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Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis
FEMS Microbiology Letters 205 (2001) 77^81
www.fems-microbiology.org
Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis î ke Forsberg, Anders Norqvist * Kerstin Kuoppa, A Swedish Defence Research Agency, Division of NBC Defence, FOI NBC-skydd, S-901 82 Umea®, Sweden Received 1 May 2001; received in revised form 10 September 2001; accepted 20 September 2001 First published online 26 October 2001
Abstract Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Reporter plasmid; Promoter; Gene expression ; Francisella tularensis
1. Introduction Francisella tularensis, the etiological agent for tularaemia, is a facultative intracellular bacterium that is capable of surviving and multiplying intracellularly [1,2]. Studies from patients and animal infection models have shown that cell-mediated host response is important to establish protective immunity [1,3]. Although a lot of knowledge has been generated in the understanding of the mechanisms behind induction of protective immunity, the basic understanding of key virulence factors and the infection strategy employed by F. tularensis is limited. Such studies have been hampered by the lack of genetic techniques in F. tularensis to enable construction of speci¢c mutants in putative virulence genes. For several bacterial pathogens the access of genetic tools has facilitated the identi¢cation of genes essential for the survival of bacteria inside the host. Methods have been developed which are based on either replicating plasmids [4^6] or on insertion via homologous recombina-
* Corresponding author. Tel. : +46 (90) 10 66 31; Fax: +46 (90) 10 68 06. E-mail address : [email protected] (A. Norqvist).
tion [7]. Identi¢cation of di¡erent genes expressed during di¡erent stages of infection is important in order to understand the strategy used by the pathogen to proliferate inside the host. The construction of a shuttle plasmid vector, which replicated both in F. tularensis and in Escherichia coli [8], opened up the possibility to study the role of di¡erent genes of F. tularensis during infection. In this work we describe the construction of a new reporter plasmid based on this shuttle vector that allows identi¢cation of the genes expressed by F. tularensis during infection. 2. Materials and methods 2.1. Bacterial strains and plasmids The live vaccine strain of F. tularensis (LVS, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA) was grown on modi¢ed Thayer^Martin agar plates [9] at 37³C in 5% CO2 . E. coli DH5K [10] was cultured in Luria^Bertani (LB) broth or on LB agar plates. E. coli TOP 10 FP (Invitrogen BV, Leek, The Netherlands) was grown according to the supplier's instructions. Plasmid pKK202 harbours the genes for tetracycline and chloramphenicol resistance and replicates both in F.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 4 6 - 3
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K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81
tularensis and E. coli [8]. The plasmids pCM7 (Amersham Pharmacia Biotech, Sweden), phagemid pBluescript0 KS+ (Stratagene, La Jolla, CA, USA) and pZErO1-1 (Invitrogen BV, Leek, The Netherlands) were also used in this study. 2.2. DNA manipulation techniques DNA manipulations were performed essentially as described by Sambrook et al. [11]. Electrocompetent cells were prepared according to the method of Zabarovsky and Winberg [12] and electroporation was carried out with a Gene Pulser (Bio-Rad Laboratories) at 2.5 kV, 25 WF and 200 6. Plasmids were introduced into F. tularensis LVS by the method of cryotransformation [8] originally described by Mokrievich et al. [13]. 2.3. Construction of reporter plasmid pKK 214 A HindIII restriction fragment of plasmid pCM7 containing a promoter-less chloramphenicol acetyltransferase gene (cat) was cloned into the HindIII site of pBluescript and transformed into E. coli DH5K. PCR ampli¢cation, using universal primers for M13, was performed to verify the insertion of the proper fragment. The resulting plasmid, designated pKK101 containing the promoter-less cat gene from pCM7, was linearised by digestion with ClaI. The linearised plasmid was incubated with Klenow DNA polymerase to generate blunt ends and thereafter digested with XbaI. The DNA fragment with an approximate size of 800 bp, containing the cat gene and an upstream cloning cassette originating from pBluescript, was puri¢ed from agarose gels using Geneclean (Bio 101, La Jolla, CA, USA).
Plasmid pKK202 was digested with AvaI, treated with Klenow DNA polymerase and then digested with XbaI. The 4-kb fragment, containing both the p15A replicon and the F. tularensis replicon and the tetracycline resistance gene, was puri¢ed and ligated to the 800-bp fragment isolated from pKK101. The resulting recombinant plasmid was denoted pKK214 (Fig. 1). 2.4. Construction of a F. tularensis promoter library Chromosomal DNA was isolated from F. tularensis LVS as described by Sambrook et al. [11]. Random F. tularensis LVS DNA fragments, obtained by partial restriction digestion with AluI, were cloned into the unique EcoRV site of pZErO1-1. The ligated plasmid DNA was introduced into E. coli, strain TOP 10 FP by electroporation. Plasmid DNA prepared from the pool of recombinant clones ( s 150 000) was digested with XbaI and PstI. Fragments of sizes 200^1600 bp were puri¢ed and ligated into pKK214 digested with XbaI and PstI upstream from the promoter-less cat gene. The ligation mix was electroporated into E. coli DH5K and clones were selected on plates containing 10 Wg ml31 of tetracycline. Plasmid DNA from a pool of recombinant clones was transformed into F. tularensis, strain LVS, and transformants were selected on plates with tetracycline. 2.5. Intracellular multiplication assay The murine macrophage-like cell line J774A.1, obtained from the American Type Culture Collection (ATCC TIB67), was routinely grown in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 10% of heat-inactivated fetal calf serum (FCS), 2 mM of L-glutamine and
Fig. 1. Construction of plasmid pKK214. For details : see MpM
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K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81
2 Wg ml31 of gentamicin (all from Gibco BRL) in a humidi¢ed 5% CO2 atmosphere at 37³C. Macrophages were seeded 20 h prior to infection in 24well tissue culture plates at a density of 2U105 cells per well in culture medium lacking antibiotics. The cells were washed once with pre-warmed wash medium (37³C), i.e. DMEM containing 1% FCS and without antibiotics. The macrophages were infected with F. tularensis LVS, harbouring the recombinant plasmids of pKK214, at a multiplicity of infection (MOI) of 50 bacteria per eucaryotic cell. After 2 h of infection, the cells were washed twice with wash medium and incubated for 1 h in the presence of gentamicin (50 Wg ml31 ) to kill extracellular bacteria. The macrophages were washed twice with wash medium and incubated for 2, 24 and 48 h in the medium containing chloramphenicol (2.5 Wg ml31 ) and gentamicin (2 Wg ml31 ). The monolayers were subsequently washed three times with phosphate bu¡ered saline (PBS) and surviving intracellular bacteria were recovered by lysis of the macrophages with 0.1% sodium deoxycholate in PBS. The number of intracellular bacteria was determined by direct colony count on non-selective plates. The intracellular bacteria recovered after 48 h incubation in the macrophages were then used for a new round of infection. These infection cycles were repeated up to four times using the surviving bacteria after 48 h as inoculum for the next infection. 3. Results and discussion 3.1. Construction of a promoter screening vector for F. tularensis The objective of this study was to construct a reporter plasmid that could be used to screen for F. tularensis promoters that are active during infection. We recently constructed a plasmid denoted pKK202 that could serve as a shuttle vector between F. tularensis and E. coli [8]. Chloramphenicol is an antibiotic that is known to be taken up by eucaryotic cells and therefore chloramphenicol resis-
79
tance can be used to select for intracellular bacteria. Promoter-less cat genes have also been successfully used as reporter genes in cloning bacterial promoters that are active inside host cells [5,6]. We decided to use a similar strategy and therefore cloned a promoter-less cat gene into the shuttle vector pKK202, thereby creating plasmid pKK214 (Fig. 1). To allow cloning of putative promoters, pKK214 has a cloning cassette with several unique restriction sites upstream from the cat gene. We noted that pKK214 conferred chloramphenicol resistance when introduced into E. coli. This is probably due to the very ATrich F. tularensis DNA that is located upstream from the cloning cassette and that this sequence serves as a promoter for the downstream cat gene in E. coli. However, in F. tularensis pKK214 did not confer any chloramphenicol resistance in vitro. F. tularensis strain LVS was unable to grow on Thayer^Martin plates containing levels as low as 2.5 Wg ml31 of chloramphenicol irrespective if the strain contained pKK214 or not. Therefore, pKK214 could be used to select for promoter activity in F. tularensis. 3.2. Construction of a F. tularensis promoter library in pKK214 pKK214 was next used to construct a library containing putative promoter sequences of F. tularensis. Random chromosomal fragments from F. tularensis strain LVS were inserted in front of the promoter-less cat gene in pKK214 and the resulting bank of putative promoter sequences was introduced into F. tularensis strain LVS. Ten thousand clones of F. tularensis containing the recombinant plasmid variants of pKK214 were isolated on plates containing 10 Wg ml31 of tetracycline. In order to estimate the frequencies of active promoter sequences in vitro and the promoter strength, 220 clones were patched on Thayer^ Martin plates containing di¡erent concentrations of chloramphenicol. Thirty-four clones (15.5%) grew on plates with 2.5 Wg ml31 chloramphenicol, 12 (5.5%) on 5 Wg ml31 and three (1.5%) on 10 Wg ml31 chloramphenicol. The recombinant plasmids from 20 colonies growing on
Table 1 Growth of recombinant F. tularensis LVS in the presence (+) or absence (3) of chloramphenicol after one and two rounds of infection Bacteria
Macrophage incubation time (h)
Number of surviving intracellular bacteria round 1 +cml
F. tul/pKK214 (negative control)
F. tul/promoter library
a
3U103 5U102 3U102 1U103 1U104 5U104
2 24 48 2 24 48
The number of intracellular bacteria was determined by viable count 2, 24 and 48 h after infection. a +cml, presence of 2.5 Wg ml31 chloramphenicol during the whole infection cycle. b 3cml, absence of 2.5 Wg ml31 chloramphenicol during the whole infection cycle.
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round 2 b
3cml
+cml
3cml
1U103 4U106 5U106 1U103 1U105 8U104
3U103 4U102 3U102 6U103 1U106 4U106
2U103 4U106 7U106 5U103 9U105 5U105
80
K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81
2.5 Wg ml31 chloramphenicol were isolated and digested with HindIII (data not shown). From the digestion pattern it could be established that the majority of the clones were di¡erent. From this we conclude that pKK214 can be used to identify promoters in F. tularensis active in vitro. 3.3. Screening for F. tularensis promoters active in macrophages The F. tularensis promoter library was then used in a screening with the aim of isolating promoters that were active inside macrophages. For this purpose, the macrophage-like cell line J774A.1 was infected with the F. tularensis promoter library in the presence or absence of chloramphenicol. F. tularensis carrying the promoter vector pKK214 was used as negative control. Up to four consecutive rounds of 48 h infections were performed (for details see Section 2). In the absence of chloramphenicol, the number of intracellular bacteria increased signi¢cantly for all the strains used including the strain containing the vector control (Table 1). After one round of infection in the presence of chloramphenicol, the number of resistant bacteria after 48 h increased 50-fold for cells infected with the promoter bank while the strain containing pKK214 was unable to grow inside the macrophage in the presence of chloramphenicol. When the bacteria isolated after one round of infection were used in a second round of infection, the number of bacteria isolated from the macrophages increased approximately at the same rate in the presence as well as in the absence of chloramphenicol (Table 1). The same results were obtained when the infection cycle was repeated one or two times more. This shows that two cycles of infection are enough to enrich for the promoters that are active inside the macrophages. In order to con¢rm that the clones isolated after passage through macrophages were di¡erent, plasmids from 20 di¡erent colonies that grew on plates containing 2.5 Wg ml31 chloramphenicol were isolated and digested with HindIII. As can be seen in Fig. 2, the majority of the clones harboured inserts with di¡erent sizes. These clones, isolated from infected macrophages, were spread on plates containing 5 or 10 Wg ml31 chloramphenicol. It was found that all clones also grew in vitro on 5 Wg ml31 chloramphenicol and that 16 clones (85%) also grew on 10 Wg ml31 chloramphenicol. When a similar test was performed on clones isolated from plates containing 2.5 Wg ml31 chloramphenicol, but not passed through macrophages, it was found that approximately 25% grew on 5 Wg ml31 chloramphenicol and 10% on 10 Wg ml31 chloramphenicol. Thus, it can be concluded that passing the promoter library in macrophages resulted in the enrichment of clones with stronger promoter activity, compared to in vitro growth. Based on our initial ¢ndings in this work, we conclude that pKK214 can be used to identify F. tularensis promoters active in vitro as well as in vivo with a strategy
Fig. 2. Results of HindIII digestion of plasmid DNA from 20 di¡erent clones isolated from recovered bacteria from macrophages after 48 h incubation. The lane labelled `C' is plasmid pKK214 lacking promoter, which was used as the negative control. Lanes 1^20 are the di¡erent clones growing on 2.5 and 5 Wg ml31 chloramphenicol. All clones except those in lanes 9, 12, 17 and 19 also grew on 10 Wg ml31 chloramphenicol. The left- and rightmost lanes in each gel contain the molecular size standard.
similar to what has been used for cells infected with Salmonella typhimurium [5,6]. A similar strategy has also been used to identify the promoters and genes active in animal infection models [4,5,14]. Therefore, it should also be possible to use the promoter library in this study to identify promoters that are active in the mouse infection model by performing the infection in the presence of chloramphenicol. Another possibility and future challenge in using this promoter screening vector for F. tularensis are to identify the genes that are not active in vitro but are uniquely expressed during infection.
References [1] Ta«rnvik, A. (1989) Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11, 440^451. [2] Fortier, A.H., Leiby, D.A., Narayanan, R.B., Asafoadjei, E., Crawford, R.M., Nacy, C.A. and Meltzer, M.S. (1995) Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth. Infect. Immun. 63, 1478^1483. [3] Ta«rnvik, A., Eriksson, M., Sandstro«m, G. and Sjo«stedt, A. (1992) Francisella tularensis ^ a model for studies of the immune response to intracellular bacteria in man. Immunology 76, 349^354. [4] Mahan, M.J., Slauch, J.M. and Mekalanos, J.J. (1993) Selection of bacterial virulence genes that are speci¢cally induced in host tissues. Science 259, 686^688. [5] Mahan, M.J., Tobias, J.W., Slauch, J.M., Hanna, P.C., Collier, R.J. and Mekalanos, J.J. (1995) Antibiotic-based selection for bacterial genes that are speci¢cally induced during infection of a host. Proc. Natl. Acad. Sci. USA 92, 669^673. [6] Staendner, L.H., Rohde, M., Timmis, K.N. and Guzma¨n, C.A. (1995) Identi¢cation of Salmonella typhi promoters activated by invasion of eukaryotic cells. Mol. Microbiol. 18, 891^902. [7] Hensel, M., Shea, J.E., Gleeson, C., Jones, M.D., Dalton, E. and Holden, D.W. (1995) Simultaneous identi¢cation of bacterial virulence genes by negative selection. Science 269, 400^403. [8] Norqvist, A., Kuoppa, K. and Sandstro«m, G. (1996) Construction of a shuttle vector for use in Francisella tularensis. FEMS Immunol. Med. Microbiol. 13, 257^260. [9] Sandstro«m, G., Ta«rnvik, A., Wolf-Watz, H. and Lo«fgren, S. (1984)
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K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81 Antigen from Francisella tularensis : nonidentity between determinants participating in cell-mediated and humoral reactions. Infect. Immun. 45, 101^106. [10] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557^580. [11] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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[12] Zabarovsky, E.R. and Winberg, G. (1990) High e¤ciency electroporation of ligated DNA into bacteria. Nucleic Acids Res. 18, 5912. [13] Mokrievich, A.N., Fursov, V.V. and Pavlov, V.M. (1994) Plasmid cryotransformation (in Russian). Problemy Osobo Opasnyh Infektsy Saratov 4, 186^190. [14] Strohmeier Gort, A. and Miller, V.L. (2000) Identi¢cation and characterization of Yersinia enterocolitica genes induced during systemic infection. Infect. Immun. 68, 6633^6642.
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www.fems-microbiology.org
Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis î ke Forsberg, Anders Norqvist * Kerstin Kuoppa, A Swedish Defence Research Agency, Division of NBC Defence, FOI NBC-skydd, S-901 82 Umea®, Sweden Received 1 May 2001; received in revised form 10 September 2001; accepted 20 September 2001 First published online 26 October 2001
Abstract Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Reporter plasmid; Promoter; Gene expression ; Francisella tularensis
1. Introduction Francisella tularensis, the etiological agent for tularaemia, is a facultative intracellular bacterium that is capable of surviving and multiplying intracellularly [1,2]. Studies from patients and animal infection models have shown that cell-mediated host response is important to establish protective immunity [1,3]. Although a lot of knowledge has been generated in the understanding of the mechanisms behind induction of protective immunity, the basic understanding of key virulence factors and the infection strategy employed by F. tularensis is limited. Such studies have been hampered by the lack of genetic techniques in F. tularensis to enable construction of speci¢c mutants in putative virulence genes. For several bacterial pathogens the access of genetic tools has facilitated the identi¢cation of genes essential for the survival of bacteria inside the host. Methods have been developed which are based on either replicating plasmids [4^6] or on insertion via homologous recombina-
* Corresponding author. Tel. : +46 (90) 10 66 31; Fax: +46 (90) 10 68 06. E-mail address : [email protected] (A. Norqvist).
tion [7]. Identi¢cation of di¡erent genes expressed during di¡erent stages of infection is important in order to understand the strategy used by the pathogen to proliferate inside the host. The construction of a shuttle plasmid vector, which replicated both in F. tularensis and in Escherichia coli [8], opened up the possibility to study the role of di¡erent genes of F. tularensis during infection. In this work we describe the construction of a new reporter plasmid based on this shuttle vector that allows identi¢cation of the genes expressed by F. tularensis during infection. 2. Materials and methods 2.1. Bacterial strains and plasmids The live vaccine strain of F. tularensis (LVS, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA) was grown on modi¢ed Thayer^Martin agar plates [9] at 37³C in 5% CO2 . E. coli DH5K [10] was cultured in Luria^Bertani (LB) broth or on LB agar plates. E. coli TOP 10 FP (Invitrogen BV, Leek, The Netherlands) was grown according to the supplier's instructions. Plasmid pKK202 harbours the genes for tetracycline and chloramphenicol resistance and replicates both in F.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 4 6 - 3
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K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81
tularensis and E. coli [8]. The plasmids pCM7 (Amersham Pharmacia Biotech, Sweden), phagemid pBluescript0 KS+ (Stratagene, La Jolla, CA, USA) and pZErO1-1 (Invitrogen BV, Leek, The Netherlands) were also used in this study. 2.2. DNA manipulation techniques DNA manipulations were performed essentially as described by Sambrook et al. [11]. Electrocompetent cells were prepared according to the method of Zabarovsky and Winberg [12] and electroporation was carried out with a Gene Pulser (Bio-Rad Laboratories) at 2.5 kV, 25 WF and 200 6. Plasmids were introduced into F. tularensis LVS by the method of cryotransformation [8] originally described by Mokrievich et al. [13]. 2.3. Construction of reporter plasmid pKK 214 A HindIII restriction fragment of plasmid pCM7 containing a promoter-less chloramphenicol acetyltransferase gene (cat) was cloned into the HindIII site of pBluescript and transformed into E. coli DH5K. PCR ampli¢cation, using universal primers for M13, was performed to verify the insertion of the proper fragment. The resulting plasmid, designated pKK101 containing the promoter-less cat gene from pCM7, was linearised by digestion with ClaI. The linearised plasmid was incubated with Klenow DNA polymerase to generate blunt ends and thereafter digested with XbaI. The DNA fragment with an approximate size of 800 bp, containing the cat gene and an upstream cloning cassette originating from pBluescript, was puri¢ed from agarose gels using Geneclean (Bio 101, La Jolla, CA, USA).
Plasmid pKK202 was digested with AvaI, treated with Klenow DNA polymerase and then digested with XbaI. The 4-kb fragment, containing both the p15A replicon and the F. tularensis replicon and the tetracycline resistance gene, was puri¢ed and ligated to the 800-bp fragment isolated from pKK101. The resulting recombinant plasmid was denoted pKK214 (Fig. 1). 2.4. Construction of a F. tularensis promoter library Chromosomal DNA was isolated from F. tularensis LVS as described by Sambrook et al. [11]. Random F. tularensis LVS DNA fragments, obtained by partial restriction digestion with AluI, were cloned into the unique EcoRV site of pZErO1-1. The ligated plasmid DNA was introduced into E. coli, strain TOP 10 FP by electroporation. Plasmid DNA prepared from the pool of recombinant clones ( s 150 000) was digested with XbaI and PstI. Fragments of sizes 200^1600 bp were puri¢ed and ligated into pKK214 digested with XbaI and PstI upstream from the promoter-less cat gene. The ligation mix was electroporated into E. coli DH5K and clones were selected on plates containing 10 Wg ml31 of tetracycline. Plasmid DNA from a pool of recombinant clones was transformed into F. tularensis, strain LVS, and transformants were selected on plates with tetracycline. 2.5. Intracellular multiplication assay The murine macrophage-like cell line J774A.1, obtained from the American Type Culture Collection (ATCC TIB67), was routinely grown in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 10% of heat-inactivated fetal calf serum (FCS), 2 mM of L-glutamine and
Fig. 1. Construction of plasmid pKK214. For details : see MpM
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K. Kuoppa et al. / FEMS Microbiology Letters 205 (2001) 77^81
2 Wg ml31 of gentamicin (all from Gibco BRL) in a humidi¢ed 5% CO2 atmosphere at 37³C. Macrophages were seeded 20 h prior to infection in 24well tissue culture plates at a density of 2U105 cells per well in culture medium lacking antibiotics. The cells were washed once with pre-warmed wash medium (37³C), i.e. DMEM containing 1% FCS and without antibiotics. The macrophages were infected with F. tularensis LVS, harbouring the recombinant plasmids of pKK214, at a multiplicity of infection (MOI) of 50 bacteria per eucaryotic cell. After 2 h of infection, the cells were washed twice with wash medium and incubated for 1 h in the presence of gentamicin (50 Wg ml31 ) to kill extracellular bacteria. The macrophages were washed twice with wash medium and incubated for 2, 24 and 48 h in the medium containing chloramphenicol (2.5 Wg ml31 ) and gentamicin (2 Wg ml31 ). The monolayers were subsequently washed three times with phosphate bu¡ered saline (PBS) and surviving intracellular bacteria were recovered by lysis of the macrophages with 0.1% sodium deoxycholate in PBS. The number of intracellular bacteria was determined by direct colony count on non-selective plates. The intracellular bacteria recovered after 48 h incubation in the macrophages were then used for a new round of infection. These infection cycles were repeated up to four times using the surviving bacteria after 48 h as inoculum for the next infection. 3. Results and discussion 3.1. Construction of a promoter screening vector for F. tularensis The objective of this study was to construct a reporter plasmid that could be used to screen for F. tularensis promoters that are active during infection. We recently constructed a plasmid denoted pKK202 that could serve as a shuttle vector between F. tularensis and E. coli [8]. Chloramphenicol is an antibiotic that is known to be taken up by eucaryotic cells and therefore chloramphenicol resis-
79
tance can be used to select for intracellular bacteria. Promoter-less cat genes have also been successfully used as reporter genes in cloning bacterial promoters that are active inside host cells [5,6]. We decided to use a similar strategy and therefore cloned a promoter-less cat gene into the shuttle vector pKK202, thereby creating plasmid pKK214 (Fig. 1). To allow cloning of putative promoters, pKK214 has a cloning cassette with several unique restriction sites upstream from the cat gene. We noted that pKK214 conferred chloramphenicol resistance when introduced into E. coli. This is probably due to the very ATrich F. tularensis DNA that is located upstream from the cloning cassette and that this sequence serves as a promoter for the downstream cat gene in E. coli. However, in F. tularensis pKK214 did not confer any chloramphenicol resistance in vitro. F. tularensis strain LVS was unable to grow on Thayer^Martin plates containing levels as low as 2.5 Wg ml31 of chloramphenicol irrespective if the strain contained pKK214 or not. Therefore, pKK214 could be used to select for promoter activity in F. tularensis. 3.2. Construction of a F. tularensis promoter library in pKK214 pKK214 was next used to construct a library containing putative promoter sequences of F. tularensis. Random chromosomal fragments from F. tularensis strain LVS were inserted in front of the promoter-less cat gene in pKK214 and the resulting bank of putative promoter sequences was introduced into F. tularensis strain LVS. Ten thousand clones of F. tularensis containing the recombinant plasmid variants of pKK214 were isolated on plates containing 10 Wg ml31 of tetracycline. In order to estimate the frequencies of active promoter sequences in vitro and the promoter strength, 220 clones were patched on Thayer^ Martin plates containing di¡erent concentrations of chloramphenicol. Thirty-four clones (15.5%) grew on plates with 2.5 Wg ml31 chloramphenicol, 12 (5.5%) on 5 Wg ml31 and three (1.5%) on 10 Wg ml31 chloramphenicol. The recombinant plasmids from 20 colonies growing on
Table 1 Growth of recombinant F. tularensis LVS in the presence (+) or absence (3) of chloramphenicol after one and two rounds of infection Bacteria
Macrophage incubation time (h)
Number of surviving intracellular bacteria round 1 +cml
F. tul/pKK214 (negative control)
F. tul/promoter library
a
3U103 5U102 3U102 1U103 1U104 5U104
2 24 48 2 24 48
The number of intracellular bacteria was determined by viable count 2, 24 and 48 h after infection. a +cml, presence of 2.5 Wg ml31 chloramphenicol during the whole infection cycle. b 3cml, absence of 2.5 Wg ml31 chloramphenicol during the whole infection cycle.
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round 2 b
3cml
+cml
3cml
1U103 4U106 5U106 1U103 1U105 8U104
3U103 4U102 3U102 6U103 1U106 4U106
2U103 4U106 7U106 5U103 9U105 5U105
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2.5 Wg ml31 chloramphenicol were isolated and digested with HindIII (data not shown). From the digestion pattern it could be established that the majority of the clones were di¡erent. From this we conclude that pKK214 can be used to identify promoters in F. tularensis active in vitro. 3.3. Screening for F. tularensis promoters active in macrophages The F. tularensis promoter library was then used in a screening with the aim of isolating promoters that were active inside macrophages. For this purpose, the macrophage-like cell line J774A.1 was infected with the F. tularensis promoter library in the presence or absence of chloramphenicol. F. tularensis carrying the promoter vector pKK214 was used as negative control. Up to four consecutive rounds of 48 h infections were performed (for details see Section 2). In the absence of chloramphenicol, the number of intracellular bacteria increased signi¢cantly for all the strains used including the strain containing the vector control (Table 1). After one round of infection in the presence of chloramphenicol, the number of resistant bacteria after 48 h increased 50-fold for cells infected with the promoter bank while the strain containing pKK214 was unable to grow inside the macrophage in the presence of chloramphenicol. When the bacteria isolated after one round of infection were used in a second round of infection, the number of bacteria isolated from the macrophages increased approximately at the same rate in the presence as well as in the absence of chloramphenicol (Table 1). The same results were obtained when the infection cycle was repeated one or two times more. This shows that two cycles of infection are enough to enrich for the promoters that are active inside the macrophages. In order to con¢rm that the clones isolated after passage through macrophages were di¡erent, plasmids from 20 di¡erent colonies that grew on plates containing 2.5 Wg ml31 chloramphenicol were isolated and digested with HindIII. As can be seen in Fig. 2, the majority of the clones harboured inserts with di¡erent sizes. These clones, isolated from infected macrophages, were spread on plates containing 5 or 10 Wg ml31 chloramphenicol. It was found that all clones also grew in vitro on 5 Wg ml31 chloramphenicol and that 16 clones (85%) also grew on 10 Wg ml31 chloramphenicol. When a similar test was performed on clones isolated from plates containing 2.5 Wg ml31 chloramphenicol, but not passed through macrophages, it was found that approximately 25% grew on 5 Wg ml31 chloramphenicol and 10% on 10 Wg ml31 chloramphenicol. Thus, it can be concluded that passing the promoter library in macrophages resulted in the enrichment of clones with stronger promoter activity, compared to in vitro growth. Based on our initial ¢ndings in this work, we conclude that pKK214 can be used to identify F. tularensis promoters active in vitro as well as in vivo with a strategy
Fig. 2. Results of HindIII digestion of plasmid DNA from 20 di¡erent clones isolated from recovered bacteria from macrophages after 48 h incubation. The lane labelled `C' is plasmid pKK214 lacking promoter, which was used as the negative control. Lanes 1^20 are the di¡erent clones growing on 2.5 and 5 Wg ml31 chloramphenicol. All clones except those in lanes 9, 12, 17 and 19 also grew on 10 Wg ml31 chloramphenicol. The left- and rightmost lanes in each gel contain the molecular size standard.
similar to what has been used for cells infected with Salmonella typhimurium [5,6]. A similar strategy has also been used to identify the promoters and genes active in animal infection models [4,5,14]. Therefore, it should also be possible to use the promoter library in this study to identify promoters that are active in the mouse infection model by performing the infection in the presence of chloramphenicol. Another possibility and future challenge in using this promoter screening vector for F. tularensis are to identify the genes that are not active in vitro but are uniquely expressed during infection.
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