INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Bacillus anthracis is the etiological agent of anthrax, a disease that affects all mammals, including humans. Key virulence factors of the bacterium include the proteins that comprise the edema and lethal toxins. These binary toxins are composed of distinct enzymatic proteins and a common protein that mediates entry into target cells. Protective antigen (PA) and edema factor (EF) comprise edema toxin, while PA plus lethal factor (LF) comprise lethal toxin. PA binds to a specific receptor on target cells and following endocytosis of the toxin-receptor complex, facilitates translocation of EF and LF across the cell membrane such that the enzymatic proteins can contact their cytosolic substrates (15, 19, 34).

The genes encoding the toxin proteins, pagA (previously known as pag) (which encodes PA), cya (which encodes EF), and lef (which encodes LF), are located noncontiguously within a 30-kb region of the 182-kb plasmid, pXO1 (12, 17, 22, 23, 29, 32). As might be expected due to the pivotal role of PA in intoxication, the pagA gene is highly expressed. When B. anthracis is cultured under optimal conditions, culture supernatants contain up to 20 mg of PA per liter, while LF and EF are present at 5 and 1 mg per liter, respectively (16). Analyses of toxin gene expression employing reporter gene fusions suggest that the steady-state level of mRNA of pagA is 4-fold higher than that of lef and 14-fold higher than that of cya (25).

Two host-related cues, CO₂-bicarbonate and temperature, are important signals for expression of all three toxin genes. During in vitro growth, optimal expression of pagA, cya, and lef is achieved when B. anthracis is cultured in buffered R medium, a defined medium containing glucose, salts, and all amino acids, or CA medium, a medium containing Casamino Acids and glucose (16, 21, 27). Toxin synthesis is greatest when cultures are incubated in elevated (5% or greater) atmospheric CO₂ or when bicarbonate is added to culture medium in a closed vessel. Toxin synthesis is also increased when cultures are incubated at 37°C compared to when they are incubated at 28°C. CO₂-bicarbonate- and temperature-controlled gene expression is at the level of transcription (2, 4, 12, 25, 31).

The atxA gene, located within the toxin gene region of pXO1, positively controls transcription of all three toxin genes and at least one gene required for capsule synthesis, capB, which is located on the 93-kb plasmid pXO2 (9, 12, 29, 30). Under all growth conditions tested, atxA is essential for transcription from the unique start sites of cya and lef and for transcription from the major start site, P1, of the pagA gene (7, 12). The mechanism by which atxA activates expression of virulence genes and the relationship(s) between host-related cues and atxA function are not known.

In an effort to identify regulatory genes that act downstream of atxA to activate toxin gene expression, we screened for CO₂-enhanced atxA-dependent loci on pXO1. We created random transcriptional lacZ fusions using transposon Tn917-pLTV3 and tested insertion mutants for CO₂-enhanced atxA-dependent -galactosidase activity. In addition to mutants harboring transposon insertions in the toxin genes, our screen yielded a number of mutants harboring apparent CO₂-enhanced atxA-regulated fusions at distinct loci on pXO1 (10). Here we characterize a 300-bp open reading frame (ORF), pagR, identified in our screen. We show that the pagR gene is cotranscribed with the toxin gene pagA and that a pagR mutant shows increased levels of pagA and atxA transcripts.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains, media, and growth conditions. Escherichia coli JM109 was used as a host for cloning. B. anthracis strains and their relevant characteristics are listed in Table 1. All B. anthracis strains are derivatives of the Weybridge strain, a noncapsulated toxigenic isolate originally obtained from the Microbiological Research Establishment, Porton Down, England. For DNA extractions, the B. anthracis strains were grown in brain heart infusion medium (Difco, Detroit, Mich.) containing 10% horse serum. For electroporation experiments, B. anthracis strains were grown in brain heart infusion medium containing 0.5% glycerol. For RNA extractions, B. anthracis strains were grown in CA broth (27) buffered with 100 mM HEPES (pH 8.0). CA medium contained 0.8% sodium bicarbonate for cultures incubated in 5% CO₂.

Plasmid and strain constructions. Plasmid DNA was extracted from B. anthracis by the method of Green et al. (8). Preparation of plasmid DNA from E. coli, transformation of E. coli, and recombinant DNA techniques were carried out by standard procedures (1). B. anthracis was electroporated with plasmid DNA from E. coli GM1684, as described previously (12). Restriction enzymes, T4 ligase, and Taq polymerase were purchased from Promega (Madison, Wis.) or New England Biolabs (Beverly, Mass.).

RNA analysis. Methods for RNA extraction, primer extension reactions, and RNase T2 protection assays have been described (12, 28). RNA was extracted from B. anthracis cultures grown to late log phase (optical density at 600 nm of 0.8 to 1.0) in CA medium. RNA was quantified spectrophotometrically and by visualization on 1.2% formaldehyde gels. Oligonucleotides used for atxA and pagA primer extensions were described previously (6). Oligonucleotides were labeled with [-³²P]ATP (6,000 Ci/mmol) (Amersham Corp., Arlington Heights, Ill.), hybridized to 20 µg of RNA, and extended by using avian myeloblastosis virus reverse transcriptase (Promega). The 5' ends of the atxA and pagA genes were sequenced by the dideoxy-chain termination method (1), using the appropriate primers and a Sequenase version 2.0 DNA sequencing kit purchased from United States Biochemical Corp. (Cleveland, Ohio). The [-³⁵S]dATP (>1,000 Ci/mmol) for sequencing was purchased from Amersham Corp. Primer extension and sequencing reaction mixtures were subjected to electrophoresis on 6% polyacrylamide and 42% urea gels. Primer extension products were quantified by using a Packard Instant Imager.

Immunoblotting. Relative levels of PA produced by parent and mutant strains were determined by assaying cell lysates for protein that reacted with polyclonal anti-PA antiserum. PA in culture supernatants is subject to varying levels of degradation by proteases secreted by B. anthracis. Therefore, for monitoring small differences in PA levels, more-reproducible data can be obtained by examining cell lysates rather than culture supernatants. B. anthracis cultures were grown in 25 ml of CA medium to an optical density at 600 nm of 0.8 to 1.0. Cells were collected on cellulose acetate membranes (pore size, 0.45 µm) (Nalge, Rochester, N.Y.), resuspended in 1 ml of buffer (1% sodium dodecyl sulfate, 200 mM dithiothreitol, 28 mM Tris HCl, 22 mM Tris OH, 2 mM phenylmethylsulfonyl fluoride), and passed through a French press minicell three times at 20,000 lb/in². The soluble fraction was obtained following centrifugation at 16,000 × g for 5 min, and protein concentrations were determined by using a Bio-Rad protein assay reagent (Bio-Rad, Hercules, Calif.). Samples (5 µg) were subjected to electrophoresis on sodium dodecyl sulfate-7.5% polyacrylamide gels.

Nucleotide sequence accession number. The complete nucleotide sequence of the pagR gene and flanking regions has been deposited in the GenBank database under accession no. AF031382.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of pagR. In experiments reported previously, we used the transposon Tn917-pLTV3 to identify B. anthracis promoters that exhibited increased expression during growth in elevated CO₂ and were atxA-dependent (10). Tn917-LTV3 carries a promoterless lacZ gene at one end and generates a transcriptional fusion when inserted downstream of an active promoter (3). In this investigation, we studied one Tn917-pLTV3 insertion mutant that showed CO₂- and atxA-dependent -galactosidase activity.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   pXO1 DNA sequence extending from the 3' end of pagA through pagR. Predicted amino acid sequences are indicated under the pagA and pagR DNA sequences. Straight arrows indicate an inverted repeat sequence, located downstream of pagA, that functions as a weak transcription terminator. A potential ribosome binding site upstream of pagR is underlined. A tyrosine residue predicted to be a tyrosine kinase phosphorylation site is boxed. Locations of a BamHI site (the site of the km-2 insertion in UT119) and a Tn917-LTV3 insertion in UT82 are as indicated.

pagR is cotranscribed with pagA. Northern hybridization experiments were performed to detect mRNA corresponding to the pagR gene. Biotinylated DNA probes corresponding to sequences internal to pagR and pagA were hybridized to RNA isolated from the parental strain, UM44, and mutants UT62 and UT119. Results are shown in Fig. 2. Hybridization of the pagR probe with UM44 RNA revealed a single band representing a 4.2-kb transcript, while hybridization of UM44 RNA with the pagA probe resulted in two bands corresponding to 4.2- and 2.7-kb transcripts. Mutations in UT62 and UT119 are shown schematically in Fig. 2C. UT62 carries an km-2 insertion that prevents transcription from the major transcription start site of pagA (12). RNA isolated from UT62 did not hybridize to the pagR or pagA probes. UT119 is a mutant containing an km-2 insertion in a BamHI restriction site (Fig. 1) between the pagA and pagR genes. No transcripts were detected when UT119 RNA was probed with pagR. When the pagA probe was hybridized to RNA from UT119, only the smaller, 2.7-kb transcript was detected. Taken together, these results indicate that transcription from the pagA promoter results in a monocistronic transcript corresponding to pagA and a bicistronic transcript corresponding to pagA and pagR (see Fig. 2C).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Northern blot detection of pagR and pagA mRNA. RNA samples (20 µg) from cells grown at 37°C in 5% CO2 were probed with biotinylated DNA probes corresponding to sequences internal to pagR (A) and pagA (B). Transcript sizes are indicated. (C) The relative locations of the pagA and pagR mRNA transcripts and the km-2 insertions in UT62 and UT119 are shown.

View larger version (44K): [in this window] [in a new window]   FIG. 3.   Mapping the 3' ends of the monocistronic pagA transcript and determining the effects of CO2 and atxA on pagR expression. RNase T2 protection assays were performed with 20 µg of RNA. (A) Locations of antisense riboprobes, the km-2 insertion in UT119, and the transcription attenuator are shown. (B) Lane 1: riboprobe 1 was hybridized to RNA from UM44 grown in 5% CO2. RNase T2-protected fragments are indicated. Lanes 2 and 3: nucleotide size markers, as indicated. (C) Riboprobe 2 was hybridized to RNA from cells grown in 5% CO2 or in air, as indicated. RNase T2-protected fragments are shown. Size markers are as indicated. The probe is 1,167 nt (including 222 nt of vector-derived sequences).

pagR represses pagA expression. Considering that pagR is cotranscribed with pagA and that the predicted PagR protein has sequence similarity to regulators of transcription, we hypothesized that pagR affects expression of pagA in a feedback control loop. Primer extension experiments were performed to measure steady-state levels of pagA transcripts in UM44 and UT119 (pagR) grown at 37°C in 5% CO₂. Results of a representative experiment are shown in Fig. 4A. In repeated experiments, the level of pagA transcript was 2.5- to 7.0-fold higher in UT119 than in UM44, indicating that pagR represses pagA expression. It is not clear why the level of repression varied in different experiments. Identical RNA samples used to measure expression of atxA (see below) did not exhibit such variation. Western blottings were performed to determine if the increase in pagA expression in the pagR mutant also led to an increase in PA protein level (Fig. 4B). Levels of PAs from UT119 lysates were 2.0-fold higher than those from UM44 lysates.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Regulation of pagA by pagR. Cultures for isolation of RNA and protein were incubated in 5% CO2. Strains are as indicated. (A) Primer extension experiments were performed with 20 µg of RNA. The end-labeled primer was complementary to a 33-bp sequence located 32 nt downstream of the first nucleotide of the pagA translational start site. Lanes G, A, T, and C correspond to the dideoxy sequencing reaction carried out with the same oligonucleotide primer. (B) Western blot showing PA detected in cell lysates.

pagR represses atxA expression. The pagA gene is positively regulated in trans by atxA (12, 29). We tested for pagR-controlled expression of atxA. Results of primer extension experiments are shown in Fig. 5. The steady-state level of atxA mRNA was increased twofold in UT119 (pagR) compared to that in UM44, indicating that pagR represses atxA at a low level.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Regulation of atxA by pagR. Primer extensions were performed as described in the legend for Fig. 4. The end-labeled primer was complementary to a 27-bp sequence located 55 bp downstream of the first nucleotide of the translational start site of the atxA gene. Strains are as indicated.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we determined that a previously unknown gene, pagR, is cotranscribed with the toxin gene pagA (previously known as pag) and negatively controls expression of the operon. Incomplete transcription termination near the 3' end of an inverted repeat sequence downstream of pagA results in mono- and bicistronic transcripts containing pagA mRNA. There is no evidence that attenuation at this site is regulated. Expression of pagR appears to mimic pagA expression. In light of our results, we refer to the structural gene for PA as pagA. Thus, the pag operon is composed of pagA and pagR.

The predicted amino acid sequence of the putative PagR protein is similar to those of regulatory proteins NolR from R. meliloti (13) and CadC from L. monocytogenes (14). NolR and CadC contain helix-turn-helix motifs and are predicted to be DNA-binding proteins. If the PagR protein binds DNA specifically, the PagR target may be within the promoter region of pagA or within the promoter region of some other gene that affects pagA expression.

A pagR mutant shows elevated expression of pagA and atxA, a trans-acting positive regulator of pagA. Yet it is unlikely that increased pagA expression in a pagR mutant is attributed simply to increased AtxA levels. Results of previous studies indicate that AtxA levels are not limiting for expression of pagA. Strains carrying multiple copies of the pagA promoter synthesize normal levels of PA (24). Furthermore, a 10-fold increase in atxA expression has a negative effect on pagA expression, resulting in a 60 to 70% decrease in PA levels (6). Thus, PagR may control expression of the pag operon independent of AtxA or in conjunction with AtxA and some other regulatory protein(s).

Our data indicate that pagR functions to limit pagA expression under culture conditions which are optimal for synthesis of protective antigen. In experiments not presented here, we tested virulence of the pagR mutant in a mouse model for anthrax. Subcutaneous inoculation of mice with high doses of spores of the toxigenic noncapsulated Sterne strain results in a lethal disease (20). In this model, the 50% lethal dose for the pagR mutant was the same as that for the parent strain (11). This result indicates that increased PA synthesis by the pagR strain does not increase virulence in the mouse model. It is also possible that pagR function during infection differs from that observed during culture of B. anthracis in vitro.

The pagA gene is the only toxin gene that is known to be transcribed in an operon. The 5' ends of transcripts corresponding to the cya and lef genes have been mapped (7); however, the sizes of the cya and lef transcripts have not been determined. Sequence analysis of regions downstream of cya and lef does not indicate that there are putative transcription regulators adjacent to these genes. A 332-bp ORF lies 286 bp downstream of cya and in the same orientation (accession no. AF003936). The predicted protein product of this ORF has sequence similarity to the purine salvage pathway enzyme adenine phosphoribosyltransferase of many organisms. A 303-bp ORF is located 1.4-kb downstream of lef and in the same orientation (accession no. AF031382). The predicted protein product of this ORF has no homology to any sequences in GenBank.

The pagA gene is coordinately expressed with the other toxin genes, cya and lef, in response to the same signals, i.e., CO₂-bicarbonate and temperature, and the same activator, atxA. It is possible that pagR also regulates the cya and lef genes. Cataldi et al. (5) examined EF activity and LF protein level in the culture supernatant of a B. anthracis strain harboring an erythromycin resistance cassette in the pagA gene. EF activity was increased twofold, while LF protein level was unchanged, compared to that in the strain without the cassette (5). If the insertion in the pagA mutant had a polar effect on pagR expression, it is possible that the observed increase in EF activity was due to increased expression of cya in the absence of pagR expression. In future experiments we will address the mechanism for pagR-mediated repression and determine whether or not pagR also regulates the other toxin genes.