminal concentration of L-alanine plus L-amino acids

To determine a role for gerH in B. anthracis germination, a gerHA-null strain was constructed from the Sterne 34F2 (pXO1⁺, pXO2^-) strain by using forward and reverse primers with 5' XmaI restriction sites (5'-TCCCCCCGGGCAAGAAGGTTTTGTAGAGGA-3', 5'-TCCCCCCGGGGATTGCATAGGCTTTTTAAC-3' ) to PCR amplify gerHA DNA (1.7 kb), which was cloned into pUC19 (New England Biolabs) and maintained in Escherichia coli XL1 Blue (American Type Culture Collection). A central section of gerHA (gerHA) was deleted and replaced by an erythromycin cassette, which had been amplified from pDG641 (Bacillus Genetic Stock Center), at the gerHA ClaI restriction site with appropriate primers (5'-CCATCGATGGGCGGTGTAGATGTTGATGA-3', 5'-CCATCGATGGACATGCTACACCTCCGGATA-3') (3). The resulting construct was transferred into the gram-positive shuttle vector pKSV7, creating pKSV7:gerHA:Erm, and maintained in E. coli GM272 (Bacillus Genetic Stock Center) (10). Electroporation of B. anthracis Sterne 34F2 was performed with polyethylene glycol-precipitated plasmid DNA (8). Transformants were plated on selective medium, and B. anthracis Sterne gerHA-null strains were obtained via allelic exchange, after curing of the plasmid vector containing wild-type gerHA (8). The identity of the null construct was confirmed by PCR and by Southern blotting.

Germinant surveys with an L-alanine-amino acid combination versus an inosine-amino acid combination. B. anthracis Sterne and gerHA-null endospores were preradiolabeled with ⁴⁵Ca as described previously but with modified G medium (6, 7). Parental and gerHA-null strains exhibited similar vegetative growth kinetics (data not shown). The percentage of germination was measured as the percentage of ⁴⁵Ca released from spores relative to the total amount of ⁴⁵Ca contained in a sample (6, 12). Next, the binary combinations of germinants known to trigger germination in B. anthracis were tested to compare profiles of wild-type and gerHA-null strain phenotypes (Tables 1 and 2) in MES (morpholineethanesulfonic acid) buffer at pH 8.0 by using 10⁶ endospores/ml of germinant solution. Slight differences between the Sterne germination profiles reported here and those for Sterne (pXO1^-, pXO2^-) reported previously may result from strain differences or from slight variations in experimental conditions (4, 6). In our studies, MES buffer was used to minimize germination enhancement by monovalent ions. The gerH locus was required for germination with inosine-His, inosine-Met, inosine-Phe, inosine-Tyr, inosine-Val, and Ala-Tyr (Tables 1 and 2). The loss of Ala-Tyr-triggered germination in gerHA-null spores indicated that gerH also influenced a non-nucleoside-dependent germination pathway (Table 1). Therefore, B. anthracis gerH facilitates germination via an inosine-amino acid pathway and, to a lesser extent, via an Ala-aromatic-amino-acid pathway. The presence of an aromatic ring structure is required for gerH-mediated germination, and Tyr cannot substitute for inosine with any amino acids other than Ala (6).

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TABLE 2. Germination of B. anthracis Sterne 34F2 in inosine plus L-amino acids

Germinant studies indicate the testing of purine promiscuity with L-alanine but not with histidine. The degree to which purines could be substituted for each other with an amino acid cogerminant was determined for gerHA-null and Sterne endospores. Purine cogerminants (adenosine, guanosine, ATP, GTP, ITP) were substituted for inosine with 1 mM Ala in parental and gerHA-null strains. Each triggered germination to similar levels, with the sole exception of GTP plus Ala, with which the gerHA-null endospores responded more dramatically than did parental endospores (Fig. 1A). It is possible that a nonproductive interaction that interferes with GTP-Ala-triggered germination in parental, but not gerHA-null, endospores occurs between GTP and GerH proteins.

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FIG. 1. B. anthracis Sterne (parental strain; grey bars) and gerHA-null (black bars) endospore germination responses to a subgerminal concentration of alanine (A) and histidine (B) with purine nucleosides and nucleoside triphosphates. We used a subgerminal concentration of L-alanine (1 mM) (A) and a 10 mM concentration of histidine (B) with a 1 mM concentration of adenosine (ADE), inosine (INO), guanosine (GUA), ATP, ITP, or GTP. Purines and amino acids alone at the concentration used stimulated no endospore germination. Percent germination was calculated at 90 min as described in the text. Experiments were performed at pH 8 with 10 mM MES. Each experiment was performed in triplicate with three independent endospore preparations. Experimental error was calculated as 1 standard deviation from the mean.

The requirement of inosine for purine-His-triggered germination is absolute. Replacing inosine with any other purine in combination with His (10 mM) resulted in a total loss of germination (Fig. 1B). The ability of the gerHA-null strain to germinate via the purine-alanine pathway shows that gerH is not required for an inosine-based response to a subgerminal concentration of alanine. These data demonstrate, for the first time, differential responses to purines of B. anthracis endospores depending on the amino acid supplied as a cogerminant. The purine-Ala pathway exhibited a higher degree of purine promiscuity than the inosine-His pathway. The purines recognized by the purine-Ala pathway and the amino acids recognized by the inosine-His pathway allow a broad recognition of germinants that likely helps B. anthracis endospores recognize varieties of hospitable environments in which to germinate.

Recently, the gerS operon in B. anthracis was characterized and was found to mediate the germination of B. anthracis endospores by germinants containing aromatic ring structures (6). Disruption of the gerS locus results in germinant profiles similar to that of the gerH-null strain. A functional relationship may also exist between gerS and gerH, though that relationship remains undefined. If the two loci are redundant, the loss of one should not abolish germination in response to a pair of cogerminants. Alternatively, it is possible that GerH and GerS are functionally redundant and that together they provide a critical number of germinant sensors required to facilitate germination.

 
We thank T. Dixon, J. Ireland, B. Thomason, S. Cendrowski, B. Heffernan, N. Fisher, and N. Bergman for their useful comments on this work.

The sequencing of B. anthracis was supported by the ONR, DOE, NIAID, and DERA. This work was supported in part by the NIH grants AI-08649 and AI-40644 and ONR grants 14-00-1-0422, 14-01-1-1044, and 14-02-1-0061 (P.C.H.).

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, 5641 Med. Sci. II, Box 0620, Ann Arbor, MI 48104. Phone: (734) 615-3706. Fax: (734) 764-3562. E-mail: pchanna{at}umich.edu.

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Journal of Bacteriology, February 2003, p. 1462-1464, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1462-1464.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weiner, M. A. Articles by Hanna, P. C. Search for Related Content PubMed PubMed Citation Articles by Weiner, M. A. Articles by Hanna, P. C.