1 Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, USA
2 Seattle Biomedical Research Institute, 4 Nickerson St, Seattle, WA 98109, USA
3 Departments of Pathobiology and Microbiology, University of Washington, Seattle, WA 98195, USA
Correspondence
Nancy E. Freitag
nfreitag{at}sbri.org
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ABSTRACT |
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Abbreviations: GUS, ß-glucuronidase; LLO, listeriolysin O
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INTRODUCTION |
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We have been using the facultative intracellular bacterial pathogen Listeria monocytogenes as a model system to define the mechanisms used by a prokaryotic parasite to regulate virulence gene expression within host cells. L. monocytogenes is responsible for serious infections in immunocompromised individuals and pregnant women (Gray & Killinger, 1966; Gellin & Broome, 1989
; Vazquez-Boland et al., 2001
). The bacterium expresses a variety of virulence factors that enable it to invade many host cell types and to gain entry into the cell cytosol where bacterial replication occurs. L. monocytogenes regulates the expression of virulence genes in response to specific host cell compartment environments, including those of the phagosome and cytosol (Bubert et al., 1999
; Freitag & Jacobs, 1999
; Moors et al., 1999
; Kreft & Vazquez-Boland, 2001
). The majority of the L. monocytogenes virulence determinants that have been thus far identified are regulated by a transcriptional activator known as PrfA, a 27 kDa site-specific DNA binding protein that is essential for L. monocytogenes pathogenesis (Mengaud et al., 1991
; Chakraborty et al., 1992
; Freitag et al., 1993
).
While it is clear that PrfA is a key regulatory element required for the control of virulence gene expression in L. monocytogenes, it is not clear what controls its activity or how prfA expression is regulated. Three distinct promoters contribute to prfA expression. Two promoters, prfAP1 and prfAP2, are located immediately upstream of the prfA coding region (Freitag & Portnoy, 1994) (Fig. 1
). The third promoter is contributed by the upstream plcA gene through the generation of a bicistronic plcAprfA transcript (Camilli et al., 1993
; Freitag et al., 1993
). PrfA positively regulates its own expression through the activation of plcA transcription, and the increase in PrfA synthesis resulting from the generation of the prfAplcA transcript is essential for spread of the bacteria from the initial infected cell to adjacent cells and for full virulence (Camilli et al., 1993
; Freitag et al., 1993
; Freitag & Portnoy, 1994
). PrfA also appears to negatively regulate its own expression from the prfAP1 and prfAP2 promoters, as transcripts directed by these two promoters are significantly increased in the absence of functional PrfA (Freitag et al., 1993
). It has been proposed that L. monocytogenes regulates the amounts of available PrfA protein through the use of a negative feedback loop that involves PrfA binding to the -35 region of prfAP2 and inhibition of transcriptional initiation at that promoter (and possibly at prfAP1) by an unknown mechanism (Freitag & Portnoy, 1994
). The existence of this negative feedback loop has been postulated to be an important facet of L. monocytogenes virulence gene expression.
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METHODS |
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Construction of L. monocytogenes prfA promoter mutants.
The introduction of targeted deletion and substitution mutations within the prfA promoter region was accomplished using the Kunkel method of site-directed DNA mutagenesis with M13 bacteriophage-derived vectors as previously described (Ausubel et al., 1991; Freitag & Portnoy, 1994
). prfA mutant constructs were sequenced to verify desired mutations and then fragments containing the mutations were subcloned into the thermo-sensitive shuttle plasmid vector pKSV7 (Smith & Youngman, 1992
). Mutations were introduced into the L. monocytogenes chromosome of strain NF-L476 [containing a transcriptional fusion of actA with the gus reporter gene (Shetron-Rama et al., 2002
)] by allelic exchange as previously described (Freitag & Portnoy, 1994
) to generate strains NF-L623 (containing plcA-
T +prfA
P2-35); NF-L625 (containing plcA-
T); NF-L627 [containing prfA
P2(hly)]; and NF-L629 (containing prfA
P2-35). Chromosomal mutations were confirmed by sequencing of PCR amplified products derived from genomic DNA isolated from each mutant strain.
Introduction of prfA-7973 (prfA*) into L. monocytogenes mutant strains.
L. monocytogenes strain NCTC 7973, a natural isolate, has been described (Park & Kroll, 1993; Millenbachs et al., 1997
; Behari & Youngman, 1998
). The NCTC 7973 prfA allele, which contains a serine in place of a glycine at position 145, produces increased expression of virulence genes in NCTC 7973 in comparison to wild-type L. monocytogenes and is thought to represent a transcriptionally active, cofactor-independent form of PrfA protein (PrfA*) (Ripio et al., 1997
). The NCTC 7973 PrfA also has a second amino acid change (in comparison to 10403S PrfA), a Cys to Tyr change at position 229; however this substitution has not been demonstrated to influence PrfA-dependent gene expression (Behari & Youngman, 1998
). pCON1-
prfA7973 was used to insertionally inactivate the wild-type prfA allele and to introduce the prfA* allele under the control of the wild-type prfA promoter (Behari & Youngman, 1998
). pCON1-
prfA7973 conjugated into 10403S derivatives NF-L476, NF-L623, NF-L625, NF-L627 and NF-L629 to generate NF-L657 (prfA* actAgusplcB); NF-L653 [prfA*
P2(hly) actAgusplcB]; NF-L654 [plcA-
T prfA* actAgusplcB]; NF-L655 (plcA-
T+prfA*
P2-35 actAgusplcB); and NF-L656 (prfA*
P2-35 actAgusplcB). Transconjugants were isolated as described by Behari & Youngman (1998)
with the following modifications: transconjugants were selected on BHI agar containing chloramphenicol (5 µg ml-1) after a 24 h incubation at 30 °C. Selected colonies were used to inoculate BHI media containing chloramphenicol and streptomycin, and grown overnight at 40 °C with shaking to force chromosomal integration of the vector. Overnight 40 °C cultures were then diluted 1 : 1000 into fresh BHI containing chloramphenicol and streptomycin, and again incubated at 40 °C overnight with shaking. Appropriate dilutions of overnight cultures were plated on BHI agar containing 5 µg chloramphenicol ml-1 and incubated at 40 °C. Integration of the vector into the chromosome and confirmation of the prfA7973 (prfA*) sequences were verified by PCR amplification of prfA from genomic DNA and sequencing of the PCR products.
ß-Glucuronidase (GUS) assays of bacteria grown in liquid culture.
For experiments using LB with and without added glucose or cellobiose, overnight cultures of bacteria grown in LB medium were diluted 1 to 10 into fresh LB buffered with 100 mM MOPS (pH 7·4) with or without glucose or cellobiose at the indicated concentrations and grown for 5 h with shaking at 37 °C. Bacterial pellets from 1 ml culture aliquots were collected following centrifugation and were quickly frozen on dry ice. Optical density (at 595 nm) was measured for each culture using a Spectronic 20 spectrophotometer (Milton Roy). For GUS enzymic assays, bacterial cell pellets were thawed, washed once with ABT buffer [0·1 M potassium phosphate (pH 7·0), 0·1 M NaCl, 0·1 % (v/v) Triton X-100] and resuspended in 200 µl ABT buffer. GUS activity was measured as described by Youngman (1987) with the substitution of 4-methylumbelliferyl ß-D-glucuronide in place of 4-methylumbelliferyl ß-D-galactoside.
Western analysis of PrfA protein levels.
Polyclonal rabbit antiserum was generated against amino-terminal histidine-tagged PrfA protein produced using the Qiagen pQE30 vector in E. coli and purified by nickel column chromatography as recommended by the manufacturer (Qiagen). For Western analysis, overnight cultures of L. monocytogenes grown in LB broth at 37 °C without shaking were diluted 1 : 100 into fresh LB broth buffered to pH 7·4 with 100 mM MOPS buffer and grown for 6 h at 37 °C with shaking. For experiments designed to measure PrfA protein levels in LB broth buffered to pH5·8 or in the presence of glucose or cellobiose, overnight cultures grown in LB were diluted 1 : 100 into fresh LB broth buffered with 100 mM MES to a pH of 5·8, or into LB broth buffered with 100 mM MOPS to pH7·4 plus 25 mM glucose or 25 mM cellobiose. A 20 ml aliquot of each culture was briefly centrifuged to recover the cell pellet; the pellet was resuspended in 500 µl Cell Lysis Buffer (50 mM Tris/HCl, pH7·5, 1 mM DTT, 0·1 % (v/v) Triton X-100). Bacterial suspensions were disrupted using a Mini-Beadbeater (Biospec Products) with 1 min cycles followed by 30 s cooling on ice, for a total of 3 cycles. Samples were then centrifuged at 13 000 g for 5 min to recover supernatants. Protein concentration was determined using the DC Protein Assay Kit as recommended by the manufacturer (Bio-Rad Laboratories). Equivalent amounts of total protein from each culture lysate were mixed with equal volumes of 2xSDS-PAGE buffer [60 mM Tris/HCl (pH6·8), 2 % (w/v) SDS, 10 % (v/v) glycerol, 10 % 2-mercaptoethanol, 0·01 % (w/v) bromophenol blue]. Samples were heated to 90 °C for 5 min prior to being subjected to SDS-PAGE on 10 % polyacrylamide gels. Proteins were visualized by staining the gel with Coomassie brilliant blue prior to transfer. Proteins were transferred to nitrocellulose membranes and incubated with a 1 : 200 dilution of polyclonal antibody generated against purified PrfA protein, followed by incubation with an alkaline phosphatase-coupled goat anti-rabbit antibody (Sigma). PrfA protein antigen was detected using the Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets, a colorimetric alkaline phosphate substrate.
For quantitative comparison of PrfA protein levels as visualized by Western analysis, 2 µg, 8 µg and 15 µg purified PrfA protein were compared with serial dilutions of protein extracts derived from the wild-type and prfA promoter mutant strains to determine the relative amount of PrfA produced by each mutant.
Assay for haemolytic activity.
Stationary phase bacteria were diluted 1 : 10 into BHI and grown at 37 °C for 5 h with shaking. The supernatant fluid was assayed for haemolytic activity as previously described (Camilli et al., 1989).
Plaque formation in L2 cells.
Plaque assays were performed as previously described by Sun et al. (1990). Plaque size was measured using a micrometer and the mean diameter of at least 10 plaques from three independent experiments was determined.
LD50 determinations.
The LD50 were determined in BALB/c mice by intravenous injection as previously described (Portnoy et al., 1988).
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RESULTS |
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Analysis of the effects of prfA promoter mutations on PrfA protein levels
The prfAP2-35 mutation has been previously demonstrated to increase levels of transcripts initiating at the prfAP1 promoter (Freitag & Portnoy, 1994
). However, PrfA protein levels were not examined in this strain, and it was important to determine whether increased mRNA levels directly correlated with an increase in protein levels. To directly compare the levels of PrfA present in each of the prfA promoter mutant strains, extracts derived from wild-type L. monocytogenes and the promoter mutants were examined by Western analysis using a rabbit polyclonal antibody directed against purified PrfA protein. As shown in Fig. 2
, all of the L. monocytogenes strains containing prfA promoter mutations produced dramatically higher levels of PrfA protein than the wild-type strain. Each of the prfA promoter mutants produced at least five times more PrfA protein as determined by comparison of extract dilutions with known amounts of purified PrfA (S. Greene & N. Freitag, data not shown). It has been previously reported that a threefold increase in PrfA protein levels occurs in bacteria that reside within the host cytosol, and that this increase appears sufficient for full induction of intracellular gene expression (Renzoni et al., 1999
). The prfA promoter mutations therefore appeared to result in increased synthesis of PrfA protein in broth-grown cultures to levels equivalent to those observed for intracellular bacteria via the alleviation of negative regulation of prfA expression.
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Despite the high levels of PrfA protein observed in each of the mutant strains, none of the mutants produced significantly increased levels of LLO (Table 1). In contrast, significant increases in the levels of actA expression were observed for all prfA promoter mutant strains following growth in LB broth, with the sole exception of mutant plcA-
T which had levels of expression equivalent to those observed for wild-type L. monocytogenes (Fig. 3
). The prfA
P2(hly) mutation resulted in the highest levels of actA expression, approximately eightfold greater than the levels observed for wild-type bacteria grown in broth culture. These results suggest that increased synthesis of PrfA protein influences a subset of PrfA-regulated genes (such as actA) and does not result in increased levels of all PrfA-dependent gene products (such as LLO).
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Intracellular growth of the prfA promoter mutants was also examined in a variety of host cell types, including mouse macrophage-like tissue culture cell lines. No defect in intracellular growth or cell-to-cell spread was detected (S. Greene & N. Freitag, unpublished data). Loss of prfA negative regulation also resulted in no significant defect in virulence following intravenous injection of mice for any of the mutant strains (LD50 2x104 c.f.u. for parent strain NF-L476, and <105 c.f.u. for each mutant).
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DISCUSSION |
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We had anticipated that the loss of prfA negative regulation would be amplified in the presence of the constitutively activated PrfA* form of the protein. The G145S substitution within PrfA* has been postulated to produce a conformational change in the protein that results in activation of PrfA in the absence of cofactor binding (Ripio et al., 1997; Vega et al., 1998
). As it has been demonstrated that an approximately threefold induction of PrfA synthesis occurs during infection of mammalian cells (Renzoni et al., 1999
), the fivefold increase in PrfA* observed for the promoter mutants might have been sufficient to induce actA expression to the levels observed in cytosolic bacteria (Shetron-Rama et al., 2002
). However, the prfA* promoter mutants did not show a significant increase in actA expression in comparison to strains containing prfA* in the presence of the native promoter, and the approximately tenfold induction of actA expression measured for the prfA* strains was still well below the greater than 200-fold induction observed for bacteria located within the host cytosol (Moors et al., 1999
; Shetron-Rama et al., 2002
). These results strongly support the premise that other factors besides PrfA are required for optimal induction of actA expression in L. monocytogenes and that the expression of these factors is likely to be PrfA-independent. Work in progress has indicated that it is possible to isolate L. monocytogenes mutants with increased actA expression in broth culture which contain mutations mapping outside of the prfA regulon (L. Shetron-Rama & N. Freitag, unpublished results). The participation of additional regulatory factors in the induction of L. monocytogenes gene expression within infected host cells further illustrates the resources used by the bacterium to ensure that specific gene products are synthesized within the proper host cell environment.
Why does negative regulation of prfA expression exist if it does not contribute to virulence in mammals? L. monocytogenes is a ubiquitous bacterium that occupies a variety of habitats. PrfA has been reported to negatively influence the expression of genes required for environmental stress, such as clpC (Ripio et al., 1998) and has been implicated in the down-regulation of stress resistance mechanisms during exponential growth (Herbert & Foster, 2001
). Genes required for bacterial motility, such as motA, have also been reported to be downregulated by PrfA (Michel et al., 1998
). It is possible that negative regulation of prfA expression plays an important role in facilitating bacterial survival outside host cells by ensuring that gene products required for survival in the extracellular environment are expressed. It is tempting to speculate, therefore, that while positive regulation of prfA expression is of paramount importance during mammalian infection, negative regulation has evolved to sustain bacterial survival in the outside environment. Confirmation of this hypothesis awaits detailed studies of factors contributing to L. monocytogenes survival within its ubiquitous habitats.
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ACKNOWLEDGEMENTS |
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This work was supported by Public Health Service grant AI41816 to N. E. F.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Behari, J. & Youngman, P. (1998). Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA. Infect Immun 66, 36353642.
Bohne, J., Kestler, H., Uebele, C., Sokolovic, Z. & Goebel, W. (1996). Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA. Mol Microbiol 20, 11891198.[Medline]
Brundage, R. A., Smith, G. A., Camilli, A., Theriot, J. A. & Portnoy, D. A. (1993). Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl Acad Sci U S A 90, 1189011894.[Abstract]
Bubert, A., Sokolovic, Z., Chun, S.-K., Papatheodorou, L., Simm, A. & Goebel, W. (1999). Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol Gen Genet 261, 323336.[CrossRef][Medline]
Camilli, A., Paynton, C. R. & Portnoy, D. A. (1989). Intracellular methicillin selection of Listeria monocytogenes mutants unable to replicate in a macrophage cell line. Proc Natl Acad Sci U S A 86, 55225526.[Abstract]
Camilli, A., Tilney, L. G. & Portnoy, D. A. (1993). Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol 8, 143157.[Medline]
Chakraborty, T., Leimeister-Wachter, M., Domann, E., Hartl, M., Goebel, W., Nichterlein, T. & Notermans, S. (1992). Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol 174, 568574.[Abstract]
Cotter, P. A. & DiRita, V. J. (2000). Bacterial virulence gene regulation: an evolutionary perspective. Annu Rev Microbiol 54, 519565.[CrossRef][Medline]
Cotter, P. A. & Miller, J. F. (1997). A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens. Mol Microbiol 24, 671685.[CrossRef][Medline]
Datta, A. R. & Kothary, M. H. (1993). Effects of glucose, growth temperature, and pH on Listeriolysin O production in Listeria monocytogenes. Appl Environ Microbiol 59, 34953497.[Abstract]
Freitag, N. E. & Jacobs, K. E. (1999). Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria. Infect Immun 67, 18441852.
Freitag, N. E. & Portnoy, D. A. (1994). Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo. Mol Microbiol 12, 845853.[Medline]
Freitag, N. E., Rong, L. & Portnoy, D. A. (1993). Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread. Infect Immun 61, 25372544.[Abstract]
Garcia Vescovi, E., Soncini, F. C. & Groisman, E. A. (1996). Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Cell 84, 165174.[Medline]
Garges, S. & Adhya, S. (1985). Sites of allosteric shift in the structure of cyclic AMP receptor protein. Cell 41, 745751.[Medline]
Gellin, B. G. & Broome, C. V. (1989). Listeriosis. J Amer Med Assoc 261, 13131320.[CrossRef][Medline]
Gray, M. L. & Killinger, A. H. (1966). Listeria monocytogenes and listeric infections. Bacteriol Rev 30, 309382.[Medline]
Harman, J. G., McKenney, K. & Peterkofsky, A. (1986). Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. J Biol Chem 261, 1633216339.
Herbert, K. C. & Foster, S. J. (2001). Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components. Microbiology 147, 22752284.
Jones, S. & Portnoy, D. A. (1994). Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect Immun 62, 56085613.[Abstract]
Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749795.[CrossRef][Medline]
Kreft, J. & Vazquez-Boland, J. A. (2001). Regulation of virulence genes in Listeria. Int J Med Microbiol 291, 145157.[Medline]
Lampidis, R., Gross, R., Sokolovic, Z., Goebel, W. & Kreft, J. (1994). The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol Microbiol 13, 141151.[Medline]
Lee, S. H. & Camilli, A. (2000). Novel approaches to monitor bacterial gene expression in infected tissue and host. Curr Opin Microbiol 3, 97101.[CrossRef][Medline]
Mahan, M. J., Tobias, J. W., Slauch, J. M., Hanna, P. C., Collier, R. J. & Mekalanos, J. J. (1995). Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host. Proc Natl Acad Sci U S A 92, 669673.[Abstract]
Marquis, H., Doshi, V. & Portnoy, D. A. (1995). The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect Immun 63, 45314534.[Abstract]
Mengaud, J., Dramsi, S., Gouin, E., Vazquez-Boland, J. A., Milon, G. & Cossart, P. (1991). Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol Microbiol 5, 22732283.[Medline]
Michel, E., Mengaud, J., Galsworthy, S. & Cossart, P. (1998). Characterization of a large motility gene cluster containing the cheR, motAB genes of Listeria monocytogenes and evidence that PrfA downregulates motility genes. FEMS Microbiol Lett 15, 341347.
Millenbachs, A. A., Brown, D. P., Moors, M. & Youngman, P. (1997). Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol 23, 10751085.[Medline]
Moors, M. A., Levitt, B., Youngman, P. & Portnoy, D. A. (1999). Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect Immun 67, 131139.
Park, S. F. & Kroll, R. G. (1993). Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Mol Microbiol 8, 653661.[Medline]
Portnoy, D. A., Jacks, P. S. & Hinrichs, D. J. (1988). Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 167, 14591471.[Abstract]
Renzoni, A., Klarsfeld, A., Dramsi, S. & Cossart, P. (1997). Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes can be present but inactive. Infect Immun 65, 15151518.[Abstract]
Renzoni, A., Cossart, P. & Dramsi, S. (1999). PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts. Mol Microbiol 34, 552561.[CrossRef][Medline]
Ripio, M.-T., Dominguez-Bernal, G., Lara, M., Suarez, M. & Vazquez-Boland, J.-A. (1997). A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes. J Bacteriol 179, 15331540.[Abstract]
Ripio, M. T., Vazquez-Boland, J. A., Vega, Y., Nair, S. & Berche, P. (1998). Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes. FEMS Microbiol Lett 158, 4550.[CrossRef][Medline]
Sheehan, B., Klarsfeld, A., Msadek, T. & Cossart, P. (1995). Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator. J Bacteriol 177, 64696476.[Abstract]
Shetron-Rama, L. M., Marquis, H., Bouwer, H. G. A. & Freitag, N. E. (2002). Intracellular induction of Listeria monocytogenes actA expression. Infect Immun 70, 10871096.
Slauch, J. M. & Camilli, A. (2000). IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues. Methods Enzymol 326, 7396.[CrossRef][Medline]
Slauch, J. M., Mahan, M. J. & Mekalanos, J. J. (1994). In vivo expression technology for selection of bacterial genes specifically induced in host tissues. Methods Enzymol 235, 481492.[Medline]
Smith, K. & Youngman, P. (1992). Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74, 705711.[CrossRef][Medline]
Smith, G. A., Marquis, H., Jones, S., Johnston, N. C., Portnoy, D. A. & Goldfine, H. (1995). The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect Immun 63, 42314237.[Abstract]
Soncini, F. C., Garcia Vescovi, E., Solomon, F. & Groisman, E. A. (1996). Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes. J Bacteriol 178, 50925099.[Abstract]
Stanley, T. L., Ellermeier, C. D. & Slauch, J. M. (2000). Tissue-specific gene expression identifies a gene in the lysogenic phage Gifsy-1 that affects Salmonella enterica serovar typhimurium survival in Peyer's patches. J Bacteriol 182, 44064413.
Sun, A. N., Camilli, A. & Portnoy, D. A. (1990). Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect Immun 58, 37703778.[Medline]
Swanson, M. S. & Hammer, B. K. (2000). Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54, 567613.[CrossRef][Medline]
Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. & Kreft, J. (2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584640.
Vega, Y., Dickneite, C., Ripio, M.-T., Böckmann, R., Gonzalez-Zorn, B., Novella, S., Gominguez-Bernal, G., Goebel, W. & Vazquez-Boland, W. (1998). Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. J Bacteriol 180, 66556660.
Wosten, M. M., Kox, L. F., Chamnongpol, S., Soncini, F. C. & Groisman, E. A. (2000). A signal transduction system that responds to extracellular iron. Cell 103, 113125.[Medline]
Youngman, P. (1987). Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other Gram-positive bacteria. In Plasmids: a Practical Approach, pp. 79103. Edited by K. Hardy. Oxford: IRL Press.
Received 23 April 2002;
revised 20 September 2002;
accepted 26 September 2002.
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