Structure of the Mycobacterium tuberculosis Antigen 88, a Protein Related to the Escherichia coli PstA Periplasmic

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1 NFECrlON AND MMUNTY, Mar. 1994, p /94/$ Copyright 1994, American Society for Microbiology Vol. 62, No. 3 Structure of the Mycobacterium tuberculosis Antigen 88, a Protein Related to the Escherichia coli PstA Periplasmic Phosphate Permease Subunit MARTNE BRABANT,' LUKAS DE WT,' PRSKA PERS,' MCHAEL KALA,1 JOSETTE OOMS,' ANNE DROWART,2 KRS HUYGEN,' AND JEAN CONTENTl* Department of Virology, nstitut Pasteur du Brabant, B-1180 Brussels,' and Service de Pneumologie, H6pital Erasme (Universite Libre de Bruxelles), 1070 Brussels,2 Belgium Received 19 October 1993/Accepted 8 December 1993 We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in Xgtll. The gene encodes a 403-amino-acidresidue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane a-helical domains and presents a significant homology to the PstA protein ofescherichia coli. n its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS ofe. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria. The development of recombinant DNA technology for efficient expression of mycobacterial genes in Escherichia coli represented an important breakthrough in the study of defined antigenic molecules from Mycobacterium tuberculosis and Mycobacterium leprae (37). The use of murine monoclonal antibodies and patient sera in the screening of Xgtll recombinant DNA libraries containing the genomes of the leprosy and tuberculosis bacilli has led to the identification of numerous antigens (see reference 36 for a review). The majority of monoclonal antibodies used in early cloning attempts had been defined in the first and second World Health Organizationsponsored workshops and generally originated from BALB/c mice immunized with mycobacterial extracts (14). Most of the products of the initially cloned genes later turned out to belong to the group of highly conserved heat shock proteins homologous among others to the products of the GroEL, GroES, and DnaK genes from E. coli (36). Subsequently, new antigenic specificities could be defined by immunizing mice with early mycobacterial culture filtrate (24), containing mostly secreted antigens, or by infecting mice with live M. tuberculosis (20) or Mycobacterium bovis BCG (18). Furthermore, BCG infection of different strains of mice was reported to generate different repertoires of antibodies against culture filtrate antigens, and genes from the major histocompatibility complex turned out to be essential in the control of the antibody repertoire (18). Sera from BCG-infected H-2b haplotype mice were found to react preferentially against a 37-kDa-38-kDa doublet protein and against a 40-kDa antigen from a BCG culture filtrate. Analysis of the antibody repertoire against culture filtrate antigens in BCG-infected C57BL/10 mice with a recombinant H-2 haplotype and in C57BL/6 mice with a mutant bml2 (major histocompatibility complex class ) haplotype has indicated that the -A het- * Corresponding author. Mailing address: Department of Virology, nstitut Pasteur du Brabant, 642 Rue Engeland, B-1180 Brussels, Belgium. Phone: Fax: Electronic mail address: jcontent@rc4.vub.ac.be. erodimer controls the recognition of this 40-kDa antigen (17). Using two potent monoclonal antibodies, i.e., 2F8-3 and 2C1-5, raised against this 40-kDa culture filtrate antigen, we were able to identify this immunodominant 40-kDa protein as antigen 88 (Ag 88) according to the reference system of Closs et al. for BCG antigens (9). We now report the cloning and sequence determination of the gene encoding Ag 88 from M. tuberculosis, and we demonstrate that it is the mycobacterial homolog of the PstA gene of E. coli, coding for a membrane-bound phosphate permease protein subunit. MATERLALS AND METHODS Screening of the Xgtll M. tuberculosis and M. bovis BCG recombinant DNA libraries. The Agtll library from M. tuberculosis (Erdman strain) kindly provided by R. A. Young (37) was screened as described previously (7, 19), with two monoclonal antibodies (2F8-3 and 2C1-5) directed against Ag 88 (17) mixed and used at a dilution of 1:10. The BCG Xgtll library was constructed earlier (13). Crude lysates from Xgtll recombinant lysogens were prepared exactly as described before (7). Southern blot analysis. Genomic DNA from M. bovis BCG was prepared and analyzed as described previously (10) by using T4 kinase 32P-labeled PstA-like (5' AGGAGATGCTC AGGTTGG) and PstC-like (5' CTCCATGCTTGG'TTTGG) probes (see Fig. 3). Cloning and sequencing. Plaque screening by hybridization, subcloning in pbluescript SK+, and restriction analysis were done according to standard procedures (19). All sequencing was performed on double-stranded plasmidic DNA on both strands by using Sanger's technique with the T7 sequencing kit (Pharmacia) and labeling with 35S-dCTP. Ambiguous regions with high GC content or compressions were resolved by using dtp instead of dgtp with the Sequenase kit and using 7-deaza-dGTP with the Taq-Track kit (Promega). Computeraided analyses of the nucleic acids and deduced amino acid sequences were performed with the DNA Strider program (25) 849

2 850 BRABANT ET AL kda *b. C1 N U~ C7 FG. 1. mmunoblotting analysis of crude lysates of E. coli lysogenized with clones Cl (M. tuberculosis) and C7 (M. bovis BCG). Shown is a Western blot with an anti-,b-galactosidase antibody (anti- 3-gal) (Boehringer, Mannheim, Germany), monoclonal antibody 2C1-5, and crude serum from BCG-infected C57BL/6 mice. Molecular mass (in kilodaltons) was estimated by comparison with a molecular weight marker (Rainbow protein molecular weight markers; Amersham). and the Genetics Computer Group program of the Belgian EMBnet Node (network facility). Homology searches in the protein sequence data banks were greatly facilitated by the use of the EMBL Blitz server, which utilizes the Smith and Waterman algorithm (27) on an MPsrch program. Nucleotide sequence accession number. The DNA sequence of Ag 88 has been deposited in the European Molecular Biology Laboratory data base under accession number X RESULTS Screening of Agtll M. tuberculosis and M. bovis BCG recombinant DNA libraries with two Ag 88-specific monoclonal antibodies. Fifty filters containing the replica of 5.0 x 106 plaques of the M. tuberculosis Agt 1l library (37) were probed with a mixture of two monoclonal antibodies which both recognize specifically the Ag 88 protein in Western blot (immunoblot) analysis of culture filtrates of M. bovis BCG and M. tuberculosis (17). One positive Agtll clone (Cl) could be isolated and expressed as a fusion protein with E. coli 3-galactosidase in E. coli lysogens. Because of this unexpected low frequency, a similar screening was performed on 2 x 106 plaques from a M. bovis BCG Xgtl1 library (13) and yielded 13 positive clones. Among these, six (C2, C7, C8, C9, C13, and C14) were similarly expressed as lysogens in E. coli Y1089, and crude lysates of these cells were analyzed by Western blotting. n all cases, a fusion protein of 150 to 155 kda could be identified with monoclonal antibody 2C1-5, suggesting that all clones contained a relatively large part of the Ag 88 gene. A representative assay is shown in Fig. 1 in which the 0) NFECT. MMUN. 1-galactosidase fusion products of Cl (M. tuberculosis) and C7 (M. bovis BCG) are revealed with the monoclonal antibody 2C1-5, with anti-1-galactosidase antibody, and also with crude serum from BCG-infected C57BL/6 mice. None of the observed lysogens expressed the unfused mature protein as such. This could be due to the inability to express the corresponding promoter in E. coli (as is often the case for mycobacterial genes) (28). Alternatively, the promoter could be too distant from the coding sequence, or the expression of Ag 88 as such could be toxic for E. coli. Since all six BCG clones and the single M. tuberculosis clone, Cl, cross hybridized and appeared to express fusion proteins with very similar molecular masses, we concentrated our efforts on the analysis of the M. tuberculosis-derived phage Cl. Restriction analyses of clones Cl and Al. Cl had an EcoR insert of 2.3 kb. This fragment was subcloned in pbluescript SK+ and characterized by restriction analysis with several endonucleases. An internal EcoRV site was used to subclone and partially sequence two smaller fragments. Since the largest open reading frame detected in Cl corresponded to a 341- amino-acid protein with an estimated molecular mass of 37,093 Da, we anticipated that this clone was incomplete at its 5' end (as expected in the product of a 1-galactosidase fusion protein). We therefore screened the same Xgtll recombinant M. tuberculosis DNA library with an oligonucleotide (TTCTT CACCTCGTTCGT) located at bp 219 of the 5' end of Cl (Fig. 2). Several Xgtl 1 recombinants were analyzed, and a restriction map of the most informative (Al) could be aligned with that of Cl. Al extends both 5' (1 kb) and 3' (0.9 kb) from Cl. From the results of subsequent sequencing, it was determined that the 5' end of Cl corresponds to a "natural" EcoR site where fusion with Xgtll B-galactosidase occurred, deleting the 62 NH2-terminal amino acids of the protein. DNA sequencing. The 1,655-bp nucleotide sequence derived from overlapping Xgtll clones Al and Cl is shown in Fig. 3. The upstream limit of the large open reading frame contained within this sequence is still uncertain since seven in-frame, potential initiation codons (ATG or GTG) exist within a very short distance (-6 to +84) (Fig. 3). However, only one of these potential initiation sites is preceded 7 nucleotides upstream by a typical E. coli Shine-Dalgarno ribosome binding site sequence (five of six residues homologous to AGGAGG), and therefore it is likely that the corresponding ATG (position 1 in Fig. 3) could be the 5' limit of a 1,209-bp open reading frame ending with a TGA codon at position The overall base composition of this DNA is 63.04% G+C, with a strong preference for G or C in position 3 of codons. No sequence resembling an E. coli promoter can be detected upstream of this open reading frame. One typical TTGACA (-35 hexanucleotide box) is found at position 1482, but it is not followed by a Pribnow box. Sequence of the Ag 88 protein. The deduced amino acid sequence of the Ag 88 gene corresponds to a protein of 403 amino acid residues with an expected molecular mass of 43,790 Da. The protein sequence does not seem to contain an NH2-terminal signal peptide as deduced from the hydropathy pattern and the SigCleave analysis program (32). However, most of the protein appears to be very hydrophobic (residues 130 to 400), displaying at least seven hydrophobic domains (which also contain potential ot-helices according to the Garnier and Chou-Fasman prediction programs) and could thus correspond to membrane-spanning domains (Fig. 4). The NH2-terminal quarter of the protein is much less hydrophobic and contains a large number of charged residues in particular in the region between amino acids 34 and 126, where 21

3 VOL. 62, 1994 EcoRV EcoR i1 Sma.1. EcoR V THE ANTGEN 88 GENE OF M. TUBERCULOSS 851 Sma EcoR Cl 11 i i i i i Al l 11 Noti Pvul Rsal Pvul Rsal _ )r _ ( EcoRV EcoR Smnal EcoRV 11 Sma EcoR 0 o.s iS kb FG. 2. Restriction map of the DNA insert in the Agtl 1 M. tuberculosis recombinant clone Cl (identified with monoclonal antibodies 2F8-3 and 2C1-5 and clone Al (selected by hybridization with an oligonucleotide located at bp 219 of the 5' end of Cl). The vertical bars represent experimentally determined restriction sites. The solid box indicates the sequenced region, and the stippled box indicates the region coding for Ag 88. O, localization of the oligonucleotide used for the screening by hybridization. positions (corresponding to 22.5% of the sequence) correspond to arginine residues. n this NH2-terminal region, there are three clusters of Arg triplicates, a fourth being present at the carboxy-terminal end. Three of them are encoded by the same sequence, CGA-CGG- CGG. A short hydrophobic domain between positions 70 and 80 is also predicted, but it is probably too short to be considered a transmembrane (TM) oa-helix and, in contrast to what is generally observed for a membrane-spanning helix, it contains several charged residues (see Fig. 6). A search of the SwissProt data bank (release 24) with the EMBL Blitz server, which uses the Smith and Waterman algorithm (27) on an MPsrch program, revealed a strong homology to several proteins belonging to the group of multisubunit TM periplasmic permeases of gram-negative bacteria (1, 2). nterestingly, among these, the highest homology score was observed for the E. coli PstA protein (29). n association with PstC, PstB, and PstS, this permease subunit is responsible for the transport of phosphate and has 35.8% identity over 279 amino acid residues (76.7% homology when conservative replacements are considered) with the corresponding sequence of Ag 88 (Fig. 5). This homology is also apparent when the hydropathy profiles of the two proteins are compared (Fig. 4). The corresponding DNA regions also show a significant homology: 54.5% over a 718-bp region (data not shown). The Prosite "signature" of the inner membrane component of binding-protein-dependent transport systems was not found in our mycobacterial protein (6). Nevertheless, a signature motive differing only by one amino acid residue (Ala-308) from the presently defined consensus was found in a region homologous to its location in the PstA protein (Fig. 5). This conserved sequence is usually located 80 to 100 residues from the C-terminal extremity (12) or, according to others, at least upstream of the last two TM domains, on the cytoplasmic side of the membrane, and it is probably the site of interaction of the protein and the ATP binding subunit of the permease complex (i.e., PstB in the case of the phosphate periplasmic permease of E. coli) (21). An additional high degree of homology (61.3% identity in a 31-amino-acid overlap, 90% including homologous residues) was found between the N-terminal 33 amino acids of Ag 88 and the E. coli subunit PstC. This region, the first TM region of Ag 88, turned out to be homologous to the fifth TM domain of PstC, including 4 amino acids of the signature domain and 11 residues of a cytoplasmic loop (Fig. 5 and 6). Southern blot analysis. n order to verify whether this cloned gene is unique or is represented by multiple copies in the mycobacterial genome, we hybridized EcoRV- and Nco- Sma-digested M. bovis BCG genomic DNA with two oligonucleotide probes corresponding to the PstA and PstC homology domains (see Materials and Methods and Fig. 3). The results of these hybridizations are shown in Fig. 7. The two probes revealed a common 1,500-bp EcoRV fragment, whereas the Nco-Sma digestion shows an expected 1,900-bp Sma fragment with the PstA-like probe and a fragment smaller than 1,500 bp hybridizing with the PstC-like probe. This pattern is in good agreement with the map of the Al Xgtll phage (Fig. 2). Of course, we have no information on sites located upstream of Al and cannot interpret further the PstC-like hybridization at present. DSCUSSON A DNA region coding for mycobacterial Ag 88 has been cloned from a Xgtll library from M. tuberculosis and M. bovis BCG and sequenced. The corresponding gene is unique in the genome of M. bovis BCG. The deduced amino acid sequence from the M. tuberculosis gene corresponds to a polytopic, highly hydrophobic protein of 43,790 Da (403 amino acids) presenting at least seven putative TM hydrophobic domains. nterestingly, it presents a very strong homology to the two hydrophobic subunits of the E. coli periplasmic phosphate permease called PstA and PstC (29). The homology to PstA (35.8% identity, 76.7% homology if conservative replacements are included over 279 residues) includes the six putative TM helices in the carboxy-terminal region. The homology to PstC (61.3% identity, 90% homology with conservative replacements over 31 residues) is located in the N-terminal region of Ag 88 and corresponds to the entire fifth TM domain of PstC and part of the adjacent connecting loop. The protein sequence does not contain the pattern of a typical signal sequence and is predicted to be an integral membrane protein.

4 852 BRABANT ET AL. NFEcr. MMUN ATGATCCTGCTCATCGTCACATCGTATAC -80 ACGCGAAGTGrrCCGGCAGACTCCGCTGATCCAAATCCAAGCACGCTGGCCATGGCGCGACGAAT66A66!A[6ACGG -1 1 AM ACC GT CTG CCA TAC GGG CGA AGC GGG LD GTC GCG GCC TCC ATC CTC GGT TTC GGG 60 1 M T V L P Y G R S G V V A A S M L G L G CGG GCT CTG GGC GAA ACC JM GCC G CTG GTC ATC CTG CGC TCG GCC GGC GCG GCC GGG R A L G E T V A V L V L R S A G A A G GAC CTG GTC GCT GTT CGA CGG CGG TTA TAC GTT CGC TTC CAA GAT CGC CTC CGC TGC TTC D L V A V R R R L Y V R F Q D R L R C F AGA ATr CAG CGA ACC GCT GCC GAC CGG AGC CTA TAT TTC GGC GGG ATT TGC GTT ATT CGT Z40 61 R Q R T A A D R S L Y F G G C V R GCT GAC GTT CCT GGT CAA TGC GGC CGC TCG CGC AAT CGC CGG CGG GAA GGT CAA CGG GTG A D V P G Q C G R S R N R R R E G Q R V AGT CCC TCA ACG AGC ATC GAG GCG CTC GAC CAG CCG GTA AAG CCG GTG GTG TTT CGT CCG S P S T S E A L D Q P V K P V V F R P CTT ACG CTG CGA CGG CGG ATC AAA AAC AGC GTC GCG ACA ACG TmT TTC TTC ACC TCG TTC L T L R R R K N S V A T T F F F T S F GTG GTC GCG TTG ATA CCG TTG GTC TGG CTG CTT TGG GTG GTG ATT GCC CGG GGT TGG TTT V V A L P L V W L L W V V A R G W F GCC GTC ACC CGA TCG GGC TGG TGG ACC CAC TCG CTG CGC GGC GTG CTG CCA GAG CAA TTC A V T R S G W W T H S L R G V L P E Q F GCC GGT GGG GTG TAT CAC GCC CTG TAC GGC ACG CTG GTG CAG GCC GGG GTG GCC GCC GTG A G G V Y H A L Y G T L V Q A G V A A V CTG GCC GTG CCG CTG GGC TTG ATG ACC GCG GTT TAC CTA GTG GAA TAC GGG ACT GCT CGA L A V P L G L M T A V Y L V E Y G T G R ATG TCG CGG GTG ACT ACC TTC ACC GTC GAC GTG CTT GCC GGC GTG CCC TCT ATC GTG GCG M S R V T T F T V D V L A G V P S V A GCG TTA TTC GTC TTC AGC CTG TGG ATC GCC ACC CTA GGA TTT CAG CAG AGC GCC TTT GCC A L F V F S L W A T L G F Q Q S A F A GTG GCG TTG GCG TTG GTC CTG CTG ATG TTG CCG GTG GTG GTT CGG GCA GGC GAG GAG ATG V A L A L V L L M L P V V V R A G E E M CTC AGG TTGiGTG CCC GAT GAA CTG CGA GAA GCC AGC TAC GCG TTA GGC GTT CCG AAA TGG L R L V P D E L R E A S Y A L G V P K W AAG ACG ATC GTG CGG ATC GCT GCC CCG ATC GCG ATG CCG GGC ATC GTG TCA GGC ATC TTG K T V R A A P A M P G V S G L TTG TCC ATC GCG CGC GTC GTC GGT GAA ACC GCA CCG GTT CTG GTG CTG GTC GGG TAC AGC S A R V V G E T A P V L V L V G Y S CAC TCC ATC AAC CTC GAC GTC TTC CAC GGC AAC ATG GCC TCG CTG CCG TTG CTG ATC TAC H S N L D V F H G N M A S L P L L Y ACC GAA CTC ACC AAT CCC GAG CAC GCC GGC TTC CTG CGC GTC TGG GGC GCG GCG CTG ACC T E L T N P E H A G F L R V W G A A L T CTG ATC ATC GTG GTC GCC ACG ATC AAC CTG GCC GCG GCG ATG ATC CGG TTC GTC GCA ACC L V V A T N L A A A M R F V A T CGA CGG CGG TGA CTCCCGTTATGACGTGAGMTCACCACTCGGTCGTTGCCGCGGTCGGCGACGTAGACGGTCCGG R R R 1275 TCGCTGTCCACTGCCACCGCGAGGGGGGTGTTGAGGCCGGTGAACGGTAGCACTGTCGAGGTGGTCGACCCGGCCAGAGT TTGACCACCTGGTTTGTGTTGTGCTCGGTGAGGTAGACGGT1CCGGCTTCGTCCACCGCGATGCCCCACGGTGCGGTGAT ATCCGTGAATGGCAGCACGACTGGTTATTCGACTCGGCCTCTAGCT!GACAACCCTGTTGTGTCGGTGTCGGTGACATA GACGCGGAGTTGTCGACCTCACCCGTGCAG 1546 FG. 3. Nucleotide and amino acid sequences of the Ag 88-containing region of M. tuberculosis. The seven potential initiation codons (ATG or GTG) are underlined. Among these, only one ATG (at position 1) is preceded, 7 nucleotides upstream, by a putative Shine- Dalgarno ribosome binding site sequence (underlined). The M. tuberculosis PstA-like and PstC-like probes used in the Southern blot analyses are underlined (positions 833 and 42, respectively). PstA and PstC are the two hydrophobic subunits of the E. coli phosphate permease belonging to the family of ATP binding cassette periplasmic transport proteins, or traffic ATPases (2). The ATP-dependent permeases are very efficient, high-affinity transport systems used by gram-negative bacteria to concentrate in the periplasm and selectively import through the inner membrane various nutrients (sugar, amino acids, peptides, etc.) (1). Each of these permeases is a multimeric complex composed of one soluble periplasmic protein binding the ligand (PstS or PhoS in the case of the phosphate permease of E. coli), one or two very hydrophobic TM proteins, and one or two nonhydrophobic ATP binding subunits (1, 2). n addition, in E. coli, the genes coding for the four subunits of the phosphate permease are located within the same operon, whose expression is coordinated with that of :-81 Ag psta , 0* _ * FG. 4. Hydropathic patterns of the M. tuberculosis Ag 88 amino acid sequence and the E. coli PstA protein. The sequences of the two proteins have been analyzed by the Kyte and Doolittle method (22). Horizontal brackets indicate the potential helices according to the Garnier and Chou-Fasman prediction programs. several other operons implicated in the control of phosphate metabolism and constituting the phosphate regulon (29, 33). We do not know at present whether such a complete complex functional system also exists in M. tuberculosis and M. bovis Ag 88 RPLTLRRRKNSVATTFFFTSFVVALPLVWLLWVVARGWFAVTRSGWWTHSLRGVLPE 1: 111:11::1 1: ::::: :1::1:1:1 :1:11 :: : : :1: :: 1: PstA RKMQARRRLKNRALTLSMATMAFGLFWLWLMSTTRG-DGMSLALFTE--MTPPPN Ag 88 QFAGGVYHALYGTLVQAGVAAVLAVPLGLMTAVYLVEYG-TGRMSRVTTFTVDVLAGVPS PstA TEGGGLANALAGSGLLLWATVFGTPLGMAGYLAEYGRKSWLAEVRFNDLLSAPS Ag 88 VAALFVFSLWATLGFQQSAFAVALALVLLMLPVVVRAGEEMLRLVPDELREMYALGV 11::11:: :1:::1:: ::1:11:1::1:::11111:1111:1111: PstA VVGLFVYTVVAQME-HFSGWAGVALALLQVPVRTTENMLKLVPYSLREA&YALnT Ag 88 PKWKTVRAADAMPGVSGLLSARVVGETAPVLVLVGYSHSNLDVFHGNMASLPL PstA : :::11::1111:111:: :::: 1:::: :1:11: PKWKMSATL.ASVSGMTGLLAARAGETAP-LLFTALSNQFWSTDMMQPANLPV Ag 88 LYTELTNPEHAGFLRV-WGAALTLVVATNLAAAM 1: ::: : 1:: :: 1:::1:::: :1: :: PstA TF-KFAMSPFAEWQQLAWAGVLTLCVLLLNLARVV kg 88 MTVLPYGRSGVVAASMLGLGRALGETVAVLVL 1:?StC RVLPFTKNGVGGMLGLGRALGETMAVTF a / -- FG. 5. Alignment of the amino acid sequence of the M. tuberculosis Ag 88 with those of the E. coli PstA and PstC proteins. dentical amino acids (1), conserved residues (:), and gaps (-) are indicated. The Prosite signature (see text) motive is underlined. A

5 VOL. 62, 1994 s 60 mmy i it1 2 Arg rich s -~~ U - - m Pstc homology PstA homology FG. 6. Putative TM helices of Ag 88. Dots, arginine residues; S, the signature motive of bacterial binding protein-dependent transport systems. Heavily stippled motives represent the six TM helices homologous to the putative E. coli PstA helices. The lightly stippled motive represents a putative helix corresponding to the fifth TM domain of the E. coli PstC protein. BCG. However, it is interesting that the periplasmic phosphate binding component (equivalent to E. coli PstS) in M. tuberculosis has previously been described (see references 3, 4, and 16 for reviews). This phosphate-binding protein has been called the 38-kDa protein, protein antigen b, Ag 78, and Ag 5 by various authors. n addition, a mycobacterial homolog of an E. coli PhoM phosphate sensor system has also been described (23). For all living organisms, a minimal phosphate supply is essential since phosphate plays a structural role in the biosynthesis of many essential components (nucleotides, nucleic acids, and phospholipids) and also a major role in the regulation of biochemical events, including signal transduction (26). A func- M. tub. probes 3.53 kb 2.02 kb kb kb kb.947 kb.831 kb.564 kb PstC-like o 0 u z PstA-like z > > E E U) W W FG. 7. Southern analysis of total genomic DNA from M. bovis BCG. Hybridization was done with PstA-like and PstC-like oligonucleotides (Fig. 3) under the conditions described in Materials and Methods. The molecular sizes of the hybridizing bands were calculated by comparison with standards. THE ANTGEN 88 GENE OF M. TUBERCULOSS 853 tional phosphate permease may be especially crucial for bacteria such as E. coli which are adapted to develop in the colon, an environment containing a very low level of phosphate (30). Mycobacteria such as M tuberculosis or M bovis BCG demonstrate intracellular multiplication within the phagolysosome, but it is not known to what extent phosphate concentration limits their growth. Recently, the mouse bcg gene, which determines natural resistance and which is responsible for the capacity of macrophages to destroy ingested intracellular parasites early during infection, has been identified and designated as Nramp. Curiously, Nramp corresponds to an integral membrane protein related to PstA and the traffic ATPase family, and it has been suggested that the gene could function as a nitrite or nitrate concentrator within the lipid bilayer of phagolysosomes (31). One striking difference between the E. coli PstA protein and Ag 88 (described here) is the presence of a PstC-homologous domain within the protein. Although very short, this domain may be very important since recent structure-function studies have suggested that in E. coli a cooperation between helices 5 of PstA and PstC probably constitutes the phosphate channel itself within the permease. Furthermore, within these two helices, three charged residues (R-220 in PstA and R-237 and E-241 in PstC) are essential and could possibly serve as relays to which the translocated anion could jump (34). nterestingly, these three residues are conserved within mycobacterial Ag 88 (Fig. 5). We do not know at present whether a complete PstC subunit as such also exists in mycobacteria. Finally, the role of the arginine-rich domain in Ag 88 is presently unknown. t could play a role in orienting the Ag 88 protein within the internal membrane, since positively charged residues are generally found in excess at the cytoplasmic side of bacterial membrane proteins (5, 11). To our knowledge, Ag 88 (identified here) is the first integral membrane protein characterized for mycobacteria, which are generally considered gram positive, that do not have a periplasmic space (8). t is interesting that this kind of permease exists in M. tuberculosis, M. bovis BCG, and other pathogenic mycobacteria (M. avium and M. kansasii) (unpublished data). The ligand-binding subunit of these permeases appears to be a lipoprotein both in gram-positive bacteria (15) and in M. tuberculosis (4, 35) which is probably anchored in the cell membrane. t is anticipated that other transport permeases exist in mycobacteria, and this certainly merits further examination since the characterization of internal membrane proteins of mycobacteria of known (or putative) function and topology could be of interest both for the pharmacological inhibition of these functions and for the presentation of immunogens in recombinant BCG vaccines. ACKNOWLEDGMENTS We are grateful to R. A. 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