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Infection and Immunity, June 2002, p. 2915-2925, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2915-2925.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Regulation of the Overlapping pic/set Locus in Shigella flexneri and Enteroaggregative Escherichia coli

Martin Behrens,^, Jalaluddin Sheikh, and James P. Nataro^*

Center for Vaccine Development, Departments of Pediatrics, Medicine, and Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 20 September 2001/ Returned for modification 6 December 2001/ Accepted 14 February 2002

	ABSTRACT

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Most strains of Shigella flexneri 2a and enteroaggregative Escherichia coli carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the setA and setB genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that pic and the setBA loci are transcribed as complementary 4-kb mRNA species. The major pic promoter is maximally activated at 37°C in exponential growth phase. Our data suggest that the setB gene is transcribed from a promoter which lies more than 1.5 kb upstream of the setB structural gene; setA may be transcribed via readthrough of the setB transcript and possibly by its own promoter. The long leader of the setB gene provides a strong silencing effect on setB transcription. The signals which provide relief from setB silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of setB expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.

	INTRODUCTION

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Shigella flexneri and enteroaggregative Escherichia coli (EAEC) are enteric pathogens implicated as agents of diarrheal disease throughout the world. Whereas multiple virulence factors and mechanisms have been elucidated for these organisms, most of the work has focused on plasmid-borne virulence genes. However, several recent studies suggest that genes on the chromosomes of these pathogens are likely to play important roles in pathogenesis (1, 4, 9, 12, 13, 22, 34).

We and others have shown that most S. flexneri 2a and EAEC strains share a chromosomal locus, designated pic/set, which encodes at least two putative virulence factors (1, 4, 9). In S. flexneri, pic/set is encoded on a pathogenicity island called SHI-1 (1); the sequence of the island encoding pic/set in EAEC has not been reported. pic/set encodes a high-molecular-weight mucinase (Pic), which is a serine protease secreted by the autotransporter mechanism (12). Pic degrades intestinal mucin and may play a role in mucosal colonization by both EAEC and S. flexneri 2a strains. Encoded on the opposite strand but completely within the pic gene is an oligomeric enterotoxin called Shigella enterotoxin 1 (ShET1), which is thought to comprise a single 20-kDa catalytic A subunit (encoded by the setA gene) and five 7-kDa B subunits (encoded by the setB gene) (9). The ShET1 mode of action has not been defined, but it does not appear to act via the traditional mediators of toxin-induced intestinal secretion, such as cyclic AMP and cyclic GMP (10).

S. flexneri and EAEC have different pathogenetic strategies. S. flexneri is a large-bowel pathogen which causes invasive and inflammatory colitis (for a review, see reference 11). Many shigellosis patients experience a watery prodromal phase, which may be a manifestation of early small-bowel involvement and to which ShET1 may contribute. In contrast, EAEC are thought to be distal small-bowel and/or colonic pathogens which typically cause watery diarrhea without evidence of invasion or frank inflammation (26). Nevertheless, recent data suggest that EAEC may induce mild inflammatory enteritis (31).

We approached the characterization of the pic/set locus with the aim of understanding when in the pathogenic sequence these loci are expressed and how this expression is controlled. Clarification of these events will provide further understanding of EAEC and S. flexneri pathogenesis and may also shed light on signals recognized by enteric pathogens in general. Our data suggest that both loci are expressed in the intestinal lumen; moreover, our results suggest a novel mechanism of ShET1 regulation and the existence of pathogen-specific regulators of pic and setBA. We also report for the first time the use of a continuous anaerobic culture to study expression of virulence genes.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Strains and plasmids used in this study are listed in Table 1. All E. coli strains were grown aerobically at 37°C in Luria-Bertani (LB) medium unless otherwise stated. Antibiotics were added at the following concentrations when appropriate: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; chloramphenicol, 50 µg/ml, and tetracycline, 50 µg/ml.

lacZ fusion analysis. To monitor transcriptional activities, defined fragments from the pic/set locus were amplified by PCR using primers carrying EcoRI and BamHI restriction sites. Fragments were directionally cloned into compatible sites in the promoterless lacZ reporter vector pRS551 (30). Clones are listed in Table 1, and primers are listed in Table 2. Cloned fragments were confirmed by plasmid isolation and restriction, PCR, and DNA sequencing. ß-Galactosidase activity, expressed in Miller units, was measured as described previously (20).

RNA extraction and RT-PCR techniques. Bacterial strains were grown to exponential phase, and whole-cell RNA was isolated using the RNeasy kit (Qiagen Inc., Valencia, Calif.) according to the manufacturer's instructions. RNA was treated with RNase-free DNase to eliminate contaminating DNA. cDNA was synthesized using sequence-specific primers (Table 2) and the Superscript enzyme from Gibco-BRL (Gaithersburg, Md.) for 10 min at 55°C. PCR was performed using standard procedures with Taq DNA polymerase (BRL). Controls for all reactions included samples without reverse transcriptase (negative control) and the relevant purified plasmid DNA (positive control).

The abundance of mRNA transcripts for pic and set genes was determined by real-time quantitative reverse transcription (RT)-PCR. RNA extracted as above was tested for DNA contamination by 40 cycles of standard PCR with the same primers used in the RT-PCR amplification. Single-step RT-PCR was performed with the Superscript enzyme in the presence of SYBR Green I dye (Roche Molecular Biochemicals, Mannheim, Germany), followed by PCR performed according to the manufacturer's instructions using AmpliTaq Gold polymerase (Roche). Fluorescence was detected with the GeneAmp 5700 sequence detection system and accompanying software. The threshold for positivity was set at 1. The constitutively expressed cat (chloramphenicol acetyltransferase) gene of E. coli O42 was used as an internal standard for reactions involving this strain; the tet gene of pACYC184 was used as the standard for experiments involving pPic1. For each primer pair, a standard curve was generated by using known amounts of purified pic/set plasmid pPic1 (12). Unless otherwise indicated, experiments were performed with exponential-phase cells grown in L-broth.

Simulated human intestinal microbial ecosystem. The simulated intestinal ecosystem was essentially that described in references 7 and 21, with the modifications described below (Fig. 1). The system comprised a continuous anaerobic culture of human feces maintained for at least 8 days at 37°C and pH 6.9 (Fig. 1). The growth medium of the culture has been shown previously to provide a stable culture of colonic bacteria and is designed to mimic the conditions of the human transverse colon (Table 3) (21). The volume of the culture was 500 ml, and flow was set to provide turnover of 1 culture volume/24 h. The inoculum consisted of 50 g of fresh feces, which was preincubated in 100 ml of fermentor medium for 6 h with stirring. Thereafter, 60 ml of this starter culture was inoculated into the main fermentor culture. The fecal inoculum was cultured quantitatively for aerobic and anaerobic bacteria as described in reference 32.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Diagram of the apparatus used for continuous culture of human fecal bacteria. See Materials and Methods for a detailed description.

After the fermentor had been stabilized for at least 7 days, a sample was withdrawn and cultured again quantitatively for aerobic and anaerobic flora. Only if the bacteriologic profile was similar to that of the fecal inoculum was the experiment continued.

To study gene regulation in the fermentor, E. coli 042 harboring different lacZ fusion plasmids was introduced into the fermentation vessel. The preinoculation baseline ß-galactosidase activity of the culture in the fermentation vessel was determined at steady state. This value was subtracted from all subsequent measurements. An overnight culture of E. coli 042 harboring a pRS551 lacZ fusion construct was then introduced into the fermentation vessel to an initial concentration of 5 x 10⁶ CFU/ml, and the ß-galactosidase activity was immediately sampled after mixing (time zero). The third and final measurement was carried out after 24 h of incubation. The number of E. coli 042 CFU was determined at time zero and after 24 h of incubation by plating dilutions on LB-agar plates containing ampicillin (100 µg/ml); cultures from the fermentation vessel without added E. coli 042 did not yield growth on these plates. Miller units were calculated according to the standard equation (20), except that the number of 042 CFU was converted to an optical density at 600 nm (OD₆₀₀) value at the ratio of 1 OD₆₀₀ = 10⁹ CFU, determined to have a protein concentration of 0.2 µg/ml.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transcriptional organization of pic and set genes. The pic/set loci from S. flexneri 2a strain 2457T and EAEC strain 042 were cloned and sequenced previously (12). Both sequences comprise the pic structural gene of 4,116 nucleotides and, on the complementary strand, the setA gene (534 nucleotides) and the setB gene (186 nucleotides) (Fig. 2A). The loci from the two strains are 99% identical.

View larger version (47K): [in this window] [in a new window]   FIG. 2. Organization of the pic/set locus. (A) Map of the chromosomal DNA fragment encoding pic and setBA and results of RT-PCR experiments to determine the initiation and termination sites of set transcription. Locations of nested set primers are indicated, along with presence (solid line) or absence (dotted line) of product generated by that primer pair. (B) Agarose gel electrophoresis demonstrating products of RT-PCR from the pic/set locus, using the primers illustrated in panel A. RNA was extracted from EAEC strain 042 and subjected to RT synthesis and cDNA amplification by standard PCR (cDNA lanes). A direct PCR step was carried out in parallel on 042 DNA to demonstrate the specificity of the PCR (DNA lanes). The primer pairs used are shown above the lanes. Molecular size markers are shown in the first lane of each gel; the sizes of some markers are indicated.

In contrast to pic, sequence analysis did not provide clues as to the likely start sites for setB or setA transcription. When subjected to RT-PCR analysis (Fig. 2) with a series of overlapping primer pairs from the upstream region of the set strand, only primer pair 21FRT and 22RRT failed to yield a product; these data suggest that the most upstream setB transcription start site lies between coordinates 4958 (the position of primer 20FRT) and 5218 (the position of primer 21FRT) in Fig. 2A, i.e., at least 1.6 kb upstream of the setB start codon and downstream of the pic stop codon on the opposite strand. Interestingly, of the setA downstream primers, only 26RRT failed to generate a product when paired with any other primer. This experiment suggests the presence of a transcriptional terminator of set mRNA upstream of the pic start codon on the opposite strand. Moreover, our RT-PCR data using overlapping primers suggested the presence of a setBA polycistronic mRNA, since primer pair 35RRT and 33FRT span the junction of the two open reading frames (ORFs).

RT-PCR experiments thus directed us toward the flanking regions of pic as the likely location of promoters for both pic and set, albeit on opposite strands. Primer extension experiments of the pic gene using automated sequencing protocols produced multiple potential start sites, without any clearly preferred site. One potential start site corresponded to the previously predicted ⁷⁰ promoter 19 bp upstream of the pic start codon (12), but additional potential sites were found upstream.

To better characterize the pic promoters, we cloned a series of pic upstream DNA fragments into the multiple cloning site of promoterless lacZ reporter vector pRS551 (Fig. 3) and electroporated the plasmids into EAEC strain 042. Three regions of interest were designated P_pic1 (between coordinates 21 and 851 of Fig. 3), P_pic2 (between coordinates 851 and 933), and P_pic3 (between coordinates 934 and 1131 and including the predicted promoter from reference 12). The transcriptional start site suggested by RT-PCR mapped between coordinates 537 and 693, within the P_pic1 region. However, lacZ fusion data suggested that this promoter was weak under the conditions tested (compare pP15/2 and pP16/2 in Fig. 3). Fragment P_pic2 yielded the highest lacZ expression levels, yet, a lacZ reporter fusion with P_pic2 alone (pP16/20) yielded threefold less ß-galactosidase activity than a fusion including both the P_pic2 and P_pic3 regions (pP16/2). A fusion construct comprising only P_pic3 (pP3/2) yielded 20-fold-higher activity than a construct that did not include any of the predicted promoter regions (pP4/2). A fusion construct comprising only the P_pic1 region (21 to 851) yielded ninefold higher activity than pRS551 alone (not shown in the figure). These data suggest that multiple promoters direct pic expression.

View larger version (34K): [in this window] [in a new window]   FIG. 3. ß-Galactosidase activity derived from pic and set promoter fusions either in vector pRS551 transformed into EAEC 042 or as single-copy chromosomal integrations in E. coli K-12 strain TE2680. Values are the results of triplicate determination + standard deviation. The plasmids are described in Table 2. The numbers above the inserts refer to nucleotide positions with respect to Fig. 2. pic fusions are expressed from left to right and set fusions from right to left with respect to the lacZ gene. ND, not determined.

Surprisingly, DNA fragments comprising the predicted P_setB promoter but also including additional downstream DNA suggested the presence of a repressing downstream regulatory element (DRE) or silencer region. A series of lacZ fusion constructs localized the setB DRE between positions 4958 and 3396 (Fig. 3). Analyzing a further series of deletions in the predicted DRE, we found that even small segments (ca. 50 to 60 bp) of this element extending to either end were sufficient to repress P_setB activity. The deletion of the entire predicted DRE (pS41/19) restored P_setBA activity almost to the levels exhibited by pS25/26.

Since all of the above fusions were constructed in high-copy-number plasmid pRS551, we transferred the lacZ fusions onto the chromosome of the E. coli K-12 strain TE2680 in single copy. The relative activities of the different fusions integrated into the TE2680 chromosome were similar to the activities observed in the extrachromosomal constructs (Fig. 3). These experiments suggest that our data were not influenced by copy number or topologic effects.

Orientation-specific regulation of pic and set genes by set silencer region. Given that the setB DRE is embedded within the pic sequence, we asked whether the set silencer acted only on the set strand or instead bidirectionally, thereby also silencing pic expression. The lacZ reporter fusion plasmids pP16/2 (comprising P_pic2,3) and pS25/26 (P_setB) were used in these experiments. The silencer region was amplified by PCR and cloned in both orientations downstream of P_pic2,3 or P_setB. Results of ß-galactosidase assays for all four constructs are shown in Fig. 4.

View larger version (31K): [in this window] [in a new window]   FIG. 4. DNA strand-specific regulation of the pic and set genes by the set DRE silencer region. Results represent ß-galactosidase measurements of at least triplicate determinations from L-broth cultures grown to late logarithmic phase; error bars represent 1 standard deviation. Test strains were EAEC strain 042 transformed with pic or set promoter DNA cloned upstream of the lacZ reporter in plasmid pRS551. Constructs used in these experiments were pS37/19 (setB promoter with the silencer in the native orientation); pS37/19-inverted (setB promoter with the silencer in the inverted orientation), pP37/19 (Ppic2,3 promoter region with the silencer in the native orientation), and pP37/19-inverted (Ppic2,3 promoters with the silencer in the inverted orientation).

Conditions affecting regulation of pic and set transcription. We sought to identify conditions under which pic and set were maximally expressed. We tested lacZ expression of fusions pP15/2 (P_pic1,2,3) and pS23/19 (P_setB with silencer) under the following conditions: carbon starvation, anaerobiosis, iron limitation, and adherence to plastic, as well as varied pH, osmolarity, temperature, and growth phase. None of the conditions yielded P_setB activity significantly higher than that in the negative control, whereas a baseline level of pic expression was observed in all conditions at 37°C. Only logarithmic growth phase produced any detectable increase in pic expression over baseline (data not shown).

To characterize further the effect of growth phase in the EAEC strain 042 background, we employed real-time quantitative RT-PCR for pic and setBA transcripts. Whole-cell RNA was isolated from strain 042 at three time points after nutrient upshift (0.5, 1.5, and 6.5 h), and cDNA was synthesized as the template for quantitative RT-PCR. For the pic gene, we detected clear growth phase-dependent expression (Fig. 5A). However, the abundance of the setBA transcript displayed no significant change during growth, confirming that different signals are likely to be responsible for regulation of the pic and setBA genes.

View larger version (16K): [in this window] [in a new window]   FIG. 5. (A) Effect of growth phase on pic and set expression as determined by real-time quantitative RT-PCR. RNA was extracted from E. coli 042 grown in L-broth for various times (0.5, 1.5, and 6.5 h) after 1:20 dilution of an overnight culture with fresh medium. cDNA was synthesized and subjected to quantitative RT-PCR as described in Materials and Methods. (B) ß-Galactosidase activity of E. coli 042 harboring plasmid pP15/2 (Ppic1,2,3), pP16/2 (Ppic2,3), or pP3/2 (Ppic3) grown in L-broth and assayed at various times after dilution of overnight cultures with fresh medium.

From these studies, we infer that (i) region P_pic1 is weak under all conditions tested, (ii) P_pic2 harbors a strong promoter inducible during growth, and (iii) the promoter within P_pic3 may be constitutively expressed but at low levels. These observations suggest that the P_pic2 promoter is the major source of pic expression.

Notably, we were unable to identify any in vitro conditions that induced expression of setB. Since silencing in E. coli typically involves binding of a histone-like protein to a DRE (3, 5, 14, 16, 18, 27, 35-38), we analyzed lacZ expression from construct pS23/19 (P_setB with silencer) in E. coli K-12 strains carrying known mutations in Fis, H-NS, or the H-NS homolog StpA. lacZ expression was not derepressed in any of these mutants compared with the E. coli K-12 parent (not shown).

Regulation of pic and set genes in EAEC, E. coli K-12, and S. flexneri. Maurelli et al. have reported that Shigella strains have undergone a large chromosomal deletion which results in augmentation of enterotoxic effects (19). This observation could be mediated in part by different regulatory controls of ShET1 operative in nonpathogenic E. coli versus Shigella spp. To address this hypothesis, lacZ reporter fusions with P_pic1,2,3 (pP15/2), P_pic2,3 (pP16/2), P_pic3 (pP3/2), P_setB (pS25/26), P_setB with the full silencer region (pS23/19), and P_setA (pS8/1) were transformed into EAEC strain 042, E. coli K-12, and S. flexneri 2457T (Fig. 6).

View larger version (21K): [in this window] [in a new window]   FIG. 6. Transcriptional regulation of pic and set in EAEC strain 042, E. coli K-12, and S. flexneri 2457T. Plasmids containing all three pic promoter regions (Ppic1,2,3), two pic promoter regions (Ppic2,3), the most downstream pic promoter region (Ppic3), the setB promoter (PsetB), the setB promoter with the DRE silencer region, or the setA promoter (PsetA) constructed in pRS551 were transformed into EAEC 042, S. flexneri 2457T, or E. coli K-12 and tested for ß-galactosidase activity. Bacterial strains were grown to exponential phase in L-broth medium at 37°C to the late logarithmic phase. Bars represent results of at least three determinations; error bars represent 1 standard deviation.

Regulation of setB transcription in a simulated human intestinal microbial ecosystem. Using conventional approaches, we were not able to identify in vitro conditions or mechanisms that resulted in relief of P_setB silencing. We therefore expected either that the DRE was continually repressed, providing constitutively low levels of the ShET1 toxin, or that the bacterium would respond to some in vivo signal to induce toxin expression.

Given that no good animal models are available for either Shigella or EAEC infection, we tested our hypothesis in a simulated human intestinal ecosystem. Continuous-flow anaerobic cultures have been used previously to study the ecology of the complex intestinal ecosystem (7, 21). Adapting these published protocols, we established such a continuous anaerobic culture, inoculated with fecal material from healthy humans. The baseline ß-galactosidase activity in the fermentor was determined before adding the test strain; the test strain, EAEC strain 042 harboring a lacZ reporter construct, was added to the vessel, and ß-galactosidase activity was measured again. The last ß-galactosidase determination was performed 24 h after addition of the test strain. Results are reported in Table 4.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mucinase Pic and Shigella enterotoxin 1 are recently described factors which may play important roles in the virulence of S. flexneri 2a and EAEC. Whereas recent research has characterized aspects of the pathogenetic schemes for both of these pathogens, significant questions remain. Among these questions is the mechanism of the watery diarrhea phase seen in many cases of shigellosis. In addition, it is notable that S. flexneri 2a is the single most prevalent Shigella species worldwide (17); the mechanism for this increased virulence or transmissibility is not known, but the presence of pic/set in this serotype is one possible explanation.

In this study, we have characterized the transcriptional regulation of the pic and set genes on the complementary strands in S. flexneri strain 2457T and EAEC strain 042. Although the major promoter could not be assigned with certainty, the region upstream of pic yielded multiple fragments with promoter activity, and overall expression of pic was highest in the exponential growth phase. Promoter region P_pic3 contains a predicted ⁷⁰ promoter and increases lacZ expression in several different constructs, but with only slightly increased expression in exponential phase. Region P_pic2 does not contain a strongly predicted ⁷⁰ promoter, but it does confer strong growth phase-dependent lacZ expression in a reporter vector. RT-PCR suggests the presence of a transcript starting further upstream (P_pic1), and expression in a promoter-screening vector suggests that there is a functional promoter in this region as well. In toto, these observations suggest that the nutrient upshift and favorable temperatures experienced by the bacteria upon entry into the human intestine may induce pic expression from the major promoter, making the mucinase available at an early stage of the infection. It is possible that a second promoter (possibly P_pic3) ensures a constitutive low level of the mucinase, perhaps to facilitate the acquisition of nutrients once the bacterium is embedded within the mucus layer.

Expression studies of setBA yielded several surprises. Our RT-PCR data were most consistent with the existence of a ca. 4-kb transcript which encodes both setA and setB. We were not, however, able to demonstrate the existence of such a transcript by Northern blot, most likely because of the weak expression levels observed during in vitro growth. setA expression may initiate from a promoter immediately upstream of the structural gene or by readthrough from the setB promoter.

We found it notable that the strongest setB promoter was located at a site downstream of the pic stop codon on the opposite strand and that transcription from P_setB is silenced by a DRE. Well-characterized examples of such a downstream prokaryotic silencer have been reported for the proU operon of Salmonella enterica serovar Typhimurium (27), the hly operon of E. coli (15), the bgl promoter of E. coli (29), and the coo operon of enterotoxigenic E. coli (24). For proU and hly, the DRE spans some 200 bp, while for coo, the DRE is at least 900 bp long. The size of the bgl DRE has not been determined precisely. At 1,562 bp (by our definition), the DRE of the set operon would be the largest prokaryotic DRE yet delimited. We have also demonstrated that the setB DRE is orientation specific; only transcription starting from the setB promoter is silenced, whereas transcription of the pic gene is not repressed in the native orientation. On the other hand, inversion of the DRE leads to silencing of pic and not setB. In contrast, the bgl DRE can be inverted without impairing its activity (29). Also notably, the proU silencer does not regulate the tac promoter when cloned in a downstream position (27), whereas the bgl silencer is able to repress other promoters (lac and lacUV5).

The E. coli histone-like proteins Fis, H-NS, and StpA are apparently not individually responsible for the silencing effect of the setB DRE. This was surprising, since Fis and/or H-NS is essential for silencing of proU (27), bgl (29), and coo (2, 23, 24, 33). It is possible that these histone-like proteins may need to act in concert at the setB DRE or that another mechanism may be involved. Deletions which retained small portions from either end of the setB silencer region resulted in effective repression; only complete deletion of the region yielded relief of the repressed phenotype. The ability of small DNA regions to effect repression strongly argues against silencing by virtue of an RNA or DNA secondary structure.

We sought to identify in vitro conditions that relieved setB silencing, principally to suggest the timing of set expression in vivo. Surprisingly, were unable to do so by conventional methods. We therefore employed a novel approach which provides multiple in vivo-like conditions simultaneously, including temperature, pH, osmolarity, low oxygen tension, mucin, and the presence of quorum-sensing signals. We found that setB silencing was relieved (i.e., increased up to 54-fold) in a fermentor simulating the human intestine. The precise set of signals provided by the simulator are not so far apparent. Simulated human intestinal microbial ecosystems have been described by several groups (7, 21), but to our knowledge the simulator has not been used previously to study gene regulation.

Use of a simulated intestinal environment offers a number of obvious advantages in the study of gene regulation. The fermentor allows exposure of the pathogen to multiple conditions that are likely to be encountered in vivo. Just as importantly, the simulator can be modified to dissect the critical signals by removing or altering characteristics singly or in combination. The system is also more easily manipulated and sampled than an animal model, and significantly, the system can be tuned to the conditions of the human intestine, thereby avoiding erroneous observations drawn from interspecies variation in the flora or other conditions of the intestine. Further characterization of the expression of pic, set, and other virulence genes in the simulator is under way.

Although the pic and set loci are nearly identical in S. flexneri and EAEC, previous data suggest that the ShET1 toxin is more active in Shigella spp. Maurelli et al. have suggested that this observation is due to the deletion of a large region of the Shigella chromosome (the so-called black hole), which results in enhanced enterotoxic effects (19). These authors have suggested further that cadaverine, a product of the lysine decarboxylase reaction, may directly inhibit toxin activity. Our data suggest that the expression of the set genes may be enhanced in Shigella spp. and EAEC compared with E. coli K-12. Whether this effect is due to the loss of a negative regulator encoded on the black hole or to the presence of an additional pathogen-specific activator is not yet clear.

Our studies have characterized a novel locus in S. flexneri 2a and EAEC. It has not escaped our notice that additional virulence genes could be encoded by the setB mRNA. Indeed, two potential ORFs (>50 amino acids) are found within the DRE region; these are currently being characterized. Further studies to elucidate the roles of pic/set will likely provide new insights into the pathogenesis of Shigella spp. and EAEC.

	ACKNOWLEDGMENTS

 
This work was supported by U.S. Public Health Service awards AI33096 and AI43615 to J.P.N.

We are grateful to Marlene Belfort for bacterial strains and Nicholas Carbonetti and Jay Mellies for critical review of the manuscript. We acknowledge the excellent technical assistance of Suzanne Davis and Thomas J. Maher III.

	FOOTNOTES

 
* Corresponding author. Mailing address: 685 W. Baltimore St., Baltimore MD 21201. Phone: (410) 706-5328. Fax: (410) 706-6205. E-mail: jnataro{at}medicine.umaryland.edu.

Editor: D. L. Burns

Present address: Limetec Biotechnologies GmbH, 16321 Bernau bei Berlin, Germany.

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