Division of Bacteriology, Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK1
Author for correspondence: J. Michael Sharp. Tel: +44 131 445 5111. Fax: +44 131 445 6111. e-mail: sharm{at}mri.sari.ac.uk
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ABSTRACT |
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Keywords: MAC, genomic insertion element, p40 gene, differential expression, gene conservation
The GenBank accession number for the p40 gene, together with 542 bp upstream sequence, is AF247653.
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INTRODUCTION |
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Recently, we described a 40 kDa protein in IS901+ strains of Mycobacterium avium (isolated from birds and domestic livestock) that is absent in IS901- strains (Nyange, 1990 ; Inglis et al., 1994
). This tight link between expression of the 40 kDa protein and the presence of IS901 was confirmed in a larger study of M. avium complex (MAC) isolates; only M. avium strains that were IS901+ expressed the 40 kDa protein (Ahrens et al., 1995
).
Experiments in mice and sheep have indicated that this protein may have an important role in vivo. Burrells et al. (1995) provided clear evidence for in vivo expression of the 40 kDa protein by demonstrating that sheep, infected experimentally with IS901+ M. avium, developed specific T-cell immune responses to the 40 kDa protein. No responses to the 40 kDa protein were detected in uninfected animals or in sheep infected with the closely related M. avium subsp. paratuberculosis, which is IS900+ but IS901-. Furthermore, experiments in mice have suggested that IS901 may enhance the pathogenicity of M. avium. In experimentally inoculated mice, IS901+ M. avium strains grew more rapidly than an IS901- strain and induced splenomegaly (Kunze et al., 1991
).
Because of the apparently absolute association between IS901 and expression of the 40 kDa protein, and because of their implication in increased pathogenicity, we initiated a study to characterize the 40 kDa gene and to determine its distribution and expression in other mycobacteria.
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METHODS |
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Disruption of mycobacterial cells.
M. avium (JD88/118) cells were harvested and washed three times in fresh PBS (0·137 M NaCl, 0·003 M KCl, 0·008 M Na2HPO4, 0·015 M KH2PO4, pH 6·9). Approximately 1 g washed mycobacterial cells was resuspended in 10 ml PBS containing PMSF at a final concentration of 1 mM. The cell suspension was transferred to the upper compartment of a pre-chilled (-70 °C) Eaton pressure chamber (Eaton, 1962 ), allowed to freeze for 34 min, and then forced through the aperture at 10000 p.s.i. (=69 MPa) in a hydraulic press. After thawing, high-Mr genomic DNA was sheared by 2x15 s rounds of sonication at 50% amplitude in a Vibra-cell model VCX 600 sonicator (Sonics & Materials), allowing 1 min cooling on ice between each sonication.
All other mycobacteria were disrupted by shaking with zirconium beads (Biospec Products). Approximately 100 mg washed mycobacterial cells was resuspended in 0·4 ml PBS containing PMSF at a final concentration of 1 mM. The cells were transferred to a screw-cap microfuge tube containing 1 ml washed 0·1 mm zirconium/silica beads. Cells were disrupted by bead-beating in a Mini Bead-Beater (BioSpec Products) (Challans et al., 1994 ). Five beating cycles of 1 min each were separated by 1 min cooling intervals on ice to avoid overheating and possible denaturation of the antigen preparation.
Protein purification and characterization.
Soluble protein concentrations in cell lysates were determined using the Micro BCA Protein Assay Reagent (Pierce-Warriner) in accordance with the manufacturers recommendations. p40 was purified from M. avium (JD88/118) cell lysates by HPLC (Burrells et al., 1995 ; Inglis, 1997
). The purity was confirmed by SDS-PAGE, using a discontinuous buffer system (Laemmli, 1970
), and by visualization of proteins by silver-staining (Morrisey, 1981
).
To determine the amino-terminal amino acid sequence of HPLC-purified p40, protein was transferred from an SDS-PAGE gel onto a PVDF membrane in 50 mM boric acid (pH 8·0), 20% (v/v) methanol, 3 mM 2-ß-mercaptoethanol at 100 V/1·0 A (constant voltage) for 60 min. Membrane-bound protein was visualized by staining with Coomassie brilliant blue and the bands were excised. The amino-terminal amino acid sequence was determined by sequential Edman degradation at the Microchemical Facility, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
Protein expression was investigated by Western transfer and immunoblotting. Electrophoretic transfer of proteins from SDS-PAGE gels to nitrocellulose membranes (Schleicher & Schuell) was carried out in accordance with the method of Herring & Sharp (1984) . Mono-specific polyclonal antibodies were affinity-purified from rabbit sera as described by Beall & Mitchell (1986)
.
Confirmation of species identity by PCR.
Cultured mycobacterial cells were mechanically disrupted by bead-beating. Lysates were centrifuged at 13000 g for 5 min to remove cell debris, and 5 µl clarified lysate was subjected to PCR analysis. The identity of M. avium subsp. paratuberculosis was confirmed by the detection of IS900 amplified using primers P90 (5'-GTTCGGGGCCGTCGCTTAGG-3') and P91 (5'-CCCACGTGACCTCGCCTCCA-3'), as described by Sanderson et al. (1992) . The identity of M. avium was confirmed by the detection of IS901 amplified using primers P102 (5'-CTGATTGAGATCTGACGC-3') and P103 (5'-TTAGCAATCCGGCGCCCT-3'). PCR conditions were as described for amplification of IS900, and amplification of a single band of 252 bp confirmed the species identity as IS901+ M. avium.
Cloning and sequencing of the p40 gene.
The entire p40 ORF was amplified by PCR from M. avium JD88/118 as follows. M. avium genomic DNA (125 ng) was added to pre-prepared reaction mixtures (100 µl final volume) comprising (x1) reaction buffer at 1·5 mM MgCl2 (Qiagen), 1·25 mM each of dATP, dCTP, dGTP and dTTP, 20 pmol each of the forward and reverse primers (forward primer: 5'-CTGCCAGCATGTGGCGTTGTG-3', positions -18 to +3; reverse primer, 5'-TCAGAACTGCAGCGCGTCGAACCGC-3', positions 1023998) and 1 U HotStar Taq polymerase (Qiagen). Hot-start PCR conditions consisted of an initial denaturation step of 15 min at 96 °C, followed by 35 cycles of 90 s denaturation at 96 °C, 1 min annealing at 65 °C and 90 s extension at 72 °C. The final cycle included an extension at 72 °C for 8 min. The 1041 bp amplification product was excised from a 0·8% (w/v) low-melting-point agarose gel, recovered using AgarAce (Promega) in accordance with the manufacturers instructions and then cloned into pGEM-T (Promega) using standard laboratory procedures (Sambrook et al., 1989 ). Competent Escherichia coli JM109 cells (Promega) were transformed in accordance with the manufacturers instructions. The identification of recombinants of interest was achieved by preparing colony arrays of 100 transformants per grid (Buluwela et al., 1989
) and screening by DNA hybridization, as described below. Small-scale preparations (minipreps) of plasmid DNA were prepared from E. coli host cells by using the QIAprep Spin Plasmid Miniprep Kit (Qiagen) in accordance with the manufacturers recommendations. Cloned DNA fragments were sequenced bidirectionally by the dideoxy chain-termination method (Sanger et al., 1977
). All nucleotide sequencing was performed by Oswel DNA Service, Southampton, UK.
Sequence analysis.
Database searches were performed using the BLAST (version 2.0) program from the National Centre for Biotechnology Information internet site covering the GenBank, EMBL, DDBJ and PDB databases, the WU-BLAST (version 2.0) program from The Institute for Genome Research (TIGR) BLAST Search Engine for Unfinished Microbial Genomes, the Sanger Centre M. bovis BLAST server, and the University of Minnesota microbial genome project BLAST server. Preliminary sequence data for M. avium were obtained from the TIGR website at http://www.tigr.org.
Amplification of the 5' flanking sequence.
The upstream sequence of the IS901+ M. avium p40 gene was isolated by a PCR-based gene-walking technique described by Britton et al. (1999) . Briefly, initial low-stringency amplification was performed using a random primer (5'-GCTCATAGCTGTGTATGTTCTG-3') in conjunction with a p40-gene-derived antisense primer (5'-GTGCGCGTGTAGAGGGAATCA-3') complementary to bases +341 to +321 relative to the p40 GTG translational start codon. Reaction mixtures contained 500 ng IS901+ M. avium genomic DNA, (x1) reaction buffer at 1·5 mM MgCl2 (Promega), 1·25 mM each of dATP, dCTP, dGTP and dTTP, 20 pmol each of the forward and reverse primers and 1 U Taq polymerase (Promega) in 50 µl reaction volumes. PCR conditions consisted of an initial denaturation step of 5 min at 94 °C, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 30 °C for 1 min and extension at 72 °C for 3 min. The final cycle included an extension at 72 °C for 7 min. The amplification product of this low-stringency PCR was then used as template in a second round of high-stringency amplification, using the same random primer as above, and a nested p40-gene-derived antisense primer (5'-CCAGGCCGCGATAGAACAGAT-3') complementary to bases +295 to +275 relative to the translational start codon. PCR conditions were as described above, except that the annealing temperature was increased from 30 °C to 57·9 °C. Second-round amplification products were separated on a 1% (w/v) agarose gel, Southern-blotted and probed at high stringency using a fluorescein-labelled (Gene Images; Amersham) p40 gene fragment corresponding to positions +22 to +295. Hybridizing bands were excised from the gel and recovered by AgarAce digestion in accordance with the manufacturers recommendations.
Preparation of a 528 bp p40-specific DNA probe.
M. avium genomic DNA (125 ng) was added to pre-prepared reaction mixtures (100 µl final volume) comprising (x1) reaction buffer at 1·5 mM MgCl2 (Qiagen), 1·25 mM each of dATP, dCTP, dGTP and dTTP, 20 pmol each of the forward and reverse primers (forward primer, 5'-CAGCGGGTCAAGGCCAATCTGTTCTA-3', positions 256281; reverse primer, 5'-CGCCAGCGCCAATCCGATGGTGTGCC-3', positions 784759), and 1 U HotStar Taq polymerase (Qiagen). Hot-start PCR conditions consisted of an initial denaturation step of 15 min at 96 °C, followed by 35 cycles of 1 min denaturation at 96 °C, 1 min annealing at 65 °C and 90 s extension at 72 °C, and a final extension step of 8 min at 72 °C. The PCR product was excised from a 0·8% (w/v) low-melting-point agarose gel, extracted with AgarAce and then labelled as described below.
Labelling and Southern hybridization of probe DNA.
High-molecular-mass genomic DNA was prepared from heat-killed organisms and digested with restriction endonuclease BamHI in accordance with a protocol described for PFGE (Hughes et al., 2001 ). Samples were electrophoresed in 1% (w/v) agarose gels and the DNA transferred to Hybond N+ nylon membrane (Amersham) as described by Sambrook et al. (1989)
. Probe DNA was labelled using the Gene Images random prime labelling module (Amersham) in accordance with the manufacturers recommendations. Hybridization was performed at 55 °C over 1516 h. The final probe concentration was 3 ng liquid block ml-1 (Amersham). Initial stringency washes (3x15 min) were carried out in 1x SSC/0·1% (w/v) SDS at 42 °C. Higher-stringency washes (3x15 min) were performed in 0·1x SSC/0·1% SDS at 55 °C. Hybridized probe DNA was detected using the Gene Images CDP-Star detection module (Amersham) in accordance with the manufacturers recommendations. Autoradiography was performed using Kodak X-OMAT AR scientific imaging film. Typical exposure times varied between 5 and 15 min at room temperature.
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RESULTS |
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p40 was purified from Eaton-pressed lysates of M. avium JD88/118 by ammonium sulphate precipitation followed by sequential size exclusion, anion exchange and hydrophobic interaction HPLC. This procedure yielded highly purified p40, as visualized on silver-stained SDS-PAGE gels (Fig. 1). Approximately 500600 µg purified p40 could be obtained from 1 g (wet wt) M. avium cells. Purified p40 was used to raise polyclonal antibodies to p40 in rabbits, and mono-specific polyclonal antibodies were affinity-purified from the rabbit antisera.
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Cloning and sequencing of the gene encoding p40 in IS901+ M. avium
To characterize the p40 gene in IS901+ M. avium in more detail, the entire p40 ORF was amplified by PCR from M. avium JD88/118 genomic DNA, using primer sequences determined from the TIGR unfinished M. avium genome sequence. This human isolate of M. avium (strain 104) is IS901-. A product of 1041 bp, including 18 bp genomic sequence upstream of the putative initiation codon (GTG), was amplified and sequenced. The complete nucleotide sequence of the p40 gene, together with 542 bp upstream sequence, is available from GenBank (accession no. AF247653). Comparative alignment of the p40 genes of IS901+ and IS901- M. avium revealed only four nucleotide substitutions at positions +291, +321, +336 and +493 (99·6% identity) (data not shown). Only one of these substitutions (position 493) translated to an amino acid substitution at residue 164, where proline, in IS901- M. avium, is replaced with alanine in IS901+ M. avium (data not shown).
Characterization of the upstream sequences of the p40 gene from IS901+ M. avium
To determine whether any features of the genomic sequences immediately upstream of the p40 genes in IS901+ and IS901- strains of M. avium might be associated with the differential expression of p40, the upstream region of IS901+ M. avium strain JD88/118 was obtained by a PCR-based gene-walking technique. A total of 814 bp was obtained, comprising 272 bp of the 5' end of the p40 gene sequence and 542 bp upstream sequence (accession no. AF247653). A BLAST search of the TIGR unfinished M. avium genome sequence revealed 99% identity with a homologous region on fragment 187. Further searches of the TIGR, GenBank, University of Minnesota and Sanger Centre databases identified the corresponding p40 upstream sequences of M. avium subsp. paratuberculosis, M. bovis, M. leprae, M. smegmatis and M. tuberculosis. Comparison of the p40 upstream regions of both IS901+ and IS901- M. avium revealed two single base pair substitutions at positions -14 and -24, relative to the GTG initiation codon, with a possible third substitution at position -25, where Y, in the IS901- M. avium sequence, denotes either C or T (Fig. 3). No copy of the IS901 insertion element was observed within the 542 bp upstream region.
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p40 gene homologues are widely distributed within the genus Mycobacterium
To determine the distribution of the p40 gene within the genus Mycobacterium, Southern blots of genomic DNAs of 23 Mycobacterium species (Table 1) were probed with a 528 bp fragment of the p40 gene of M. avium (strain JD88/118). Genes homologous to the p40 gene in IS901+ M. avium were detected in all of the species tested, with eight exceptions (Table 1
and Fig. 4
). Seven of these (M. chelonae subsp. chelonae and subsp. abscessus, Mycobacterium fortuitum, Mycobacterium kansasii, M. marinum, Mycobacterium phlei and Mycobacterium terrae) were checked by PCR using p40 consensus primers. A p40-specific product was amplified from M. kansasii, M. marinum, M. phlei and M. terrae but not from M. fortuitum, M. chelonae subsp. chelonae or subsp. abscessus. The M. leprae p40 gene homologue is currently available in the EMBL database and was not tested by PCR.
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DISCUSSION |
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Database searches identified complete p40 ORFs in M. bovis, M. leprae, M. smegmatis, M. tuberculosis and, in particular, IS901- M. avium and M. avium subsp. paratuberculosis, whose respective p40 gene homologues share 99·6 and 98·9% identity with that of IS901+ M. avium. However, because almost all of the mycobacteria included in this study were propagated in vitro, our observations cannot exclude in vivo expression of p40. Nevertheless, the absence of p40 expression in M. leprae, together with the observation of specific T-cell immune responses to the p40 antigen in sheep experimentally infected with IS901+ M. avium but not in sheep infected with M. avium subsp. paratuberculosis (Burrells et al., 1995 ), support the absence of in vivo expression in other mycobacteria and indicate that differences in gene regulation may account for the unusual expression.
To ascertain whether species-specific expression of p40 might be attributable to differences in nucleotide sequence at promoter level, the upstream regions of p40 gene homologues from all seven of the above Mycobacterium species were aligned and compared. Preliminary analysis of these upstream sequences revealed -10 and -35 hexamers of a putative promoter region. Notably, IS901+ M. avium and M. smegmatis were observed to differ from the other species at the -14 position, where T replaces C at the first position of the putative -10 region. In addition, TGN motifs, which have been reported to enhance the transcriptional activity of some mycobacterial promoters (Bashyam & Tyagi, 1998 ), were noted to immediately precede the -10 hexamer in all cases except M. smegmatis, in which a single nucleotide (A) separated the TGN motif and the first base of the -10 region. Single base pair mutations in -10 regions or their TGN extensions have been reported to exert marked effects on the efficiency of gene transcription in mycobacteria and enterobacteria (Bashyam & Tyagi, 1998
; Timm et al., 1994
; Youderian et al., 1982
). However, in the absence of laboratory confirmation, no firm conclusions can be drawn from currently available data. Further work is under way to establish whether expression of the p40 gene in IS901+ M. avium is the result of only a single base pair substitution in the -10 region of the putative promoter.
Differences in the promoter region, as discussed above, do not explain the absolute concordance between expression of p40 and IS901. The insertion sequence itself does not contain an ORF for a p40 homologue, nor does it possess outward-facing promoters that are capable of switching on expression of adjacent genes, as described for IS10/Tn10 (Dale, 1995 ). It seems unlikely, therefore, that IS901 is acting in cis to upregulate p40; this is supported by our failure to amplify a product using primers derived from the 3' end of IS901 and the 5' end of the p40 gene (unpublished).
An alternative mechanism that could possibly account for constitutive expression of p40 only in IS901+ M. avium would be if expression of the p40 gene were normally tightly regulated and under the control of a repressor mechanism in Mycobacterium species that lack IS901. Such regulatory mechanisms have been reported for a number of mycobacterial genes (Dhandayuthapani et al., 1997 ; Durbach et al., 1997
; Dussurget et al., 1996
, 1999
; Manabe et al., 1999
; Movahedzadeh et al., 1997
; Schmitt et al., 1995
; Sun et al., 1998
). If a similar repressor protein is responsible for regulating expression of the p40 gene, constitutive expression of p40 would be a direct consequence of integration of IS901 within either the repressor gene itself or the p40 gene repressor binding site. Such unregulated expression of p40 could be manifested as a phenotypic change or altered pathogenicity of IS901+ M. avium. It has been demonstrated that IS901+ M. avium (type A/I) is more virulent than IS901- M. avium (type A) in experimentally inoculated BALB/c mice (Kunze et al., 1991
; Pedrosa et al., 1994
). Although the increased virulence was attributed to the presence of IS901 in M. avium type A/I strains, it is conceivable that unrepressed expression of p40 might be involved. Whatever the nature of the association between IS901 and expression of the p40 gene, it is clear that the presence of multiple copies of IS901 appears to divide M. avium into two distinct subtypes with differing host ranges and pathogenicity. It should be emphasized, however, that in the absence of a clearly defined molecular mechanism, there is currently no firm evidence upon which a direct link between these differences and the presence of IS901 can be based. Nevertheless, it is intriguing that the presence of the closely related insertion element IS900 in M. avium subsp. paratuberculosis (Green et al., 1989
) also supports differences in host range and pathogenicity relative to strains of M. avium that possess neither IS901 nor IS900. With the implications for comparative functional genomics in mind, it would clearly be of great interest to identify and characterize the genes affected by the insertion of IS901 and IS900 in M. avium and M. avium subsp. paratuberculosis, respectively. The characterization of most of the IS900 loci in M. avium subsp. paratuberculosis was reported recently (Bull et al., 2000
). Furthermore, completion of the ongoing M. avium and M. avium subsp. paratuberculosis genome sequencing projects should facilitate our understanding of how the acquisition and chromosomal integration of genomic insertion elements might affect the biology of these organisms.
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ACKNOWLEDGEMENTS |
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Received 29 September 2000;
revised 2 January 2001;
accepted 5 February 2001.
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