Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil1
Biotechnology Centre, Federal University of Pelotas, Pelotas, Brazil2
Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil3
School of Biological Sciences, University of Surrey, Guildford, Surrey, UK4
Author for correspondence: Douglas McIntosh. Tel: +55 21 2598 4284. Fax: +55 21 2260 4727. e-mail: mcintosh{at}bio.fiocruz.br
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ABSTRACT |
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Keywords: mycobacteria, shuttle vectors, expression, stability
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INTRODUCTION |
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Initial surrogate cloning hosts included Escherichia coli and Streptomyces lividans (Clarke-Curtiss et al., 1985 ; Kieser et al., 1986
). However, it was only following the discovery of a transformation efficient (ept) mutant strain of the rapid growing species Mycobacterium smegmatis, i.e. strain mc2155 (Snapper et al., 1990
), that the cloning and study of genes from pathogenic and non-pathogenic mycobacteria became a relatively common practice. The utility of this bacterium as a cloning host is well established, and its characteristics and applications to the study of mycobacterial genetics have recently been reviewed (Jacobs, 2000
). Certainly, the possible use of other species of mycobacteria as cloning hosts has received some attention (Parish & Stoker, 1997
; Hermans & de Bont, 1996
). In particular, Mycobacterium aurum has been employed in a number of studies (Hermans et al., 1990
, 1993
; Lazraq et al., 1991
). This species was considered to be attractive for the study of the genetic mechanisms involved in sensitivity and resistance to the antibiotics used in the treatment of both tuberculosis and leprosy (Lazraq et al., 1991
).
In addition, Mycobacterium vaccae, specifically the so-called Stanford strain (NCTC 11659), has been employed as a cloning host for the expression of recombinant forms of mycobacterial proteins including superoxide dismutase (Garbe et al., 1994 ), major extracellular proteins (Harth et al., 1997
) and the 19 kDa lipoprotein of M. tuberculosis (Abou-Zeid et al., 1997
), and for the development of an epitope delivery system for use with recombinant mycobacteria (Hetzel et al., 1998
). Interestingly, Golanska et al.(1998)
reported a biotechnological application for M. vaccae, where the mutant B3805 was used for cloning and expression of genes involved in steroid degradation pathways. This species has also attracted interest for use in immunotherapy of tuberculosis (Corlan et al., 1997
; Onyebujoh et al., 1995
).
A review of the literature concerning transformation of mycobacteria reveals that the overwhelming majority of vectors used with this genus contain a gene encoding resistance to kanamycin as a selectable marker (Pashley & Stoker, 2000 ; Parish & Stoker, 1997
; Hermans & de Bont, 1996
). However, some species including M. vaccae (NCTC 11659) have been reported to be non-transformable using this selection method, possibly due to inefficient expression of the aminoglycoside phosphotransferase gene involved in kanamycin resistance (Garbe et al., 1994
). Consequently, vector systems containing a gene encoding resistance to hygromycin have been developed that allow the transformation of this species (Garbe et al., 1994
; Parish & Stoker, 1997
). Hygromycin resistance is considered attractive in providing a second selectable marker for genetic experiments with mycobacteria and in providing a resistance marker that avoids the use of a clinically applicable drug (Garbe et al., 1994
). Yet, the use of hygromycin instead of kanamycin suffers from a number of disadvantages including expense (hygromycin is approximately 15 times more expensive than kanamycin), low-level mammalian toxicity and the need to use elevated concentrations of this antibiotic to select E. coli transformants (200 µg ml-1 instead of 20 µg ml-1 for kanamycin). Finally, and perhaps most significantly, almost all available mycobacterial vectors contain a kanamycin resistance gene. The possibility exists that these factors may have contributed to the relatively limited interest shown for the use of M. vaccae as a surrogate cloning host, despite indications that it may represent a valuable alternative to the more commonly employed M. smegmatis mc2155.
In this report we describe the successful transformation of M. vaccae (ATCC 15483) with a range of vectors encoding resistance to kanamycin as a selectable marker. Thereafter, our objective was to extend this observation through a comparative evaluation of this strain of M. vaccae and M. smegmatis mc2155 as cloning hosts, with emphasis on the functional activity of promoter sequences used in expression vectors, the production of heterologous proteins and stability (both functional and structural) of vector constructs.
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METHODS |
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Plasmid construction.
A schematic representation of the steps involved in the design of the pUS series of vectors is provided in Fig. 1. Plasmid pUS971 was constructed by cloning a 1·6 kb PCR fragment from pAL5000, corresponding to nt 43111100 and containing the mycobacterial origin of replication (oriM), into pUC18 digested with EcoRI/AflIII and treated with the Klenow fragment of DNA polymerase I. This construct was subsequently digested with BmpI/HindIII to excise the ampicillin resistance gene and PCR-generated DNA fragments representing aph (kanamycin resistance gene) from Tn903 or the hygromycin resistance gene of Streptomyces hygroscopicus were cloned in its place to produce pUS972 and pUS982 respectively. The vector pUS972 contains a kanamycin resistance marker, includes both an E. coli and mycobacterial origin of replication and a multiple cloning site, and formed the basis for the production of all subsequent plasmids of the pUS series developed in this study. Sequences corresponding to the hsp60 promoter of M. bovis BCG and the PAN promoter derived from IS900 of Mycobacterium paratuberculosis were generated by PCR, digested with BamHI/KpnI then cloned into pUS972 cut with the same enzymes, giving rise to two expression vectors i.e. pUS973 (Phsp60) and pUS977 (PAN), suitable for the production of recombinant proteins. A promoter-probe vector, pUS986, was developed by cloning a 3083 bp PCR-generated fragment, digested with HindIII, of the E. coli lacZ gene sequence derived from pMC1871, into HindIII-digested pUS972. This vector allows quantitative assessment of promoter activity in vitro based on the measurement of ß-galactosidase activity derived from the promoterless lacZ sequence. To generate promoter-containing derivatives of pUS986, DNA fragments corresponding to the hsp60 promoter of M. bovis BCG, the hsp70 and the 18kDa promoter of M. leprae and the PAN promoter were produced by PCR, digested with BamHI/KpnI and subsequently cloned into pUS986 cut with the same restriction endonucleases. This procedure resulted in the vectors pUS985, pUS989, pUS990 and pUS993. A fifth vector (pUS995) was produced by digestion of the vector pUS985 with BamHI/BstEII and subsequent introduction of a PCR-derived amplicon, digested using the same enzymes and representing the 16S rRNA promoter of M. tuberculosis, designed to replace the -35 and -10 regions of the hsp60 promoter while leaving the ribosome binding site and start codon of the hsp60 promoter in place. These constructs allowed a meaningful comparison of the relative strengths of different promoters within the same vector.
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ß-Galactosidase assay.
ß-galactosidase activity was assayed in E. coli and mycobacteria as described by Miller (1992) . Duplicate 1 ml volumes of test cultures (OD600 0·81·2) were collected by centrifugation in 2 ml screw cap microcentrifuge tubes (Bio-Rad), the supernatant was removed by aspiration and 500 µl glass beads (0·1 mm diameter; Biospec Products) and 1 ml Z buffer (Miller, 1992
) were added to each tube. Cells were disrupted on a mini beadbeater apparatus (Biospec Products) by mixing for 1 min or 1·5 min (M. bovis BCG) with 30 s pulses, with tubes held on ice before and between pulses. After separation of the beads by centrifugation, aliquots (0·5 ml) of the resulting supernatants were transferred to 1·5 ml microcentrifuge tubes and held at 28 °C for 5 min. Thereafter, 0·1 ml 4 mg ONPG ml-1 was added and incubation was continued at 28 °C until sufficient yellow colour had developed, at which point the reaction was stopped by adding 250 µl 1 M Na2CO3 and the incubation time was recorded. The OD420 of each reaction mix was measured and ß-galactosidase activity was calculated using the formula reported by Dellagostin et al. (1995)
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Expression and detection of recombinant S1 subunit of pertussis toxin in mycobacteria.
Mycobacteria transformed with the vectors pUS973, pUS977, pPB10 and pPB12 were grown to a density of approximately 108 cells ml-1 in liquid medium containing 25 µg kanamycin ml-1. Volumes (2 ml) of each culture were sedimented at 10000 g in 2 ml screw cap microcentrifuge tubes and prepared for Western blotting analysis by washing once in PBS, followed by resuspension in 100 µl lysis buffer (10 mM Tris/HCl, pH 7·5, 1 mM EDTA, 2 mM PMSF) in the presence of 50 mg glass beads (0·1 mm diameter). Cells were disrupted by two 30 s pulses in a mini beadbeater, mixed with 33 µl 4x concentrated SDS-PAGE sample buffer (Laemmli, 1970 ) then boiled for 10 min. The beads were allowed to settle and samples of the uncleared cell lysate were analysed by SDS-PAGE and Western blots (Towbin, 1979
). The anti-S1 monoclonal antibody (3CX4PT, kindly donated by Dr Drusilla Burns, Food and Drug Administration, Bethesda, MD, USA) was employed at a dilution of 1:6000 and protein bands reacting with this antibody were visualized using alkaline phosphatase conjugated goat anti-mouse IgG (Sigma), with subsequent incubation with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) for colour development.
Shuttle vector stability in mycobacteria and E. coli.
Mycobacterial transformants were cultured in liquid medium containing the appropriate antibiotic over three consecutive subcultures, while E. coli transformants were grown over 10 consecutive subcultures, diluting cultures 1/1000 in fresh medium each time. Total DNA was collected from the mycobacterial cultures using the small scale method reported by Santos et al. (1992) . Plasmid DNA was produced from E. coli using the Flexiprep kit (Amersham Pharmacia-Biotech), according to the manufacturers instructions. Electrocompetent cells of E. coli DH5
were transformed by electroporation with 2 µl total mycobacterial DNA and were plated on LB agar containing 25 µg kanamycin or 200 µg hygromycin B ml-1 as appropriate. Three individual colonies from each transformation were collected and used to produce plasmid DNA that was digested with the restriction endonucleases BamHI, EcoRI and HindIII (Gibco BRL). Electrophoresis of digested plasmids was performed in 1% agarose gels and banding patterns were visualized with ethidium bromide under UV illumination. The presence of gross structural modifications was assessed by comparison with the banding patterns generated from the plasmid preparations used to produce the original transformants.
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RESULTS AND DISCUSSION |
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Transformation of mycobacteria by electroporation
Details of representative transformation efficiencies obtained using the test vectors for the transformation of M. bovis BCG, M. smegmatis mc2155 and M. vaccae ATCC 15483 by electroporation are provided in Table 2, along with values obtained using M. vaccae and the vectors pMV261 and pBEN. Transformation efficiencies were calculated as the number of antibiotic resistant colonies recovered from 100 µl electrocompetent cells per µg vector DNA. It was observed that all three species could be transformed with each of the vectors examined, with the highest transformation efficiencies being recorded for M. smegmatis. An exception to this was the vector pUS982 and M. vaccae, where the level of transformation was 10-fold higher than that observed for mc2155 with the same vector. The pre-treatment of cultures with glycine (final concentration 1·5% during the last 24 h growth) did not have a marked effect upon the observed efficiency of transformation for any species with any vector (data not shown). In contrast, the use of an elevated temperature (25 °C), instead of the more commonly used 4 °C for the preparation of cells for the electroporation process was found to greatly reduce the number of transformants of both M. smegmatis and M. vaccae for all of the vectors examined (data not shown).
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In the study of Garbe et al. (1994) , data provided for transformation efficiencies were based on a comparison of the vectors pBAK14 (kan) and p16R1 (hyg). These vectors differed not only in terms of their selectable marker, but also with respect to size and the composition of their basic structure. As such, it is not clear if the resistance gene component was the true reason behind the inability of the pBAK14 vector to transform M. vaccae NCTC 11659. In an attempt to clarify this issue, we performed a comparison of the vectors pUS972 and pUS982, which are essentially identical in all aspects except their resistance marker. This experiment revealed that the presence of the hyg sequence resulted in a vector with an improved capacity to transform M. vaccae. Indeed, this vector produced the highest transformation efficiencies recorded in this study and an argument could be made for using hygromycin resistance as the marker of choice with M. vaccae. However, it should be emphasized that the efficiencies recorded with the other vectors are more than sufficient for most applications. Furthermore, in contrast to the observations of Garbe at al. (1994)
, we found the vectors conferring kanamycin resistance to be superior to pUS982 in terms of the efficiency of transformation achieved with BCG and M. smegmatis mc2155, and we did not detect any marked difference in the growth rate of the two types of transformants.
Comparison of promoter activity in different cloning hosts
The ability to readily transform M. vaccae with previously described cloning vectors and with those developed in this study was considered a significant observation. Therefore, experiments were performed to evaluate the suitability of this species as a cloning host in comparison with M. smegmatis mc2155. Initial experiments examined the functional activity of a range of previously characterized mycobacterial promoters in three mycobacterial cloning hosts and in E. coli. The promoters used in the present study have been employed by other workers for the development of mycobacterial expression vectors or in the study of gene regulation in mycobacteria (Murray et al., 1992 ; Das Gupta et al., 1993
; Yasutomi et al., 1993
; Dellagostin et al., 1995
). Yet, to our knowledge no study has compared the functional activities of these promoters when cloned in a unique expression system and there is no information on their ability to regulate gene expression in M. vaccae.
The promoters used were the hsp60 promoter of M. bovis BCG, the hsp70 and the 18kDa promoters of M. leprae, the PAN promoter of M. paratuberculosis and the 16S rRNA promoter of M. tuberculosis. The vectors pUS986 (promoterless), 985 (hsp60), 989 (PAN), 990 (hsp70), 993 (18kDa) and 995 (16S rRNA) were used to transform M. bovis BCG, M. smegmatis mc2155, M. vaccae ATCC 15483 and E. coli by electroporation. The transformation efficiencies recorded for these vectors were similar to those reported in Table 2, with the exception of pUS985 which routinely gave rise to 10- to 100-fold fewer transformants than the other constructs, this effect being most noticeable in M. smegmatis mc2155.
Functional activities (Miller U ß-galactosidase activity) of the test promoters in the three species of mycobacteria and in E. coli are shown in Table 3. All promoters showed activity in the three species of mycobacteria, although the relative strengths varied as a function of the species in which they were cloned. Thus, the hsp60 promoter of M. bovis was seen to be the strongest promoter in BCG where it was approximately five times more active than the hsp70, PAN and 18kDa promoters, and 56 times more active than the 16S rRNA promoter of M. tuberculosis (Table 3
). In M. smegmatis mc2 155, the hsp60, hsp70 and 18kDa promoters exhibited similar levels of activity which were approximately fourfold lower than that of the hsp60 promoter in BCG (Table 3
). The PAN promoter was found to be the strongest promoter in this species of mycobacteria, where it showed almost twice the level of activity recorded in BCG. In the case of M. vaccae, the PAN promoter was also found to have the highest activity among the test promoters, showing almost three times the activity of the two heat-shock gene promoters and approximately two times more activity than the 18kDa promoter (Table 3
). In common with the situation in BCG, the 16S rRNA promoter was found to function relatively poorly in both M. smegmatis and M. vaccae.
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Data obtained with E. coli revealed that the majority of the test promoters functioned poorly in this genetic background. A notable exception was the 18kDa promoter of M. leprae which showed the highest activity (11310 Miller U) for any promoter in any cloning host examined (Table 3). Similarly, the 16S rRNA promoter was found to function optimally in E. coli, where it was almost four times as strong as in M. vaccae and 25 times more active than in M. smegmatis mc2155. The relatively low activity in mycobacteria was surprising in the light of previously reported high activity of this promoter in both BCG (Glaxo) and M. smegmatis LR222, when cloned in the integration-proficient expression vector pDK20 (Das Gupta et al., 1998
).
The experiments investigating the functional activity of promoters in the different cloning hosts were performed with the objective of determining whether existing mycobacterial expression systems could be employed with M. vaccae and this was found to be the case. However, it should be noted that no attempt was made to investigate the possible influence that differences in plasmid copy number may have exerted upon the levels of ß-galactosidase activity recorded in the three species. In the absence of such data it is not possible to conclusively state that the differences in enzyme activity observed with some promoters were solely the result of differences in promoter function in a given host. Previous studies have indicated that pAL5000-based vectors are present at between 2 and 5 copies in M. smegmatis (Pashley & Stoker, 2000 ; Stolt & Stoker, 1996
); it would be of value for future studies to address this issue of plasmid biology in other cloning hosts including BCG and M. vaccae.
Cloning and expression of the S1 subunit of pertussis toxin in different mycobacterial hosts
The development of vaccines based on expression of recombinant antigens in M. bovis BCG (rBCG) is an ongoing area of research in numerous laboratories worldwide (Ohara & Yamada, 2001 ; Nascimento et al., 2000
; Leung et al., 2000
; Langranderie et. al., 1998
). Due to the extended growth time of BCG, a commonly used preliminary step in the analysis of test constructs involves the transformation of M. smegmatis mc2155 to confirm antigen expression and evaluate vector stability in a mycobacterial environment (OGaora, 1997
). To determine if M. vaccae could also be used for this purpose, we prepared two constructs, pPB10 and pPB12, each containing a sequence encoding the entire S1 subunit of pertussis toxin, which has been proposed as a candidate antigen in rBCG whooping cough vaccines (Abomoelak et al., 1999
; Nascimento et al., 2000
). These vectors were used to transform the three mycobacterial cloning hosts and duplicate cultures of each recombinant were processed and analysed by Western blotting. Expression of a strongly immunoreactive band with a molecular mass of 31·7 kDa (Fig. 2
) in combination with the presence of a second immunoreactive band with a molecular mass of 28·7 kDa was observed in all pPB10 and pPB12 recombinants but was absent in control recombinants transformed with pUS973 or pUS977 (Fig. 2
). The smaller band showed the same mobility as the S1 subunit control and as such is likely to represent mature S1 protein. The larger band is thought to represent a pro-form of the S1 subunit. The levels of antigen expression were equivalent in the three species and were estimated to be around 40 ng per 106 cells, a value obtained based on comparison of the band intensities produced from known quantities of pertussis toxin which were included in all blots. This result was somewhat surprising given that the two expression vectors contained promoters that differed substantially in terms of their ability to drive the expression of recombinant ß-galactosidase.
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Analysis of vector stability
The issue of vector stability, both structural and functional, within mycobacteria has received relatively little attention hitherto but has begun to achieve greater prominence particularly in the light of its implications for vaccine development.
Plasmid DNA was recovered from mycobacterial transformants as described in Methods and was used to transform E. coli DH5 by electroporation. Vector DNA was purified and subjected to restriction digestion with BamHI, HindIII and EcoRI. The resulting restriction patterns were compared with those of the vectors originally used to transform the mycobacterial hosts. Gross modifications in vector structure were observed in mc2155 (pUS973, 985, 989, 995, pPB10 and pPB12) and in M. vaccae (pUS972 and pUS985). In the majority of cases the modifications were represented by deletions but insertions were occasionally encountered.
A more detailed analysis of this phenomenon was performed using the vectors pUS985 and pUS989. The pUS985 construct was found to be highly unstable in mc2155 with 90% of clones losing the ability to express ß-galactosidase within two subcultures in liquid medium containing kanamycin. All of these clones were subsequently found to contain structurally modified vectors (Fig. 3). Thirty percent (3 of 10) of M. vaccae clones transformed with this vector were found to suffer structural modifications (Fig. 3
) and loss of ß-galactosidase expression within three subcultures. In contrast, the pUS989 vector was observed to be more stable in M. smegmatis mc2155 with only one clone out of 10 suffering gross structural modifications over three subcultures. However, 40% (4 of 10) of the clones lost the ability to express ß-galactosidase within three subcultures. In the case of M. vaccae, 20% (2 of 10) of the clones transformed with pUS989 lost the ability to express ß-galactosidase but no evidence of gross structural modifications was detected. Finally, six individual E. coli clones transformed with either pUS985 or pUS989 were subcultured 10 times and no evidence of gross modifications was found. The ability to allow stable maintenance and expression of heterologous DNA is an essential feature of a cloning host and M. vaccae appears to fulfil these criteria adequately. Indeed, data from this study indicate that M. vaccae is less likely to induce either structural or functional plasmid instability than M. smegmatis. However, this will only be confirmed through a more detailed examination of this topic, using a wider range of vectors.
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Existing cloning hosts and vector systems have limitations
Immunological and biochemical studies of subcellular components of the pathogenic mycobacteria have been hampered by the difficulty in producing sufficient contaminant-free quantities of these molecules directly from such slow-growing organisms (Harth et al., 1997 ; Horn et al., 1999
). Conventional recombinant protein technology has been applied in an attempt to resolve this problem, but suffers from the fact that many mycobacterial proteins are not faithfully produced in common cloning hosts such as E. coli (Hermans & de Bont, 1996
; Herrmann et al., 1996
). Consequently, expression systems for use with M. smegmatis were developed for the production of recombinant forms of mycobacterial proteins (Zhang et al., 1991
; Harth et al., 1997
; Triccas et al., 1998
; De Smet et al., 1999
), although this approach has not proved to be completely satisfactory. In this context, it was reported that the expression of M. tuberculosis superoxide dismutase in M. smegmatis mc2155 generated hybrid enzymes, whilst expression in M. vaccae allowed the recovery of the recombinant enzyme apparently in the native form (Garbe et al., 1994
). Moreover, production of recombinant 45/47 kDa molecules (Apa) of M. tuberculosis by M. smegmatis resulted in antigen with a different mannosylation pattern and reduced capacity to stimulate T lymphocyte responses in comparison to that observed for the native antigen (Horn et al., 1999
). These observations and those of the present study demonstrate that the expression of recombinant proteins in mycobacteria is influenced by a number of factors including vector structure, promoter strength and the nature of the cloned gene.
The identification of M. smegmatis mc2155 as a surrogate cloning host represented a landmark in the study of mycobacterial genetics. The applications and properties of this strain have recently been reviewed (Jacobs, 2000 ), and it is beyond doubt that it has served as a powerful tool for the advancement of our knowledge in numerous areas of mycobacterial biology. Indeed, the successful use of mc2155 virtually ended the search for alternative cloning hosts (Hermans & de Bont, 1996
). Yet, it has been argued by some authors (Garbe et al., 1994
; Hermans & de Bont, 1996
) that although M. smegmatis mc2155 is a useful model system for mycobacterial genetic studies, there exists a need for additional cloning hosts which may be more useful for specific aspects of mycobacterial biology and which would provide increased flexibility to the field of recombinant protein technology for mycobacteria. In this context M. vaccae, specifically ATCC 15483, represents an alternative mycobacterial cloning host that has been largely overlooked to date. We have determined that, in contrast to the findings of Garbe et al. (1994)
using M. vaccae NCTC 11659, M. vaccae ATCC 15483 is capable of being transformed with a range of commonly used vectors that carry a gene encoding kanamycin resistance as a selectable marker. A comparison of M. vaccae ATCC 15483 with M. smegmatis mc2155 as cloning hosts revealed that a range of characterized mycobacterial promoters cloned in the same promoter probe vector showed broadly similar activities, in terms of expression of the reporter enzyme ß-galactosidase, in these two hosts and in BCG, demonstrating that existing expression systems can readily be employed with M. vaccae. These results were expanded through the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those seen with rBCG and M. smegmatis. Significantly, M. vaccae appears to offer the advantage of being less likely to induce either structural or functional plasmid instability than M. smegmatis, at least with the constructs examined in this study. Taken as a whole, these data clearly show that M. vaccae ATCC 15483 represents a versatile tool for use in the study of mycobacterial genetics.
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ACKNOWLEDGEMENTS |
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Received 17 October 2001;
revised 11 March 2002;
accepted 12 March 2002.
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