The gene encoding P27 lipoprotein and a putative antibiotic-resistance gene form an operon in Mycobacterium tuberculosis and Mycobacterium bovis

F. Bigi1, A. Alito2, M. I. Romano1, M. Zumarraga1, K. Caimi1 and A. Cataldi1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
P27 is an antigenic membrane lipoprotein synthesized by members of the Mycobacterium tuberculosis complex. Northern blotting and RT-PCR experiments indicated that the genes encoding P27 and a putative antibiotic transporter (P55) form an operon. A promoter region was identified and characterized by deletion analysis in Mycobacterium smegmatis. Two transcription initiation points were mapped in Mycobacterium bovis BCG by primer extension analysis to 76 bp and 87 bp upstream of the ATG initiation codon. Putative -10 and -35 promoter consensus sequences associated with these showed 66% similarity to previously identified mycobacterial promoters. These results suggest that the P27/P55 operon is transcribed from two promoters in M. bovis BCG.

Keywords: Mycobacterium tuberculosis, Mycobacterium bovis, promoter, operon, lipoprotein


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The identification and characterization of mycobacterial antigenic proteins are essential for the development of new diagnostic tests and vaccines, and for the understanding of the pathogenic mechanisms of mycobacteria and the immune responses to them. Numerous mycobacterial antigens have been described (Young et al., 1992 ); many of them have been expressed in Escherichia coli and their recognition by immune cells and antibodies determined. However, little is known concerning their cellular function in mycobacteria. Gene expression regulation studies may help to understand their physiological function, but there is slow progress in this field. Only a few mycobacterial promoters have been studied (Bashyam et al., 1996 ; Mulder et al., 1997 ), and many of these are from phage or antibiotic-resistance genes, which might not be similar to the genes involved in virulence. In addition, most mycobacterial promoters described seem to be different from the E. coli consensus -10 and -35 elements (Dale & Patki, 1990 ). Also, little is known about to what extent mycobacterial genes form parts of operons. Some operons have been experimentally demonstrated (Papavinassasundaram et al., 1997 ; Berthet et al., 1998 ), but most were postulated based on nucleotide sequence examination (Braibant et al., 1996a , b ; DeMaio et al., 1997 ; Cole et al., 1998 ).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, and growth and electroporation conditions.
All cloning steps were performed in E. coli DH5{alpha}. Expression studies were done in Mycobacterium smegmatis mc2155. Cultures of E. coli were grown in LB broth or on LB agar. For the promoter deletion experiments, M. smegmatis cells were grown to late-exponential phase at 37 °C, either in liquid or on solid LB medium and in Middlebrook 7H11 medium supplemented with OADC (albumin, dextrose, catalase and oleic acid; Difco). When necessary, appropriate antibiotics were added at the following concentrations: ampicillin, 100 µg ml-1; streptomycin, 10 µg ml-1; kanamycin, 50 µg ml-1 for E. coli or 20 µg ml-1 for M. smegmatis. M. bovis BCG Pasteur P1732 was grown to saturation in Middlebrook 7H9/0·05% Tween 80 supplemented with OADC (M7H9-OADC-Tw). Then, 2 ml culture was transferred to 100 ml M7H9-OADC-Tw broth and cultured further for 10 d. Mycobacterial strains were electroporated using the method of Wards & Collins (1996) . Electroporations were performed using a Bio-Rad Gene Pulser. For use in transformation, M. smegmatis mc2155 was grown to mid-exponential phase in M7H9-OADC-Tw. Following electroporation, M. smegmatis was cultured on Middlebrook 7H11 medium supplemented with OADC and containing antibiotic.

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 manufacturer’s instructions (Promega). DNA probes for Southern blot hybridization were labelled with [{alpha}-32P]CTP using the Oligolabelling random priming kit according to the manu-facturer’s 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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Table 1. Plasmids used in the present study

 
A 1·5 kb SacI fragment was obtained by digestion of pMBA21 and cloned into SacI-linearized pGEM-5Zf (Promega), generating pP27G. A 1·2 kb PstI fragment of pP27G, corresponding to the 5' non-coding region followed by the sequence encoding P27, was cloned into the PstI-digested pSUM39 shuttle vector. The resulting plasmid was named pMBA27.

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{Delta}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{Omega}. The PstI fragment from pMBA27{Delta}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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Table 2. Oligonucleotide primers

 
PCR amplification and sequencing.
Primers used are listed in Table 2.

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 manufacturer’s 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 [{gamma}-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 [{gamma}-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 [{alpha}-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Determination of the transcription initiation point
The start point of P27 transcription was determined based on the primer extension assay using total M. bovis BCG RNA treated with DNase. An antisense primer whose 5' end corresponds to the nucleotide -25 relative to the A in the start codon was used. This reaction yielded two products, a larger one (P1), with a weaker signal consistent with a start at the A situated at 87 nt from the start codon, and a shorter product (P2), with the stronger signal, and corresponding to the most likely transcription start point, mapping to a G 76 nt upstream of the start codon (Fig. 1a, lane 2; Fig. 1b). However, the possibility that these extension products may result from a transcriptional stop of the reverse transcriptase in the assay due to RNA secondary structures cannot be excluded. Primer extension reactions were repeated three times, and the same banding pattern was observed. Ribonuclease treatment abolished the observed extension product (Fig. 1, lane 1).



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Fig. 1. Determination of transcriptional start points for the P27 gene. (a) Primer extension reactions were carried out with end-labelled L358P27 primer annealing at nucleotide position -25 relative to the A residue of the initiation codon of the P27 gene. This primer was then annealed to total RNA isolated from M. bovis BCG, and extended with reverse transcriptase. Reactions were analysed on urea-6% gels. Sequencing reactions were performed with the same oligonucleotide on pMBA21 and run alongside the primer extension reaction. Primer extension reactions were carried out in the presence (lane 1) or absence (lane 2) of ribonuclease (20 µg ml-1). (b) Genetic arrangement upstream from the P27 gene. The transcriptional start points (P1 and P2) are indicated; the -35 consensus sequence is boxed and the -10 consensus sequence is boxed and underlined. The essential promoting region is shaded; the putative Shine–Dalgarno sequence is underlined; the ATG translational start codon is indicated in bold.

 
P27 and P55 genes are organized in an operon
The close proximity of an ORF encoding P55 in the same transcriptional orientation downstream of the P27 gene suggests that the P27 and P55 genes form an operon. To investigate this possibility, Northern blot hybridization and RT-PCR reactions using total M. bovis BCG RNA were performed. For Northern blots, hybridizations were carried out using a specific radiolabelled antisense oligonucleotide probe, L358P27, which targets to nucleotide position -25 on the P27 mRNA relative to the ATG start codon. A single band, heavier than 2 kb, was detected in M. bovis BCG RNA (Fig. 2a). The specificity of the oligonucleotide was demonstrated by the absence of detectable hybridization in total RNA from M. smegmatis. M. bovis BCG RNA treated with ribonuclease did not react, indicating that genomic DNA was not present. The possibility of cross-hybridization of the P27 probe to the rRNA species was excluded by stripping the blot and rehybridizing with a 16S rRNA gene of Bacillus subtilis. RNA bands (one band of 1·5 kb for the small rRNA; and a weakly cross-reactive band of 3·2 kb for the large rRNA) showed different molecular masses than those observed with the P27 probe (Fig. 2b). All samples were previously treated with DNases to avoid hybridization to DNA. Thus the P27 mRNA transcript size is consistent with polycistronic transcription.



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Fig. 2. Analysis of the P27/P55 mRNA transcript. Total RNA (10 µg) extracted from M. bovis BCG (lanes 1 and 2) and M. smegmatis (lane 3) was separated on 0·8% agarose gels supplemented with formamide/formaldehyde, and processed for Northern blotting as described by Ausubel et al. (1996) in the presence (lane 1) or absence (lanes 2 and 3) of ribonuclease (20 µg ml-1). Hybridization was carried out using either the L358P27 [{gamma}-32P]ATP antisense oligonucleotide probe (a) or a radiolabelled 16S rRNA probe (b). Autoradiography was performed for 7 d or 24 h, respectively. (c) Total RNA (3 µg) from M. bovis BCG was analysed by RT-PCR. cDNA amplification was performed with random hexamer primers. (d) Primer positions of 2-1Dir/Av4 and 2-1Dir/2-1Rev used in PCR reactions.

 
For RT-PCR assays, a population of cDNA was obtained by the use of a hexanucleotide mix in the retrotranscription reaction of M. bovis BCG RNA (treated with DNase), which was used in a second step as template in PCR reactions. Two combinations of primers were used for the amplification reaction: 2-1Dir/2-1Av4 and 2-1Dir/2-1Rev. See Fig. 2(d) for the position of each primer. RT-PCR produced amplification products of the expected size for polycistronic mRNA (1·617 kb with the primers 2-1Dir/2-1Rev and 898 bp with the primers 2-1Dir/2-1Av4) (Fig. 2c). The fidelity of the reaction was confirmed by Southern blotting using pMBA21 as a probe (data not shown). In ribonuclease-treated RNA samples no bands were observed in agarose gels or by Southern blotting, excluding the possibility that the products were amplified from genomic sequences.

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 pMBA27{Delta}P. 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{Delta}P (Fig. 3, lanes 1–5). 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 ({Omega}) interposon was cloned into the plasmid pMBA2792 downstream of the plac promoter, resulting in plasmid pMBA2792{Omega}. It has been demonstrated that insertion of an {Omega} 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{Omega} (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{Delta}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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Fig. 3. Western blot of cell extracts from M. smegmatis clones carrying deletions of the 5' non-coding region of P27. Lanes: 1, M. smegmatis/pMBA27; 2, M. smegmatis/pMBA2741; 3, M. smegmatis/pMBA2792; 4, M. smegmatis/pMBA27{Delta}P; 5, M. smegmatis/pSUM41; 6, M. smegmatis/pMBA2792{Omega}; 7, M. smegmatis/pMBA28. The lanes were incubated with anti-P27 polyclonal serum.

 
Deletion analysis showed that a sequence important for promoter activity lay between 28 and 110 nt upstream of the start codon (Fig. 4). Analysis of the region upstream of transcriptional start point P2 revealed two potential -10 hexamers, one (GAACAT) 8 bp upstream which has 3/6 matches with the typical E. coli -10 consensus sequence (TATAAT), 4/6 matches with the mycobacterial superoxide dismutase gene promoter (GATTAT) (Zhang et al., 1991 ) and 4/6 matches with the promoter of a gene encoding the 38 kDa protein of Mycobacterium tuberculosis (GAAATT) (Andersen & Hansen, 1989 ), and another (CATCGC) 5 bp upstream of the transcription start point which has 4/6 matches with the -10 promoter sequence of the mpb70 gene (CATCAG) (Matsuo et al., 1995 ). In addition, we found a -35 hexamer positioned at 26 nt from the transcription start point which has 4/6 matches with the -35 consensus sequence of the hsp60 promoter of M. bovis BCG (TTGCAC) (Shinnick, 1987 ; Thole et al., 1987 ).



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Fig. 4. Determination of promoter activity of the 5' non-coding region in M. smegmatis. Genetic elements are indicated. The stem–loop indicates a possible transcriptional terminator. Plasmids harbouring different 5' non-coding deletions were obtained by cloning of PCR amplification products into pSUM41. Expression of the P27 recombinant protein is indicated.

 
However, at 7 bp upstream of the longer extension product (P1) there is an AACAAT motif that matches five of six positions of the -10 (AAGAAT) promoter sequence of the hsp60 gene of M. bovis BCG. Around the corresponding -35 position, we identified several potential consensus sequences which are homologous to published promoter sequences of M. bovis and M. tuberculosis. However, these -35 elements are outside of the minimal promoting region as determined by the deletion experiment, indicating that they are not essential to the transcription of the P27 gene under the culture conditions used in this study.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To study the expression and transcriptional features of the P27 gene, we characterized its 5' non-coding region. We mapped the P27 gene promoter region by the progressive 5' deletion of the putative regulatory sequence. Our results revealed that there is a minimal promoter region between bases -28 and -110 relative to the start codon. We defined this promoter region as an essential sequence to transcribe the P27 gene under the culture conditions used in this work. Two possible transcription initiation points were identified, but only one (P2), which maps 76 bases upstream of the proposed ATG initiation codon, had associated potential promoter sequences placed in the minimal region with promoter activity. Probably, a second promoter (P1) is localized in the deleted region. Operons with more than one promoter have been described in mycobacteria which are active under different physiological conditions (Suzuki et al., 1991 ; Movahedzadeh et al., 1997 ; Verma et al., 1999 ). It could be possible that the P1 promoter is dominant under other conditions and the promoter region may be dynamic depending on the growth conditions.

The recognition of promoter sequences in prokaryotes is mainly mediated by the {sigma} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by the Centro Argentino Brasileño de Biotecnología (CABBIO) and the Programa de Cooperacion Hispano-Argentina. We thank Haydee Gil for technical help. The valuable suggestions of Cecilia Vazquez Rovere are gratefully acknowledged.

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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 17 June 1999; revised 3 December 1999; accepted 20 December 1999.