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Journal of Bacteriology, February 2003, p . 1462-1464, Vol . 185, No . 4
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 . Hanna1*
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
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 .
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
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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
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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.
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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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