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1 Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
2 Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Correspondence
Oksana V. Barysheva
ksyusha{at}bact.med.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The bacteria infect humans via aerosols, and replicate inside alveolar macrophages causing a severe form of pneumonia called Legionnaires' disease (Horwitz & Silverstein, 1983).
The L. pneumophila icm (intracellular multiplication)/dot (defective organelle trafficking) genes, located in two chromosomal regions, are required for intracellular multiplication and host cell killing (Segal & Shuman, 1998). Chromosomal region I harbours the genes icmV, W and X, and dotA, B, C and D (Marra et al., 1992; Vogel et al., 1998), while region II contains the genes icmT, S, R, Q, P, O, N, M, L, K, E, G, C, D, J, B and F (Andrews et al., 1998; Horwitz, 1987; Segal & Shuman, 1997; Vogel et al., 1998). Most of these genes are also required for intracellular growth in the protozoan host Acanthamoeba castellanii (Segal & Shuman, 1999). The icm/dot system serves as a translocation system that delivers effector proteins to host cells (Nagai et al., 2002). However, there is not much information regarding regulation of L. pneumophila virulence, or expression of the icm/dot genes.
Investigating changes in gene expression is critical for understanding the adaptive responses of Legionella to environmental stress and during infection. Among the more commonly used reporter genes are those for the following proteins: chloramphenicol acetyltransferase, β-galactosidase, β-glucoronidase and luciferase. Green fluorescent protein (Gfp) obtained from the jellyfish Aequorea victoria has been used as an optimal bacterial reporter. Gfpmut3* is one of a number of Gfps; it is a very stable form of the protein, and gives off a very bright green fluorescence when expressed in bacteria. However, due to its long half-life, Gfpmut3* is not suitable as a reporter for studying real-time gene expression in individual cells. Furthermore, L. pneumophila strains carrying this type of Gfp did not multiply in the J774.1 macrophage cell line in our preliminary experiment. To expand the use of Gfp as a reporter protein, new variants have been constructed by insertion of a mutation into the chromophore, and by the addition of a short peptide sequence to the C-terminal end of intact Gfp (Cormack et al., 1996). The latter variant renders Gfp susceptible to the action of indigenous housekeeping proteases. The new Gfp variants should be useful for in situ studies of temporal gene expression. Therefore, to elucidate L. pneumophila gene expression during infection, the unstable type of Gfp was examined.
Unstable Gfp variants and their application have been described for Escherichia coli, Pseudomonas putida and Mycobacterium smegmatis (Andersen et al., 1998; Blokpoel et al., 2003). It has been shown that specific C-terminal amino acid tails can make stable proteins that are susceptible to degradation by tail-specific proteases. This protein degradation system is based on ssrA-mediated tagging. The ssrA transcript (known as 10Sa RNA or tmRNA) is a stable RNA molecule with tRNA properties and an internal reading frame encoding a ‘tag’ peptide. This peptide can be attached to the C-terminus of the protein, rendering it susceptible to specific proteases (Muto et al., 1998). The half-lives of tagged proteins in E. coli are dependent on the last 3 aa of the C-terminal tail (Keiler & Sauer, 1996). Andersen et al. (1998) have shown that the half-lives of Gfp with four different amino acid tails vary between E. coli and P. putida, but are significantly shorter than those of stable Gfp. It has been shown that L. pneumophila has an ssrA gene (Chien et al., 2004). This indicates that ssrA-mediated tagging could be involved in protein degradation in this species.
After examination of the stability of Gfp variants in L. pneumophila, we elucidated that one of the Gfp variants, Gfp-AAV, could be used for measuring gene expression of L. pneumophila. This variant of unstable Gfp has been used for measuring the intracellular level of gene expression of the icmS, icmT and icmQ genes that are related to the type IV secretion system of L. pneumophila. These genes are specific for the type IV secretion system of L. pneumophila, and they have homologies with the type IV secretion system of C. burnetii only (Sexton et al., 2004; Zamboni et al., 2003). The icmT and icmQ genes are involved in the pore-forming process (Dumenil et al., 2004; Ninio et al., 2005). Moreover, icmQ and icmS genes are essential for the growth of L. pneumophila. However, it is not known when and how these genes are expressed. Thus, we tried to identify the moment when icmS, icmT and icmQ genes are highly expressed during infection in J774.1 macrophage-like cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. As required, antibiotics were added to the media at the following concentrations: 25 µg kanamycin ml–1 for L. pneumophila; 30 µg kanamycin ml–1 and 50 µg ampicillin ml–1 for E. coli.
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Plasmid construction
The plasmids used in this study are listed in Table 2.
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, and T1 is the transcriptional terminator from the rrB operon of E. coli) were taken from plasmids pJBA27, pJBA45, pJBA110, pJBA112 and pJBA113, respectively. The fragments were inserted into the NotI site of the pOKM66EH vector under a lacIq IPTG-inducible promoter to create pTOM1, pTOM2, pTOM3, pTOM4 and pTOM5, which differ only in the sequence of the gfp gene (Fig. 1). The resulting plasmids were introduced via electroporation into the L. pneumophila AM511 strain, using a Gene Pulser (Bio-Rad Laboratories).
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). The resulting plasmids (pKmS3, pKmT3 and pKmQ3) were transformed into the L. pneumophila AM511 strain using an electroporation Gene Pulser (Bio-Rad Laboratories), and they were used for measuring Legionella gene expression kinetics.
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In vitro phagocytosis, and intracellular growth assay.
In vitro phagocytosis was done as follows. L. pneumophila strains were grown in BYE broth until the early stationary phase. Approximately 2x109 bacteria ml–1 were pelleted, and resuspended and diluted (1 : 1000) in RPMI 1640 tissue culture medium. The bacteria were then added to J774.1 cells (2x105 cells per well) in 24-well dishes to give a m.o.i. of about 10. The infected cells were incubated at 37 °C under 5 % CO2 for 1 h, and washed three times with PBS to remove extracellular and non-adherent bacteria. To measure bacterial internalization, 1 ml sterile distilled water was added to the wells to release intracellular bacteria from the host cells, and the number of c.f.u. was determined by plating dilutions on BCYE agar plates. To each of the remaining wells, 0.5 ml fresh tissue culture medium was added, and the culture was continued. At 24 h intervals, the intracellular and extracellular bacteria in each well were combined, and the total number of c.f.u. was determined by plating the dilutions on BCYE agar plates.
Electron microscopy.
To provide confirmation of the results of the intracellular growth assay, J774.1 macrophages were infected with the wild-type L. pneumophila AM511, L. pneumophila AM511 carrying gfpmut3*, and L. pneumophila AM511 with icm-gfp(AAV) fusions. In vitro phagocytosis was performed as described above, using a 50 ml conical tube and a m.o.i. of 10. At 20 h post-infection, the cells were fixed, and processed for electron microscopy, as previously described (Wai et al., 1998). Briefly, infected macrophages were fixed with 2 % glutaraldehyde, and then 1 % OsO4, and then dehydrated with ethanol, and embedded in Epon. Ultrathin sections were stained with uranyl acetate, followed by lead citrate, and examined by electron microscopy in a JEM 2000EX instrument (JEOL).
Flow cytometric analysis.
In vitro phagocytosis was performed as described above, using 9 cm Petri dishes (Eiken Chemical) and a m.o.i. of 10. J774.1 cells did not adhere to the Petri dishes, and the floating infected J774.1 cells were analysed directly by a FACScan flow cytometer (Becton Dickinson). To permeabilize macrophages, saponin was added at a final concentration of 0.1 %.
The flow cytometric analysis was carried out with the following detector settings: FSC, E01; SSC, 458; and FL1, 600; using logarithmic gains. Quadrant location was set at FL1 (y-axis), 229; FSC (x-axis), 250; and the cells infected with L. pneumophila containing icmS-gfp(AAV), icmT-gfp(AAV) and icmQ-gfp(AAV) were counted in the upper-right area. The FL1-FSC plot was analysed using BD CellQuest Pro (v2.6.8, f7b; BD Biosciences).
RNA isolation from intracellular bacteria.
In vitro phagocytosis was performed as described above, using a 150 cm2 culture flask and a m.o.i. of 10. The infected cells were incubated at 37 °C under 5 % CO2 for 1 h, and washed three times with PBS to remove extracellular bacteria. Cells were lysed directly in a 150 cm2 culture flask by adding 20 ml TRIzol Reagent (Invitrogen), and shaking for about 5 min at room temperature, and then they were transferred to a 50 ml tube for incubation (overnight at 4 °C). Chloroform was added to induce separation of the organic and aqueous phases, and the phases were separated by centrifugation (15 min, 4 °C, 14 000 g). RNA was precipitated using 2-propanol, and samples were centrifuged for 10 min at 4 °C and 14 000 g. Pellets were washed with 75 % ethanol, which was removed after further centrifugation (5 min, 4 °C, 14 000 g). The pellets were dried, and dissolved in 20 µl RNase-free water. RNA was treated with DNase I (Roche Diagnostics) for 1 h to ensure complete DNA removal.
Real-time RT-PCR.
The oligonucleotides of the forward and reverse primers, and the TaqMan probe for L. pneumophila icmS, icmT and icmQ, were designed using Primer Express software (Applied Biosystems). The nucleotide sequences of the oligonucleotides were as follows. icmS (GenBank accession number AY543435): forward primer, 5'-ATTGCCAAGAGAGCGGATCAA-3'; reverse primer, 5'-CGTCATAAGTTGCTCCCAAACCATA-3'; reporter probe, CCCAAACACAAGGAACACA. icmT (GenBank accession number AY543439): forward primer, 5'-TGGTTTCACTGTCCCGGTTTT-3'; reverse primer, 5'-CCACCAAGGTCTAGCAGATCTAAC-3'; reporter probe, CTGGCTCAGAAGTTTTT. icmQ (GenBank accession number AY543402): forward primer, 5'-CCGCTAGTCAGGCCAAGTTAA-3'; reverse primer, 5'-ACCACTCCTTAAGGCAATTCGATT-3'; reporter probe, AAGCAGAGTCTCATCTTGCA. Expression of mRNA for L. pneumophila icmS, icmT and icmQ was measured in J774.1 cells, and the total RNA of samples was measured by real-time PCR, using TaqMan gene expression assays on an ABI PRISM 7000 sequence detection system (Applied Biosystems). A 5 µg sample of the total RNA of each sample was used to generate cDNA using the ABI High Capacity cDNA Archiving kit (Applied Biosystems), and real-time PCR reactions were carried out using the manufacturer's protocol. On each plate, an endogenous control gene (16S rDNA) and a no-template control were also run in duplicate.
Laser scanning confocal microscopy.
In vitro phagocytosis was performed as described above using RPMI 1640 medium without phenol red (Sigma), and supplemented with 10 % fetal bovine serum (Gibco), in a 35 mm glass-base dish (Iwaki), at a m.o.i. of 10. Confocal fluorescence and time-lapse images were obtained with an LSM 510 META laser scanning confocal microscope (Carl Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Stability of Gfpmut3* and the four variants was measured as relative fluorescence against time, after the removal of IPTG by washing. The time course of degradation of the different types of Gfp samples is shown in Fig. 3. All of the four variants, Gfp(AGA), Gfp(ASV), Gfp(LAA) and Gfp(AAV), were fluorescent in L. pneumophila, and, 7 h after the removal of IPTG, they exhibited relative fluorescence values of 75, 50, 48 and 8 %, respectively. Fluorescence measurements for all the Gfp variants were compared with a value of 90 % for Gfpmut3*. From the results, one of the tagged Gfp variants, Gfp(AAV) type, which had the shortest half-life, was chosen for measurement of L. pneumophila intracellular gene expression.
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).
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). At 20 h post-infection, the wild-type (Fig. 5a) and L. pneumophila carrying Picm-gfp(AAV) (Fig. 5c) had replicated in the phagosomes, while L. pneumophila carrying gfpmut3* did not multiply in J774.1 cells (Fig. 5b).
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). The expression levels of the genes during infection were found to be different from one another. At 0 h of infection, expression of icmS, icmT and icmQ was high, particularly that of icmS and icmQ. At 3 h, all the genes were expressed at high levels, and expression of icmT was higher than that at 0 h. At 6 h, expression of the icmS gene showed a further increase, while expression of icmT and icmQ genes showed a decrease. From 20 to 23 h after infection, expression of icmT and icmQ genes was at a low level, whereas the expression of icmS showed a gradual increase.
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). mRNA was purified from bacteria growing intracellularly. At 0 h of infection, the expression levels of all three genes were observed to be very low. However, from 3 h after infection, the transcriptional level of the icmS gene significantly increased, and the transcriptional levels of the icmT and icmQ genes were higher compared with the levels at 0 h, but they were still relatively low.
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, the icm genes were expressed during infection, and this was observed as fluorescence. The icmS promoter was highly active at 20 h after infection (Fig. 8a), while the icmT promoter was active during the first 5 h, most notably at 2 h after infection (Fig. 8b). icmQ was active at 1 h after infection (Fig. 8c).
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DISCUSSION |
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Although the multifaceted potential of stable Gfp for analysis of fundamental biological phenomena has been described in detail for several organisms (Chalfie et al., 1994; Corish & Tyler-Smith, 1999; Wang & Hazelrigg, 1994), limited information is available on unstable Gfp usage in bacteria. The Gfp has some intrinsic advantages over other reporter systems: formation of the fluorescent chromophore is species independent, and does not require any additional cofactors. However, bacteria carrying gfpmut3*, which gives strong Gfp expression, do not multiply within J774.1 cells. Accumulation of Gfp in bacterial cells is thought to inhibit intracellular growth. We found that bacteria carrying gfpmut3* showed an increase in intracellular proliferation of only 10-fold during the first day, and they did not grow during the following days of incubation in J774.1 cells (Fig. 4). However, the unstable type of Gfp does not accumulate in cells, and represents only recent expression of gfp. Hence, we can suppose that this property of unstable Gfp permits L. pneumophila to multiply within host cells. The intracellular growth assay (Fig. 4) revealed that bacteria carrying the unstable type of Gfp multiplied to the same degree as wild-type L. pneumophila, and this was confirmed by electron microscopy (Fig. 5).
gfp(AAV) was chosen from the four unstable gfp genes available because the Gfp(AAV) protein was most susceptible to the action of indigenous housekeeping proteases, resulting in a protein variant with a half-life of 60 min when synthesized in L. pneumophila (Fig. 3). To study the kinetics of L. pneumophila gene expression within infected cells, transcriptional fusion vectors based on unstable Gfp were constructed, and a mutated gfp(AAV) gene was used in the present experiment.
To examine the availability of the gfp(AAV) gene as a reporter gene, icmS, icmT and icmQ genes were chosen because these genes are located most upstream of region II of the icm/dot gene clusters. It is known that the IcmT protein is essential for pore formation and intracellular trafficking of L. pneumophila within Acanthomoeba polyphaga (Molmeret et al., 2002). The IcmQ protein is also involved in the pore-forming process (Feldman et al., 2005). The IcmS protein has been identified as a chaperone of the IcmW protein, and has also been demonstrated to help export SidE. SidE is secreted very early during infection in macrophages. It plays an important role in the initial formation of the replicative phagosome, and is completely dependent on IcmS (Bardill et al., 2005). Pore formation is thought to take place when bacteria infect the host cells. As expected, we observed Gfp-mediated fluorescence from bacteria containing PicmT-gfp(AAV) and PicmQ-gfp(AAV) fusions during the early (0–6 h) stage of infection (Fig. 6). As for icmS, expression was strong during the early (0–6 h) and late (20–23 h) stages of infection. This finding was supported when fluorescent intracellular legionellae were observed by laser scanning confocal microscopy hourly for 24 h (Fig. 8, and data not shown), and Gfp-expressing bacteria containing PicmS-gfp(AAV), PicmT-gfp(AAV) and PicmQ-gfp(AAV) fusions were not found during the interval from 6 to 20 h (date not shown).
Flow cytometric analysis revealed that unstable Gfp expression could be detected on or in infected cells (Fig. 6), and it showed that expression of the icm genes was high at 0 h of infection. It was thought that at this point in time, the fluorescent bacteria adhered to the host cells. At 3 h after infection, the expression levels of icmT and icmQ genes were very high. As both IcmT (Molmeret et al., 2002) and IcmQ (Feldman et al., 2005) proteins are involved in the pore-forming process, the results obtained from 3 h after infection could correlate with these investigations, and it appears that these proteins act in the same manner during infection of J774.1 cells. High expression of icmS during the 6 h after infection can be explained by the role of the IcmS protein in the formation of replicative phagosomes within J774.1 cells. The icmS gene is more highly expressed than the icmT and icmQ genes during 23 h of infection (Fig. 6). It has been shown that IcmS-LvgA (Legionella virulence gene A) protein complex is important for Dot/Icm-dependent intracellular growth (Bardill et al., 2005; Ninio et al., 2005; Vincent & Vogel, 2006). The fact that expression of the icmS gene increased during intensive multiplication of L. pneumophila in J774.1 cells suggests the importance of IcmS in L. pneumophila multiplication and survival in host cells.
RT-PCR was performed to determine the dynamics of bacterial gene expression during infection in J774.1 cells (Fig. 7). Compared with the results of the flow cytometric analysis (Fig. 6), gene expression in the early stage of infection was very low in the icm genes. This difference may be because intracellular bacteria were harvested from the host cells and used in RT-PCR assay, while both intracellular bacteria and bacteria adhering to cell surfaces were detected in the flow cytometric assay. Thereafter, only icmS expression increased gradually. The fact that the promoter activity of icmS, icmT and icmQ genes was found to be different over the course of 23 h of infection may give a hint regarding their role in the intracellular process.
The reasons why we did not wash cells after in vitro phagocytosis in the flow cytometric assay are as follows. First, because bacterial transcription is a very rapid process, we had to save time between the point of infection to the point of observation. Second, chromophore formation of Gfp is dependent on temperature and oxygen. Third, the half-life of unstable Gfp is short, and it is degraded by indigenous housekeeping proteases. Finally, the washing procedure, including centrifugation and pipetting, may physically affect cells containing fluorescent bacteria. Furthermore, J774.1 cells are usually cultured in cell culture plates, and detached using trypsin. However, in order to prevent possible chemical influence by trypsin, ordinary Petri dishes were used during the flow cytometric experiment. As mentioned above, several procedures during the infection experiment were omitted in the flow cytometric experiment. Therefore, the result obtained in the flow cytometric analysis from 0 to 6 h is derived from intracellular fluorescent bacteria, and also from the fluorescent bacteria that adhered to the cells.
Bacteria expressing unstable Gfp within J774.1 cells were readily identified by laser scanning confocal microscope at different time points after infection. Phagocytosed gfp-expressing bacteria were observed hourly for up to 24 h after infection. A brighter green light was observed at 20 h after infection for the icmS promoter, at 2 h after infection for the icmT promoter, and at 1 h after infection for the icmQ promoter (Fig. 8). The signal of unstable Gfp was perhaps weaker than the signal of stable Gfp, but it was still visible.
Many investigations are being performed to elucidate the structure and function of known proteins, but there are not enough studies being carried out on the transcriptional programme of the human pathogen L. pneumophila during host infection. Identification of genes that are upregulated in vivo, and not in vitro, may explain their contribution to in vivo growth of L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes that are predicted to promote transmission and manipulation of a new host, thereby priming it for the next attack. Substrates of the Dot/Icm secretion system, and other factors associated with invasion and virulence, and motility and type IV pilus machineries, and more than 90 proteins, have yet to be characterized. How L. pneumophila alternates between replicative and transmissive cell types, and which host factors are exploited during the replication phase, are only partly understood. Using unstable gfp as a reporter gene, we were able to show that the icmS, icmT and icmQ genes were highly expressed at different time points in infection. This suggests the suitability of unstable Gfp for quantitative determination of promoter activities in L. pneumophila.
Our work should allow us to look at the function of other essential genes by constructing conditionally expressing strains. Here, we report, for what we believe is the first time, the expression of unstable Gfp in L. pneumophila. The advantage of this system is the ability of L. pneumophila, carrying an unstable type of Gfp, to multiply in host cells. This opens up the exciting possibility of being able to control L. pneumophila gene expression in vivo. The studies presented here using promoter-gfp fusion constructs demonstrate the utility of our Gfp reporter system in in vitro phagocytosis. The regulation of other genes of L. pneumophila may be performed using this functional method that is amenable to assay. Monitoring gene expression during the infection cycle will provide a valuable insight into how L. pneumophila responds to its environment, and makes the changes necessary for adaptation and infection. Moreover, this reporter system could play a central role in identifying and characterizing regulatory elements in L. pneumophila. Moreover, in contrast to stable reporters such as LacZ that can only monitor an increase in gene expression, the unstable Gfp variant can report both ‘on’ and ‘off’ states, and thus provide the basis for an even more accurate monitoring of gene expression (Southward & Surette, 2002).
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ACKNOWLEDGEMENTS |
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Edited by: T. P. Hatch
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 10 September 2007;
revised 13 December 2007;
accepted 9 January 2008.
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| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
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, pTOM1(gfpAGA); 
, pTOM2(gfpLAA); 
, pTOM3(gfpAAV); 
, pTOM4(gfpASV) and 
, pTOM5(gfpmut3*).
