Instituto de Biotecnología1 and Instituto de Patobiología2, CICV-INTA, PO Box 77, Castelar, Argentina
Author for correspondence: A. Cataldi. Tel: +54 11 4621 0199. Fax: +54 11 4481 2975. e-mail: acataldi{at}inta.gov.ar
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ABSTRACT |
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Keywords: Mycobacterium tuberculosis, Mycobacterium bovis, promoter, operon, lipoprotein
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INTRODUCTION |
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P27 is a novel Mycobacterium bovis antigen, identified from an expression library using sera from naturally infected cattle (Bigi et al., 1997 ). Sequence analysis indicated that P27 has a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). Cellular fractionation experiments suggested that P27 is an integral membrane protein. Downstream of the P27 gene there is an ORF for a 55 kDa protein (P55) which is highly homologous to membrane proteins related to antibiotic resistance in Streptomyces and other bacteria. These genes seem to be organized as an operon, because only six bases separate the end of the P27 gene from the start of the P55 gene. The present study demonstrates the polycistronic nature of both genes, as well as the location of the promoter.
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METHODS |
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DNA manipulations.
Standard methods were used for restriction-endonuclease digestion of plasmids, DNA ligations, Southern blotting, and other manipulations (Ausubel et al., 1996 ). Plasmid DNA isolation was performed using a Wizard Minipreps SV kit, according to the manufacturers instructions (Promega). DNA probes for Southern blot hybridization were labelled with [
-32P]CTP using the Oligolabelling random priming kit according to the manu-facturers instructions (Pharmacia).
Mycobacterial DNA preparation.
DNA from M. bovis and M. smegmatis was prepared according to van Soolingen et al. (1991) .
Plasmid constructions.
Plasmids used are described in Table 1.
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To obtain plasmids carrying different deletions in the 5' non-coding sequences of the P27 gene, four DNA fragments containing the P27 gene plus different DNA extensions upstream of the ATG start codon were obtained by PCR, using chromosomal DNA from M. bovis BCG as template and the 2-1Av3 primer in combination with the 2-1Dir, U292 and U241 primers (Table 2). The PCR products were cloned into the pGEM-T plasmid (Promega), generating pGdir27, pG292 and pG241, respectively. Subsequently, the PstI fragments obtained by digestion of pGdir27, pG292 and pG241 were cloned in the PstI site of pSUM41 vector, generating pMBA27
P, pMBA2792 and pMBA2741, respectively. In parallel, pHP45 was digested with HindIII to isolate the streptomycin-resistance gene (2·0 kb), which was subsequently ligated into HindIII-linearized pMBA2792. This plasmid was called pMBA2792
. The PstI fragment from pMBA27
P was subcloned into the unique PstI site of pMV261, downstream of the hsp60 promoter. The resulting plasmid was named pMBA28.
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PCR amplification was performed with Taq DNA polymerase (Promega) under standard conditions in a volume of 50 µl. The concentration of dNTPs was 0·2 mM each, and 20 pmol of each primer was used. Cycling conditions were: one cycle of 95 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 49 °C (for 2-1Av3/U292 primer set), 53 °C (for 2-1Av3/U241 primer set) or 54 °C (for 2-1Av3/2-1Dir primer set) for 1 min, 72 °C for 1 min, and finally 72 °C for 10 min. A total of 0·1 ng plasmid or 2 ng genomic M. bovis DNA was used as template. Sequencing was performed by PCR-mediated Taq-cycle sequencing using the fmol sequencing kit (Promega) according to the manufacturers instructions. Sequencing products were developed on 6% polyacrylamide gels
RNA preparation.
Total RNA from M. bovis BCG and M. smegmatis was isolated following the method of Bashyam et al. (1996) . The two rRNA species and a band of lower molecular mass, corresponding to tRNA, were visible after staining agarose gels with ethidium bromide, indicating that the RNA preparations were of high integrity.
Northern blotting.
RNA samples were run on 0·8% agarose gels containing 2·2 M formaldehyde (Ausubel et al., 1996 ), and transferred by vacuum in 10xSSC (3 M NaCl, 0·3 M sodium citrate) to a nylon membrane. RNA was fixed to the membrane by UV light cross-linking. Membranes were pre-hybridized for 3 h in 500 mM sodium phosphate (pH 7·2)/7% SDS at 55 °C. Hybridization was performed as described by Harth et al. (1996)
in the same buffer at 55 °C for 48 h with an antisense oligonucleotide (L358P27) labelled with [
-32P]ATP at 108 c.p.m. µg-1. Membranes were then washed three to five times with 50 mM sodium phosphate (pH 7·2)/0·1% SDS, and exposed to Kodak X-ray film (Biomax MR) at -70 °C.
Primer extension.
Primer extension was performed using the L358P27 primer. Ten picomoles of the non-phosphorylated primer was labelled with T4 polynucleotide kinase (Promega) in the presence of [-32P]ATP. RNA (6 µg) and the labelled primer (0·1 pmol) were mixed in a volume of 7 µl containing 50 mM Tris/HCl (pH 8·3) and 0·1 M KCl. The reaction was then incubated for 1 min at 94 °C, 10 min at 55 °C and 15 min on ice. The volume of the mixture was adjusted to 12 µl by the addition of 1 µl of a mixture containing dNTPs (2·5 mM each), 0·5 µl RNAsin (Promega), 2·2 µl reverse transcriptase 5xbuffer (0·25 M Tris/HCl, pH 8·3; 0·2 M KCl; 36 mM magnesium acetate, 0·01 M DTT), 0·8 µl diethylpyrocarbonate-treated water and 0·5 µl avian myeloblastosis virus reverse transcriptase (RT; Promega). Reverse transcription was performed at 42 °C for 45 min and stopped by addition of 5 µl stop buffer.
Samples were electrophoresed in a 6% polyacrylamide gel containing 8 M urea alongside the sequencing products obtained with the same oligonucleotide primer. Gels were fixed in 5% (v/v) methanol/5% (v/v) acetic acid, and exposed to X-ray film (Kodak X-Omat RS) for 24 h at -70 °C.
RT-PCR.
Synthesis of the first strand of cDNA was performed using 3 µg total M. bovis BCG RNA as template and random hexamers as primers, following the indications in the SUPERScript Preamplification System for First Strand cDNA Synthesis kit (Life Technologies). Ribonuclease-treated RNA samples were used as a negative control. A 5 µl aliquot of the cDNA synthesis reaction was amplified with primers 2-1Dir/2-1Av4 and 2-1Dir/2-1Rev. Amplification conditions were denaturation at 94 °C for 2 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min. Amplification products were detected in 1% agarose gels. The specificity of the amplified bands was determined by Southern blotting using [-32P]dCTP pMBA21 as probe.
SDS-PAGE and Western blotting.
Cells were harvested by centrifugation, washed and resuspended in PBS buffer, and lysed by boiling in loading buffer (2% SDS; 0·125 M Tris/HCl, pH 6·8; 1% 2-mercaptoethanol; 0·02% bromophenol blue; 10% glycerol) for 10 min. Proteins (50 µg) were separated by electrophoresis in 15% SDS-PAGE gels by the technique of Laemmli (1970) , and electrotransferred onto nitrocellulose using a Bio-Rad Trans-Blot Cell tank transfer unit at 150 mA for 2 h in 25 mM Tris/HCl (pH 8·0), 0·19 M glycine and 20% (v/v) methanol. Transfer yield was visualized by transient staining with Ponceau Rouge. Non-specific sites in the blot were blocked by incubation for 1 h with 5% dried non-fat powdered milk in 20 mM Tris/HCl (pH 7·5), 0·5 M NaCl buffer (TBS) at room temperature. Nitrocellulose membranes were incubated overnight at 4 °C with a 1:300 dilution of anti-P27 polyclonal serum followed by an alkaline-phosphatase-conjugated secondary antibody for 2 h at room temperature. Colour reaction was developed for 30 min by the addition of 5-bromo-4-chloro-3-indolyl phosphate and toluidinum nitro blue tetrazolium as substrates.
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RESULTS |
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Deletion analysis of the P27 gene promoter
In order to map the P27 gene promoter, deletion derivatives of the 5' non-coding region were generated. Four DNA fragments containing the P27 gene plus different-sized DNA extensions (410, 161, 110 and 28 bp) of the 5' non-coding region were amplified by PCR and inserted into the pSUM41 vector. The resulting plasmids were named pMBA27, pMBA2741, pMBA2792 and pMBA27P. All these constructions were introduced by electroporation into M. smegmatis mc2155. P27 expression was assessed in cell extracts by Western blotting using an anti-P27 serum (Fig. 3
). P27 expression was detected in M. smegmatis/pMBA27, M. smegmatis/pMBA2741 and M. smegmatis/pMBA2792 strains, while none was observed in M. smegmatis/pSUM41 and M. smegmatis/pMBA27
P (Fig. 3
, lanes 15). To discard the possibility that the plac promoter of the pSUM vector drives P27 gene transcription, a transcription terminator was added to the construction carrying the minimal promoter region. The omega (
) interposon was cloned into the plasmid pMBA2792 downstream of the plac promoter, resulting in plasmid pMBA2792
. It has been demonstrated that insertion of an
fragment into DNA leads to termination of RNA synthesis in M. smegmatis (Timm et al., 1994
). In this study, P27 expression was detected in M. smegmatis/pMBA2792
(Fig. 3
, lane 6). To test the integrity of the P27 gene in the shorter insert (that showing no expression), the P27 coding sequence in the pMBA27
P plasmid was subcloned downstream of the hsp60 promoter into the pMV261 vector, to give the pMBA28 plasmid. P27 expression was observed in M. smegmatis/pMBA28 (Fig. 3
, lane 7), confirming that the insert has a potentially functional gene.
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DISCUSSION |
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The recognition of promoter sequences in prokaryotes is mainly mediated by the subunit of RNA polymerase. In most bacteria, these promoters are constituted by hexamers placed in positions -10 and -35 or in their vicinity. In E. coli, the -10 and -35 consensus sequences and the distance that separates them have been extensively studied. However, this information is not directly applicable to M. tuberculosis or M. bovis, because, in general, mycobacterial promoters function poorly in E. coli, indicating that mycobacteria may possess different transcriptional signals. Despite their importance as microbial pathogens and heterologous expression systems, little is known about the structure of mycobacterial gene promoters. Several promoters were predicted by either computer analysis or their distances from experimentally determined transcription start points, using conventional E. coli promoter spacing and sequences as a guideline, but only in some cases was promoter activity experimentally confirmed. The low homology among the promoter sequences reported makes the establishment of a specific consensus sequence for mycobacterial promoters difficult. However, a number of mycobacterial consensus sequences have been proposed. Bashyam et al. (1996)
used mycobacterial promoter sequences isolated from M. tuberculosis and M. smegmatis to generate the following probable consensus sequence for the -10 region: T (100%), A (93%), T (50%), A (57%), a (43%) and T (71%) for M. smegmatis and T (80%), A (90%), Y (60%), g (40%), A (60%) and T (100%) for M. tuberculosis. However, they were unable to find a single strongly conserved sequence in the -35 regions. Using all the putative mycobacterial sequences published, Mulder et al. (1997)
calculated the mycobacterial promoter consensus sequence as follows: -35: T (92%), T (53%), G (62%), A (44%), C (60%), G (31%)/A (26%); and -10: T (68%), A (76%), T (41%), A (36%)/G (26%), A (34%)/C (28%), T (76%). During analysis of the 5' untranslated P27 gene region two overlapping putative -10 (GAACAT, CATCGC) sequences were identified. The GAACAT -10 hexamer proposed conserves the second and the last two positions on the Mulder et al. (1997)
consensus. We also identified a putative -35 sequence (TTCCTC) which shares 66% identity with other mycobacterial promoters. Other putative promoters were described in our previous paper (Bigi et al., 1997
). However, these potential promoters as well as the -35 element associated with transcriptional start point P1 are outside the essential promoting region. The future characterization and mapping of the P2 promoter are necessary to understand the regulation mechanisms of P27/P55 operon transcription. It is possible that the promoters are recognized by different RNA polymerase holoenzymes, and they are utilized to different extents during growth.
The spacing between the putative -10 and -35 regions is in agreement with other mycobacterial promoters reported. It has been demonstrated that the mycobacterial promoter can accommodate a large variety of sequences between the -35 and -10 regions. Such an analysis was performed by Kremer et al. (1995) , who observed that the distances from 4 bp to 64 bp are functional in the 85A promoter.
Since the mapping of the P27 gene promoter was performed in M. smegmatis for reasons of convenience, it would be important to confirm these results in a native producer species (M. bovis and M. tuberculosis). However, Bashyam et al. (1996) suggested that the basic transcriptional machineries of M. smegmatis and M. tuberculosis have transcriptional specificity determinants in common. Thus M. smegmatis can be safely used as a surrogate host for expression of at least the constitutively expressed genes from slowly growing pathogenic mycobacteria.
The proximity of an ORF downstream of the P27 gene led us to suppose the existence of a polycistronic messenger which would be the transcription product of both genes. Although polycistronic messengers are often found in prokaryotes, to our knowledge only a few mRNAs of this type have been identified in M. bovis, e.g. ESAT-6 antigen, which was found to be encoded in a transcriptional unit formed by two genes (Berthet et al., 1998 ). Many more polycistronic transcripts should exist in M. bovis and M. tuberculosis, since analysis of the genome sequence of M. tuberculosis (Cole et al., 1998
) allows the identification of various potential operons. Using Northern blotting and RT-PCR assays, we demonstrated that P27 and a gene encoding a putative antibiotic transporter (P55) belong to the same transcriptional unit. Such a genetic organization suggests that a functional relationship exists between the products of both genes. The search for the role of this operon in M. bovis is currently under way.
Interestingly, three putative membrane proteins from M. tuberculosis, LprA, LprF and LppX (accession nos Z77137, Z81011 and Z83858, respectively), share high homology with P27. The ORFs encoding these three proteins as well as the P27 version of M. tuberculosis (lprG; accession no. Z80108) were identified during the M. tuberculosis genome sequencing project (Cole et al., 1998 ) but in contrast to the P27 gene, they are not adjacent to genes encoding drug transporters and do not seem to be organized in an operon-like structure. Immunoblot studies indicated that P27 shares epitopes with LprA (data not shown). The function of P27 and these related sequences is not known, but the close identity suggests that they may belong to a novel membrane antigenic protein family.
Future studies will help determine whether this conserved sequence reflects a similarity in cellular function of these proteins.
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ACKNOWLEDGEMENTS |
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F.B., M.I.R. and A.C. are fellows of the National Research Council of Argentina (CONICET). K.C. is the recipient of a CONICET fellowship.
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Received 17 June 1999;
revised 3 December 1999;
accepted 20 December 1999.