Advertisement

Molecular Basis Defining Human Chlamydia trachomatis Tissue Tropism

A POSSIBLE ROLE FOR TRYPTOPHAN SYNTHASE*
      Here we report the cloning and sequencing of a region of the chlamydiae chromosome termed the “plasticity zone” from all the human serovars of C. trachomatis containing the tryptophan biosynthesis genes. Our results show that this region contains orthologues of the tryptophan repressor as well as the α and β subunits of tryptophan synthase. Results from reverse transcription-PCR and Western blot analyses indicate that the trpBA genes are transcribed, and protein products are expressed. The TrpB sequences from all serovars are highly conserved. In comparison with other tryptophan synthase β subunits, the chlamydial TrpB subunit retains all conserved amino acid residues required for β reaction activity. In contrast, the chlamydial TrpA sequences display numerous mutations, which distinguish them from TrpA sequences of all other prokaryotes. All ocular serovars contain a deletion mutation resulting in a truncated TrpA protein, which lacks α reaction activity. The TrpA protein from the genital serovars retains conserved amino acids required for catalysis but has mutated several active site residues involved in substrate binding. Complementation analysis inEscherchia coli strains, with defined mutations in tryptophan biosynthesis, and in vitro enzyme activity data, with cloned TrpB and TrpA proteins, indicate these mutations result in a TrpA protein that is unable to utilize indole glycerol 3-phosphate as substrate. In contrast, the chlamydial TrpB protein can carry out the β reaction, which catalyzes the formation of tryptophan from indole and serine. The activity of the chlamydial Trp B protein differs from that of the well characterized E. coli andSalmonella TrpBs in displaying an absolute requirement for full-length TrpA. Taken together our data indicate that genital, but not ocular, serovars are capable of utilizing exogenous indole for the biosynthesis of tryptophan.
      EB
      elementary body
      RB
      reticulate body
      LGV
      lymphogranuloma venereum
      IFN
      interferon
      IGP
      indole glycerol 3-phosphate
      MEM
      minimal essential medium
      FCS
      fetal calf serum
      nt
      nucleotide(s)
      RT
      reverse transcription
      IFU
      inclusion-forming units
      Members of the genus Chlamydia are obligate intracellular bacteria that possess a unique biphasic developmental cycle consisting of an extracellular, infectious, but metabolically inactive elementary body (EB)1 and an intracellular, non-infectious, replicative form called the reticulate body (RB). Chlamydial infection involves the attachment of the EB to a host cell and its subsequent internalization into a membrane-bound vesicle known as the chlamydial inclusion. Within this inclusion the EB differentiates into an RB, which then multiplies by binary fission. The daughter RBs then redifferentiate into EBs that are able to initiate new rounds of infection after release by host cell lysis (
      • Moulder J.W.
      ).
      Chlamydia consists of three species that are important pathogens of humans. Chlamydia psittaci strains are primarily pathogens of birds and lower animals, but humans are occasional hosts of avian-acquired psittacosis (
      • Schachter J.
      ,
      • Schachter J.
      ). The two major pathogens of humans are Chlamydia trachomatisand Chlamydia pneumoniae. C. pneumoniae is an important cause of community-acquired pneumoniae (
      • Kuo C.C.
      • Jackson L.A.
      • Campbell L.A.
      • Grayston J.T.
      ) and has been linked to the etiology of chronic heart disease (
      • Campbell L.A.
      • Rosenfeld M.
      • Kuo C.C.
      ,
      • Kuo C.
      • Campbell L.A.
      ,
      • Grayston J.T.
      • Kuo C.C.
      • Campbell L.A.
      • Wang S.P.
      • Jackson L.A.
      ). C. trachomatiscomprises a family of antigenically related yet divergent organisms serologically classified into 15 distinct serovars based on antigenic variation of the major outer membrane protein of the organism (
      • Stephens R.
      ). Curiously, the 15 different C. trachomatis serovars exhibit an extraordinary specificity in tissue tropism. For example, serovars A, B, Ba, and C are pathogens of the eye, where they infect columnar epithelial cells of the conjunctivae causing trachoma, a chronic inflammatory disease that is the leading cause of preventable blindness in the world (
      • Schachter J.
      ,
      • Schachter J.
      ,
      • Mabey D.
      • Bailey R.
      ). The trachoma serovars are rarely isolated from the genital tract. On the other hand, serovars D-K are sexually transmitted pathogens that infect columnar epithelial cells of the genital tract (
      • Schachter J.
      ,
      • Schachter J.
      ). These infections are the most common bacterial cause of sexually transmitted disease and in females cause pelvic inflammatory disease. The sexually transmitted disease serovars can cause neonatal conjunctivitis but have not been associated with blinding trachoma. Furthermore, although infections with both ocular (A-C) and genital serovars (D-K) are non-invasive and are restricted to the mucosal epithelium, those caused by the sexually transmitted lymphogranuloma venereum (LGV) serovars (L1, L2, and L3) are invasive (
      • Schachter J.
      ,
      • Schachter J.
      ). The LGV strains penetrate the submucosal tissue, infect monocytes and macrophages, and disseminate to the local draining lymph nodes, where they produce a chronic granulomatous disease. The factor(s) that controls the non-invasive/invasive properties of these genital serovars has been correlated with the production of a chlamydial cytotoxin (
      • Belland R.J.
      • Scidmore M.A.
      • Crane D.D.
      • Hogan D.M.
      • Whitmire W.
      • McClarty G.
      • Caldwell H.D.
      ); however, virulence factors that decide the distinctive ocular and genital tract tissue tropisms have yet to be discovered.
      T cells play an important role in the development of adaptive immunity against C. trachomatis mucosal infection, and interferon γ (IFN-γ) is key to this protective function (
      • Brunham R.C.
      ,
      • Kim S.K.
      • DeMars R.
      ,
      • Loomis W.P.
      • Starnbach M.N.
      ). The mechanism by which IFN-γ controls infection in vitro is by interfering with the replicative capacity of the parasite (
      • Beatty W.L.
      • Morrison R.P.
      • Byrne G.I.
      ,
      • Beatty W.L.
      • Belanger T.A.
      • Desai A.A.
      • Morrison R.P.
      • Byrne G.I.
      ). Through binding of the IFN-γ receptor, IFN-γ transcriptionally activates the expression of indoleamine-2,3-dioxygenase, which degradesl-tryptophan to l-kynurenine (
      • Taylor M.W.
      • Feng G.S.
      ,
      • Boehm U.
      • Klamp T.
      • Groot M.
      • Howard J.C.
      ). This cytokine-mediated host cell response deprives intracellular chlamydial RBs of tryptophan, which ultimately prevents their growth and replicative capabilities. Treatment of epithelial cells with high levels of IFN-γ completely inhibits growth, whereas subinhibitory concentrations induce the development of morphologically aberrant viable RB forms that have been implicated in the development of persistence (
      • Beatty W.L.
      • Belanger T.A.
      • Desai A.A.
      • Morrison R.P.
      • Byrne G.I.
      ).
      The complete genomic sequence of several Chlamydiaceae has been determined, including C. trachomatis serovars D (
      • Stephens R.S.
      • Kalman S.
      • Lammel C.
      • Fan J.
      • Marathe R.
      • Aravind L.
      • Mitchell W.
      • Olinger L.
      • Tatusov R.L.
      • Zhao Q.
      • Koonin E.V.
      • Davis R.W.
      ) and MoPn (
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • Gill S.R.
      • Heidelberg J.F.
      • White O.
      • Hickey E.K.
      • Peterson J.
      • Utterback T.
      • Berry K.
      • Bass S.
      • Linher K.
      • Weidman J.
      • Khouri H.
      • Craven B.
      • Bowman C.
      • Dodson R.
      • Gwinn M.
      • Nelson W.
      • DeBoy R.
      • Kolonay J.
      • McClarty G.
      • Salzberg S.L.
      • Eisen J.
      • Fraser C.M.
      ), C. pneumoniae strains CWL029 (
      • Kalman S.
      • Mitchell W.
      • Marathe R.
      • Lammel C.
      • Fan J.
      • Hyman R.W.
      • Olinger L.
      • Grimwood J.
      • Davis R.W.
      • Stephens R.S.
      ), AR39 (
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • Gill S.R.
      • Heidelberg J.F.
      • White O.
      • Hickey E.K.
      • Peterson J.
      • Utterback T.
      • Berry K.
      • Bass S.
      • Linher K.
      • Weidman J.
      • Khouri H.
      • Craven B.
      • Bowman C.
      • Dodson R.
      • Gwinn M.
      • Nelson W.
      • DeBoy R.
      • Kolonay J.
      • McClarty G.
      • Salzberg S.L.
      • Eisen J.
      • Fraser C.M.
      ), and J138 (
      • Shirai M.
      • Hirakawa H.
      • Kimoto M.
      • Tabuchi M.
      • Kishi F.
      • Ouchi K.
      • Shiba T.
      • Ishii K.
      • Hattori M.
      • Kuhara S.
      • Nakazawa T.
      ). and C. psittaci strain GPIC (www.tigr.org). In addition, partial sequence information is available for C. trachomatis serovar L2 (chlamydia-www.Berkeley.edu:4231). The gene order and content among these organisms are remarkably similar, with the exception of a region termed the plasticity zone, which has undergone genetic reorganization to a greater extent than the rest of the chromosome (
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • Gill S.R.
      • Heidelberg J.F.
      • White O.
      • Hickey E.K.
      • Peterson J.
      • Utterback T.
      • Berry K.
      • Bass S.
      • Linher K.
      • Weidman J.
      • Khouri H.
      • Craven B.
      • Bowman C.
      • Dodson R.
      • Gwinn M.
      • Nelson W.
      • DeBoy R.
      • Kolonay J.
      • McClarty G.
      • Salzberg S.L.
      • Eisen J.
      • Fraser C.M.
      ). Genes encoding enzymes required for the biosynthesis of tryptophan are found within the plasticity zone. However, the complement of trp genes within this region varies among the chlamydial species characterized to date. C. psittaci GPIC contains all of the genes of the tryptophan biosynthesis pathway with the exception of the first two enzymes encoded by trpE/G. In contrast, C. trachomatis serovar MoPn and C. pneumoniae do not encode any trp genes in the plasticity zone. Interestingly,C. trachomatis serovars D and L2 contain only a subset oftrp genes in their plasticity zone, trpR, encoding a putative tryptophan repressor, and trpA andtrpB, respectively, encoding homologues of the α (TrpA) and β (TrpB) subunits of tryptophan synthase (Fig.1). This is an unusual circumstance in that most other organisms studied to date, both prokaryotic and eukaryotic, have either the full complement of trp genes or lack them altogether. A further heterogeneity within thetrpA gene of C. trachomatis has been identified by Shaw et al. (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ) in that serovar A and C appear to encode a truncated version of TrpA compared with serovars D and L2 (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ). The differences in the trp gene complement among the chlamydiae characterized thus far suggest that the ability to synthesize tryptophan de novo is not required and raises the possibility that these genes are in the process of being lost from the genome. Alternatively, these differences may be important with respect to the unique tissue tropism of chlamydial strains and permit serovar-specific survival or growth within different microenvironments of the host.
      Figure thumbnail gr1
      Figure 1Reaction scheme for the biosynthesis of tryptophan. Genes in bold are located in the plasticity zone and are common to both C. trachomatis serovar D andC. psittaci strain GPIC. C. psittaci strain GPIC has all genes with the exception of trpE and trpG.
      Tryptophan synthase is a tetramer consisting of two α subunits and two β subunits (
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Miles E.W.
      ). This bifunctional enzyme catalyzes the two final steps in the biosynthesis of tryptophan (Fig. 1), which are the cleavage of indole glycerol 3-phosphate (IGP) to indole and glyceraldehyde 3-phosphate (termed the α reaction and catalyzed by TrpA) followed by the β-replacement reaction of indole with serine to form tryptophan (the β reaction, catalyzed by TrpB). Extensive characterization of the Escherichia coli andSalmonella enzymes has demonstrated a large degree of allosteric regulation and cooperativity between the α and β subunits (
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Miles E.W.
      ). In fact, TrpA and TrpB exhibit little activity in their respective reactions in the absence of the other subunit (
      • Lane A.N.
      • Kirschner K.
      ,
      • Miles E.W.
      • McPhie P.
      ,
      • Crawford I.P.
      • Yanofsky C.
      ). Given the association of IFN-γ with chlamydial infections and its effect on tryptophan levels in the host cell and, thus, on chlamydial growth, encoding functional tryptophan synthase may be a survival factor for intracellular chlamydiae. However, IGP substrate for the α reaction in E. coli and Salmonella is supplied by the sequential activity of the other genes of the tryptophan biosynthesis pathway (TrpE, D, FC) (Fig. 1). C. trachomatis does not encode orthologues of TrpE, G, D, or C, although paradoxically, it does have an orthologue of TrpF, the gene for which lies outside the plasticity zone (
      • Stephens R.S.
      • Kalman S.
      • Lammel C.
      • Fan J.
      • Marathe R.
      • Aravind L.
      • Mitchell W.
      • Olinger L.
      • Tatusov R.L.
      • Zhao Q.
      • Koonin E.V.
      • Davis R.W.
      ,
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • Gill S.R.
      • Heidelberg J.F.
      • White O.
      • Hickey E.K.
      • Peterson J.
      • Utterback T.
      • Berry K.
      • Bass S.
      • Linher K.
      • Weidman J.
      • Khouri H.
      • Craven B.
      • Bowman C.
      • Dodson R.
      • Gwinn M.
      • Nelson W.
      • DeBoy R.
      • Kolonay J.
      • McClarty G.
      • Salzberg S.L.
      • Eisen J.
      • Fraser C.M.
      ). Because mammalian cells lack the ability to biosynthesize tryptophan and C. trachomatis appears to lack the capability of IGP synthesis, it is unclear what the substrate for chlamydial TrpA would be.
      The present work involved a study of the diversity within thetrp region among all 15 C. trachomatis serovars and characterization of the functionality of the tryptophan synthase encoded therein. Here we report that all the C. trachomatistype strain serovars, with the exception of B and MoPn, encode homologues of trpB and trpA, that the gene products are found in both EBs and RBs, and that the ability to synthesize tryptophan differs among ocular and genital serovars. Furthermore, we provide evidence that the α subunit of C. trachomatis tryptophan synthase differs from that of other Gram-negative bacteria with respect to the utilization of IGP as a substrate.

      MATERIALS AND METHODS

       Bacterial Strains, Plasmids, and Antibodies

      The bacterial strains and plasmids utilized in this study are listed in TableI. E. coli strains were grown in Luria-Bertani (LB) broth or on LB agar and in the presence of 100 μg ml−1 ampicillin in the case of pQE-80L transformants.C. trachomatis serovars were propagated in the HeLa 229 cervical carcinoma cell line (ATCC) maintained in minimal essential medium (MEM, Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS) as described previously (
      • Tipples G.
      • McClarty G.
      ). For growth of C. trachomatis under tryptophan-free conditions, dialyzed FCS was used. C. trachomatis EBs were purified by density gradient centrifugation according to established procedures and stored in sucrose phosphate glycerol medium at −80 °C (
      • Caldwell H.D.
      • Kromhout J.
      • Schachter J.
      ). Polyclonal antiserum against C. trachomatis TrpA was raised in rabbits by immunization with purified recombinant serovar L2 TrpA. Mouse ascites polyclonal anti-TrpB was raised against recombinant serovar L2 TrpB by following the procedure of Lacy and Voss (
      • Lacy M.J.
      • Voss E.W., Jr.
      ).
      Table IBacterial strains and plasmids used in this study
      In vitropassages
      Number of in vitropassages of laboratory strains in either embryonating eggs (Y), McCoy (M), or HeLa 229 (H) cells.
      Source
      Bacterial strain
      C. trachomatis
        A/Har-13Y27H28Harlan Caldwell
        B/TW-5E9H52Harlan Caldwell
        Ba/AP2Y5H10Harlan Caldwell
        C/TW-3E6H45Harlan Caldwell
        D/UW-3E10H17Harlan Caldwell
        E/BourY15H6Harlan Caldwell
        F/IC Cal-3Y5H6Harlan Caldwell
        G/UW-524H16Harlan Caldwell
        H/UW-4E7H26Harlan Caldwell
        IUW-12E9H18Harlan Caldwell
        J/UW-36E5H34Harlan Caldwell
        K/UW-31M2H42Harlan Caldwell
        L1/LGV-440Y5H5Harlan Caldwell
        L2/LGV-434E10H48Harlan Caldwell
        L3/LGV-404Y5H7Harlan Caldwell
      GenotypeSource
      Bacterial strain
      E. coli
        KS463F, LAM, trpA33, IN(rrnD-rrnE)1,rha-7EGSC
      EGSC, E. coli Genetic Stock Center.
        BW7622Hfr, e14-, trpB114∷Tn10,relA1, spoT1, thi-1, rph-1, IN(rrnD-rrnE)1EGSC
        CY15077F, LAM, DE(trpA-trpE)872, rph-1,tna-2EGSC
      DescriptionSource
      Plasmid
       pQE-80LE. coliexpression vector, AmpRQiagen
       pCFG2pQE-80L expressing E. coli trpThis study
       pCR1pQE-80L expressing C. trachomatis ser. L2trpAThis study
       pCR2pQE-80L expressing C. trachomatis ser. L2 trpBThis study
       pCR3pQE-80L expressing C. trachomatis ser. L2trpBAThis study
       pCFG5pQE-80L expressingC. trachomatis ser. A trpAThis study
       pCFG9pQE-80L expressing C. trachomatis ser. AtrpBThis study
       pCFG6pQE-80L expressingC. trachomatis ser. A trpBAThis study
       pCFG7pQE-80L expressing C. trachomatis ser. AtrpB and ser. L2 trpAThis study
       pCFG8pQE-80L expressing C. trachomatis ser. L2trpB and ser. A trpAThis study
       pCR4pQE-80L expressing C. trachomatis ser. ItrpAThis study
       pCR5pQE-80L expressing C. trachomatis ser. I trpBThis study
       pCFG13pQE-80L expressing C. trachomatis ser. ItrpBAThis study
      1-a Number of in vitropassages of laboratory strains in either embryonating eggs (Y), McCoy (M), or HeLa 229 (H) cells.
      1-b EGSC, E. coli Genetic Stock Center.

       Sequence Analysis of C. trachomatis trp Genes

      DNA between CT175 and CT167 of serovar D (
      • Stephens R.S.
      • Kalman S.
      • Lammel C.
      • Fan J.
      • Marathe R.
      • Aravind L.
      • Mitchell W.
      • Olinger L.
      • Tatusov R.L.
      • Zhao Q.
      • Koonin E.V.
      • Davis R.W.
      ), containing the C. trachomatis trp genes, was amplified from chromosomal DNA of all 15 C. trachomatis serovars using primers 00.11 and JHC258 (TableII) and Expand High Fidelity polymerase according to manufacturer's instructions (Roche Molecular Biochemicals). After gel purification, the PCR products were cloned into pCR-XL-TOPO using the kit from Invitrogen, and the constructs were transformed into DH10B cells for propagation. Cloned insert DNA was sequenced by a commercial company (SeqWright, Houston, TX). The nucleotide (nt) and deduced amino acid sequences were aligned using ClustalW version 1.8. The following sequences have been submitted to GenBankTM: accession numbers AY096805 (serovar Atrp)AY096806 (Batrp), AY096807 (Ctrp), AY096808 (Dtrp), AY096809 (Etrp),AY096810 (Ftrp), Y096811 (Gtrp), AY096812 (Htrp), AY096813 (Itrp),AY096814 (Jtrp), AY096815 (Ktrp), AY096816 (L1trp), AY096817 (L2trp), and AY096818 (L3trp).
      Table IIPrimer sequences for nucleotide sequencing RT-PCR and cloning
      PrimerSequence (5′ → 3′)
      Sequencing
       00.11CAT TTG CTT CCG TTC TTG GGT AG
       JHC258TGA CAG ATC GCA ATC CGC
      RT-PCR
       5′-RT16SRNAGGA GAA AAG GGA ATT TCA CG
       3′-RT16SRNATCC ACA TCA AGT ATG CAT CG
       5′-RTtrpBAGCA TTG GAG TCT TCA CAT GC
       3′-RTtrpBAACA CCT CCT TGA ATC AGA GC
      Cloning
       5′-CttrpACCC CGG TAC CAT GAT GAA ATT AAC C
       3′-CttrpACCC CGT CGA CTT ATC CAG GAA TAA AC
       5′-CttrpBCCC CGG TAC CAT GTT CAA ACA TAA AC
       3′-CttrpBCCC CGT CTT ACT CAT AAA TTC C
       5′-EctrpAAGC GGA TCC GAA CGC TAC GAA TCT
       3′-EctrpAGGG GTA CCT AAG CGA AAC GGT AAA

       RT-PCR Analysis of trp Gene Expression

      Monolayers of HeLa 229 cells in T-175 flasks were infected with C. trachomatisEBs at a multiplicity of infection (m.o.i) of 3–5 inclusion-forming units (IFU) cell−1, as previously described (
      • Tipples G.
      • McClarty G.
      ). As a negative control, a mock-infected flask was prepared in the same manner but without the addition of EBs. The cells were incubated for 24 h at 37 °C, and then total RNA was prepared using Trizol reagent according to the manufacturer's instructions (Invitrogen). After treatment with amplification-grade DNase I (Invitrogen), 1 μg of RNA was reverse-transcribed using random hexamer primers and Thermoscript reverse transcriptase (Invitrogen) and then treated with RNase H (Invitrogen). Primers specific for 16 S rRNA and thetrpB-trpA junction (Table II) were used to amplify products in PCR reaction mixtures containing 2 μl of cDNA, 0.2 μm primers, 0.2 mm dNTPs, 1.5 mmMgCl2,Taq reaction buffer, and 5 units ofTaq DNA polymerase (Invitrogen). The cycling program was 3 min at 95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1.5 min at 72 °C. Products were separated on a 1.5% agarose-Tris-buffered EDTA gel and visualized by ethidium bromide staining.

       Western Blot Analyses

      Purified EBs were lysed by suspension in Laemmli buffer followed by incubation at 95 °C for 10 min. Soluble proteins were fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and then incubated with anti-TrpA antiserum followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin or with anti-TrpB ascites followed by goat anti-mouse HRP. Bound antibodies were detected by enhanced chemiluminescence according to manufacturer's instructions (Amersham Biosciences).

       Expression Cloning of trpB and trpA

      C. trachomatis and E. coli trp genes were amplified by PCR from purified chromosomal DNA using the reagent concentrations described for RT-PCR and the cycling program 3 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 30 s at 50 °C, and 2 min at 72 °C. The PCR primer sequences are listed in Table II and were designed to include unique restriction sites for cloning. For construction of plasmids to co-express trpB andtrpA, the 5′-CttrpB and the 3′-CttrpAprimers were used in the PCR reaction. The PCR products were gel-purified, restricted with KpnI and SalI (forC. trachomatis) or BamHI and KpnI (forE. coli) and ligated to expression vector pQE-80L (Qiagen) cut with the corresponding restriction enzymes. Constructs were transformed into DH5α for screening, purified by miniprep, and then used to transform E. coli mutant strains for complementation assays. Constructs co-expressing serovar A trpB with serovar L2 trpA and vice versa were prepared as follows. TheC. trachomatis trpA gene has a unique SpeI site 73 bp downstream of the start codon in a region of sequence identity among all of the serovars. Plasmids pCFG6 (serovar A trpBA) and pCR3 (serovar L2 trpBA) were restricted withSpeI and KpnI, and the fragments were gel-purified. The trpA-containing fragment from pCR3 was then ligated to the trpB-containing fragment from pCFG6 to generate pCFG7. Similarly, pCFG8 was constructed by ligating thetrpA-containing fragment from pCFG6 to thetrpB-containing fragment from pCR3.

       Complementation Assays

      The cells from stationary phase cultures of E. coli trp transformants were harvested by centrifugation and washed three times with sterile phosphate-buffered saline. The cell suspensions were then streaked onto minimal agar (1× M9 salts, 0.2% glucose, 0.2% casamino acids, 2 mm MgSO4, 0.2 mml-serine, 100 μg ml−1 ampicillin, and 50 μg ml−1 each thiamine, cysteine, and uracil) containing 100 μm indole, 50 μg ml−1l-tryptophan or without additional supplements. The plates were incubated for 48 h at 37 °C and then photographed.

       Preparation of Cell Lysates for Enzyme Assays

      Five ml of stationary phase cultures of CY15077 trp transformants were used to inoculate 50 ml of LB broth containing 100 μg ml−1 ampicillin. After incubation with aeration for 2 h at 37 °C, the cultures were cooled to 18 °C, isopropyl-1-thio-β-d-galactopyranoside (Invitrogen) was added to a final concentration of 100 μm, and the cultures were incubated with aeration for a further 18 h at 18 °C. The cells were then harvested by centrifugation, resuspended in 3 ml of 10 mm Tris-HCl, pH 7.8, and lysed by sonication on ice. Cell debris was removed by centrifugation, and the cleared lysates were kept on ice. Protein concentration was determined by Bradford assay using a commercial kit (Bio-Rad).

       Enzyme Assays

      One unit of activity is defined as the appearance of 0.1 μmol of product (α reaction) or the disappearance of 0.1 μm substrate (β and αβ reactions) in 20 min at 37 °C. The α reaction assays and the αβ reaction assays were performed using the methods of Smith and Yanofsky (
      • Smith O.H.
      • Yanofsky C.
      ). The α reaction mixture contained 0.3 μmol of IGP, 100 μmol of phosphate buffer, pH 7.0, 2 μmol of NH2OH, and 70 μl of cell lysate in a final volume of 0.5 ml. The αβ reaction mixture contained 0.4 μmol of IGP, 80 μmol of l-serine, 0.03 μmol of pyridoxal phosphate, 100 μmol of Tris buffer, pH 7.8, 30 μl of saturated NaCl, and 70 μl of cell lysate in a final volume of 1 ml. The β reaction assays were performed using the method of Miles (
      • Miles E.W.
      ). The β reaction mixture contained 0.1 μmol of indole, 20 μmol of l-serine, 0.0075 μmol of pyridoxal phosphate, 25 μmol of Tris buffer, pH 7.8, 7.5 μl of saturated NaCl, and 50 μl of cell lysate in a final volume of 250 μl. Specific activity (units mg−1) is reported as the average of triplicate determinations.

       C. trachomatis Growth Assays

      Monolayers of HeLa 229 cells in 6-well plates were infected with C. trachomatis EBs at an m.o.i. of 3–5 IFU cell−1 in MEM plus 10% dialyzed fetal bovine serum supplemented with tryptophan (10 mg liter−1) lacking tryptophan or lacking tryptophan but supplemented with indole (100 μm) or varying concentrations of anthranilate. For tryptophan-free conditions, HeLa cells were incubated for 6 h in tryptophan-free MEM plus 10% dialyzed fetal bovine serum before infection with C. trachomatis to ensure depletion of endogenous tryptophan. Infected monolayers were incubated at 37 °C for 48 h (LGV serovars) or 72 h (genital and ocular serovars), after which time the medium was collected, and the cells were lysed in cold, distilled water. Aliquots of the combined HeLa cell lysates and culture medium were used to infect fresh HeLa cell monolayers. Recoverable IFU were enumerated as previously described (
      • Caldwell H.D.
      • Kromhout J.
      • Schachter J.
      ).

       Indole Incorporation Assays

      HeLa cell monolayers in 6-well plates were infected with C. trachomatis EBs at an m.o.i. of 3–5 IFU cell−1 in the absence or presence of tryptophan (10 mg liter−1) in MEM plus 10% dialyzed fetal bovine serum supplemented with 100 μm[14C]indole (0.1 μCi μm−1). Where indicated, the cells were incubated for 6 h in tryptophan-free MEM plus 10% dialyzed fetal bovine serum before infection. After incubation for 36 h (serovar L2) or 48 h (serovars A, D, and I) at 37 °C, the medium was removed, the cells were washed with Hanks' buffered saline solution, and then the cells were lysed in cold, distilled water. Proteins in the cell lysate were precipitated with 10% trichloroacetic acid, and incorporated14C was quantified by scintillation counting (Beckman LS 5000). Data are expressed as dpm incorporated per 104cells.

      RESULTS

       Sequence Analysis of trp Genes from C. trachomatis Ocular and Genital Serovars

      Chlamydiae sequence data are available for both tryptophan synthase subunits from the serovar D genome sequencing project (
      • Stephens R.S.
      • Kalman S.
      • Lammel C.
      • Fan J.
      • Marathe R.
      • Aravind L.
      • Mitchell W.
      • Olinger L.
      • Tatusov R.L.
      • Zhao Q.
      • Koonin E.V.
      • Davis R.W.
      ). Serovar D TrpB contains 392 amino acids, giving a calculated molecular mass of 42.6 kDa, similar to the E. coli TrpB (
      • Crawford I.P.
      • Nichols B.P.
      • Yanofsky C.
      ). A comparison of the complete amino acid sequence of the serovar D TrpB with that of representative TrpBs in the public databases (Fig. 2A) indicates that the proteins are ∼54% identical. Most importantly amino acid residues identified as essential for enzyme activity, indole binding, and pyridoxal phosphate-Lys87 Schiff base complex formation in E. coli TrpB (His86, Lys87, Glu109, Arg148, Leu188, Cys230, Asp305, Phe306, Glu350) (
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Fluri R.
      • Jackson L.E.
      • Lee W.E.
      • Crawford I.P.
      ,
      • Tanizawa K.
      • Miles E.W.
      ,
      • Miles E.W.
      • Kawasaki H.
      • Ahmed S.A.
      • Morita H.
      • Nagata S.
      ,
      • Higgins W.
      • Miles E.W.
      • Fairwell T.
      ,
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      ) are conserved in serovar D TrpB.
      Figure thumbnail gr2
      Figure 2Comparison of the C. trachomatis(CtD) serovar D tryptophan synthase with that from E. coli (Ec) (
      • Crawford I.P.
      • Nichols B.P.
      • Yanofsky C.
      ,
      • Nichols B.P.
      • Yanofsky C.
      ), Bacillus subtilis (Bs) (
      • Kunst F.
      • Ogasawara N.
      • Moszer I.
      • Albertini A.M.
      • Alloni G.
      • Azevedo V.
      • Bertero M.G.
      • Bessieres P.
      • Bolotin A.
      • Borchert S.
      • Borriss R.
      • Boursier L.
      • Brans A.
      • Braun M.
      • Brignell S.C.
      • Bron S.
      • Brouillet S.
      • Bruschi C.V.
      • Caldwell B.
      • Capuano V.
      • Carter N.M.
      • Choi S.K.
      • Codani J.J.
      • Connerton I.F.
      • Danchin A.
      • et al.
      ), and Methanococcus jannaschii (Mj) (
      • Bult C.J.
      • White O.
      • Olsen G.J.
      • Zhou L.
      • Fleischmann R.D.
      • Sutton G.G.
      • Blake J.A.
      • FitzGerald L.M.
      • Clayton R.A.
      • Gocayne J.D.
      • Kerlavage A.R.
      • Dougherty B.A.
      • Tomb J.F.
      • Adams M.D.
      • Reich C.I.
      • Overbeek R.
      • Kirkness E.F.
      • Weinstock K.G.
      • Merrick J.M.
      • Glodek A.
      • Scott J.L.
      • Geoghagen N.S.
      • Venter J.C.
      ). A, TrpB alignment. Critical conserved residues identified as necessary for TrpB activity including His86, Lys87, Glu109, Arg148, Leu188, Cys230, Asp305, Phe306, and Glu350 are inbold. B, TrpA alignment. Conserved catalytic amino acids Glu49 and Asp60 are inbold and underlined. Critical residues in the active site that have been shown to be necessary for TrpA activity, most of which are not conserved including Phe22, Thr183, Gly211, Gly213, Gly234, and Ser235 are in bold. Amino acids in loop 6 are overlined, and residues that are typically invariant but not conserved in chlamydial TrpA are inbold and italics. See “Results” for details.
      Serovar D TrpA protein contains 253 amino acids, a size similar to that of E. coli TrpA (
      • Nichols B.P.
      • Yanofsky C.
      ). A comparison of the complete amino acid sequence of serovar D TrpA with that of representative TrpAs in the public databases shows that the overall level of homology is low (27% identity, Fig. 2B). The two amino acids identified as essential for catalytic activity Glu49and Asp60(
      • Nagata S.
      • Hyde C.C.
      • Miles E.W.
      ,
      • Milton D.L.
      • Napier M.L.
      • Myers R.M.
      • Hardman J.K.
      ,
      • Shirvanee L.
      • Horn V.
      • Yanofsky C.
      ), are conserved in serovar D TrpA. Surprisingly several amino acids which form the active site pocket and/or have been identified by mutagenesis as essential for TrpA activity in E. coli(Phe22, Thr183, Gly211, Gly213, Gly234, Ser235) (
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      ,
      • Allen M.K.
      • Yanofsky C.
      ,
      • Yanofsky C.
      • Helinski D.R.
      • Maling B.D.
      ,
      • Yanofsky C.
      • Horn V.
      ,
      • Weyand M.
      • Schlichting I.
      ) are not conserved in serovar D TrpA. Interestingly, of all the TrpA sequences deposited in the public databases, only the chlamydial TrpAs show these amino acid changes in the active site.
      The unusual primary structure of the serovar D TrpA and the previously reported truncation of TrpA in serovars A and C (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ) prompted us to extend our investigations on tryptophan synthase to all human C. trachomatis serovars. We sequenced the trpB andtrpA genes from the laboratory-type strains of all 15C. trachomatis serovars to determine the diversity within this region. Consistent with the results of Shaw et al.(
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ), we were unable to amplify products from serovar B chromosomal DNA, indicating a deletion of the trp region from this isolate. The sequences of the trpB genes from the 14 serovars are remarkably similar, with only 6 single nucleotide polymorphisms present in 1179 nt (data not shown). One point mutation (nt 1017) does not alter the amino acid sequence (Asn339), a second point mutation (nt 206) is conservative, resulting in a change from Arg to Lys at position 69 in serovars H, J, L2, and L3, a third point mutation (nt 696) results in a change from Ser to Phe at position 232 in serovar L1, and a fourth point mutation (nt 1143) results in a change from Pro to Ser at position 381 in serovar Ba. The remaining two point mutations at nt 107 and 179 result in the conversion of Ser36 to Asn in serovars H, J, L2, and L3 and Asn60 to Ser in serovars A and C, respectively.
      Examination of the nucleotide alignment of the 14 C. trachomatis trpA sequences revealed both single nucleotide polymorphisms and deletion mutations in the 762-nt gene (all numbering is based on genital serovar sequences unless otherwise noted). Of the 11 point mutations identified, 4 (nt 10, 120, 477, and 699) are silent, 2 (nt 110 and 344) result in conservative amino acid changes, Gln → Arg and Ala → Val, in serovars H, J, L2, L3, and three (nt 39) result in changes, Leu → Pro in serovars D and K (data not shown). The remaining mutations illustrated in Fig.3 are more likely to have effects on enzyme structure and/or function. The point mutations at nt 499 and 511 result in non-conservative amino acid substitutions and cluster the serovars into two groups. Thus, all of the ocular serovars encode His167 and Leu171, whereas all of the genital serovars encode Tyr and Phe at the corresponding positions. The ocular and genital serovars also differ in sequence at nt 408–410; these nt are deleted in the ocular serovars, resulting in the loss of Phe136 from the protein encoded by these genes (Fig. 3). The trpA genes of the ocular serovars also have a single nt deletion at position 528 (ocular serovar numbering) that results in a frameshift generating a putative stop codon at nt 550–552 (ocular serovar numbering). These deletion mutations were previously reported for serovars A and C (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ); here we demonstrate that they are also found in serovar Ba. The deletion mutation found at nt 528 in the ocular serovars lies within a mutation “hotspot” for trpA. Thus, in the genital serovars there are two point mutations found within this same region at nt 530 and 532 encompassing two codons at amino acid positions 177 and 178. The net result of these mutations is that all LGV serovars encode Tyr177Glu178, serovars D, K, and E encode Cys177-Gln178, and serovars G, F, I, H, and J encode Tyr177-Gln178(Fig. 3).
      Figure thumbnail gr3
      Figure 3Alignment of partial sequences fromtrpA gene (A) and protein (B) of the 14 reference serovars for whichtrpA gene could be amplified by PCR. ClustalW alignment of nucleotide and the corresponding protein regions containing sequence polymorphisms are illustrated. The ocular serovar specific 3 base deletion (nt 408–410), missense mutation (nt 499 and 511), and single nucleotide deletion that results in a truncated protein (stop codon nt 550–552) are in bold anditalics. Genital serovar specific missense mutations (nt 530 and 532) that result in amino acid changes (177 and 178) in loop6 of TrpA are shown in bold. See “Results” for details.

       Expression of trp Genes during HeLa Cell Infection and in Purified EBs

      To determine whether the trpB and trpAgenes were expressed in the various C. trachomatis serovars, RT-PCR and Western blot analyses were carried out on type strains representative of the LGV (L2), ocular (A), and genital serovars (D and I). For RT-PCR analysis, RNA was prepared from mid-phase (24 h) infected HeLa cell cultures. After reverse transcription, the cDNA was amplified using a forward primer complementary to the 3′ end oftrpB and a reverse primer complementary to the 5′ end oftrpA. PCR products of the expected size were amplified from cDNA derived from HeLa cells infected with serovars L2, A, I, and D as well as from a plasmid (pCR3) containing full-length L2trpB and trpA (Fig.4A). These primers were specific for C. trachomatis-derived mRNA, as no product was amplified from cDNA prepared from mock-infected HeLa cells. Similarly, primers specific for C. trachomatis 16 S rRNA only amplified products from C. trachomatis-infected HeLa cell cDNA and a plasmid control but not the mock-infected sample. These data indicate that type strains representative of the serovars causing human disease all express trpB and trpAand that these genes are transcribed as an operon. Furthermore, the single base deletion mutation found in serovar A trpA does not appear to affect transcription of trpBA mRNA.
      Figure thumbnail gr4
      Figure 4Expression of trpB andtrpA in C. trachomatis serovars L2, A, I, and D. A, RT-PCR analysis of trp transcript expression. HeLa cells were infected with C. trachomatisreference serovars, and RNA was isolated 24 h post-infection. The RNA was reverse-transcribed using random hexamer primers, and the cDNA was amplified with primers specific for C. trachomatis 16 S rRNA or sequences flanking thetrpB-trpA junction. As a negative control, RNA from non-infected HeLa cells was analyzed (mock), and as positive control, plasmid DNA containing L2 trpBA or 16 S rRNA genes were amplified in the PCR reaction. B, Western blot analysis of TrpB and TrpA expression in purified EBs. EBs were lysed in Laemmli buffer, and proteins were separated by SDS-PAGE (12%) and then transferred to nitrocellulose. Proteins were detected using polyclonal antibodies raised against recombinant (recomb) L2 TrpB or TrpA. The respective recombinant proteins were included as positive controls.
      Western blot analyses were used to determine whether the trpgene messages expressed by serovars L2, A, I, and D were translated to protein products. As shown in Fig. 4B, immunoreactive material of the same electrophoretic mobility as recombinant L2 TrpB was detected in purified EBs from serovars L2, A, I, and D. Similarly, material with the same mobility as recombinant L2 TrpA was detected in EB lysates of serovars L2, I, and D. Serovar A EBs also had anti-TrpA immunoreactive material but of lower molecular weight than that of the other serovars. This result indicates that the frameshift mutation in serovar A trpA results in the production of a truncated version of TrpA, consistent with the observations of Shaw et al. (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ). No immunoreactive material of the appropriate size for either TrpB or TrpA was detectable in EBs from serovar B or biovar MoPn (data not shown), consistent with our inability to amplify products from these isolates using trpB- or trpA-specific primers (data not shown) and the absence of trpBA genes in the MoPn genome (
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • Gill S.R.
      • Heidelberg J.F.
      • White O.
      • Hickey E.K.
      • Peterson J.
      • Utterback T.
      • Berry K.
      • Bass S.
      • Linher K.
      • Weidman J.
      • Khouri H.
      • Craven B.
      • Bowman C.
      • Dodson R.
      • Gwinn M.
      • Nelson W.
      • DeBoy R.
      • Kolonay J.
      • McClarty G.
      • Salzberg S.L.
      • Eisen J.
      • Fraser C.M.
      ).

       Genetic Complementation and in Vitro Enzyme Assays

      To determine whether the TrpB and TrpA proteins expressed by C. trachomatis were catalytically active, a heterologous complementation system was utilized. The trp genes from serovars L2, A, and I were cloned into an E. coli expression vector and transformed into E. coli mutants lacking various components of the tryptophan biosynthesis pathway. The ability of theE. coli mutants expressing C. trachomatis trp genes to grow on minimal medium was then assessed (Fig.5). The E. coli mutant KS463 expresses a non-functional TrpA but expresses active TrpB. KS463 cells transformed with either expression vector alone or constructs expressing C. trachomatis (serovars L2, A, or I) or E. coli trpA were able to grow on minimal medium supplemented with indole. These data are consistent with published observations indicating that E. coli TrpB can utilize indole in the absence of functional TrpA (
      • Lane A.N.
      • Kirschner K.
      ,
      • Miles E.W.
      • McPhie P.
      ,
      • Crawford I.P.
      • Yanofsky C.
      ). Similarly, all KS463 transformants were able to grow on minimal medium supplemented with tryptophan, as expected. However, KS463 failed to grow on minimal medium when transformed with either the expression vector alone or any of the constructs expressing C. trachomatis trpAregardless of the serovar of origin. In contrast, KS463 transformed with the E. coli trpA construct did grow on minimal medium. These data suggested that C. trachomatis TrpA could not efficiently utilize the IGP produced by KS463, either due to a loss of catalytic activity for this substrate or due to an inability to interact with and, thus, be activated by E. coli TrpB.
      Figure thumbnail gr5
      Figure 5Analysis of C. trachomatisTrpB and TrpA enzymatic function by genetic complementation. trp genes from C. trachomatis serovars L2, A, and I were cloned into the E. coli expression vector pQE-80L either individually or together (trpBA). E. coli trpA was also cloned for use as a positive control in the KS463 mutant. Constructs were transformed into E. colimutants KS463 (trpA33), BW7622 (trpB::Tn10), or CY15077 (ΔtrpE-A), and growth of the transformants was assessed on M9 minimal agar containing 100 μm indole, 50 μg ml−1tryptophan or no additional supplements. *, vector containstrpB from serovar A and trpA from serovar L2. **, vector contains trpB from serovar L2 and trpAfrom serovar A.
      To distinguish between these two possibilities, the E. coli trpB transposon mutant BW7622, which does not expresstrpB or trpA, was transformed with constructs co-expressing C. trachomatis trpB andtrpA. This eliminated the requirement for C. trachomatis TrpA to interact with a heterologous TrpB. There was no detectable growth of any of the transformants on minimal medium, whereas expression of tryptophan synthase derived from serovars L2 and I complemented the growth of BW7622 on indole-supplemented media (Fig.5). These data suggest that C. trachomatis tryptophan synthase is unable to utilize IGP or does so at levels insufficient to complement growth of BW7622. Furthermore, efficient utilization of indole by C. trachomatis TrpB appeared to require the presence of full-length TrpA since serovar A tryptophan synthase expression did not rescue the growth of BW7622 on indole, although this transformant was able to grow on tryptophan-supplemented media. This conclusion was confirmed by complementation experiments carried out in an E. coli deletion mutant, CY15077, that lacks the entire tryptophan biosynthesis operon. Thus, transformation of CY15077 with constructs expressing only C. trachomatis trpB did not complement growth on minimal medium supplemented with indole. However, co-expression of trpB and trpA from serovars L2 and I did rescue the growth of CY15077 on indole-supplemented media. Similar to the results observed with BW7622, co-expression of serovar AtrpB and trpA did not allow for growth of CY15077 on indole. This was likely due to the inability of serovar A TrpA to activate serovar A TrpB, since the co-expression of serovar A TrpB with serovar L2 TrpA permitted the growth of CY15077 on indole. In contrast, expression of serovar L2 TrpB with serovar A TrpA failed to rescue the growth of CY15077 on indole.
      In addition to the genetic complementation studies, in vitroactivity in the α, β, and αβ reactions was determined for cellular extracts prepared from E. coli CY15077 co-expressing C. trachomatis trpB and trpA (TableIII). As positive control, activity in all three reactions was detected for purified tryptophan synthase fromSalmonella enterica ser. Typhimurium. No activity was detected in any of the assays using cellular extracts from CY15077 cells transformed with the expression vector alone. As expected from the results of the complementation studies, no activity for the α or αβ reactions was detectable in any of the lysates of cells expressing C. trachomatis proteins. In contrast, activity for the β reaction was readily detectable in lysates containing TrpB and TrpA from serovars L2 and I, whereas the β reaction activity of the lysate of serovar A-expressing cells was very low. Taken together, results from the complementation studies and in vitro enzyme assays suggest that unlike the Salmonella and E. coli enzymes, tryptophan synthase from C. trachomatisis unable to efficiently catalyze the conversion of IGP to indole (α reaction) and reaction of indole with serine to form tryptophan (β reaction) requires the presence of a full-length TrpA. Because the specific activity in the β reaction for L2 and I extracts was low compared with the purified Salmonella enzyme, further studies with purified C. trachomatis enzymes will be required to confirm whether there is indeed no α activity or whether it is just too low to be detected in cellular extracts.
      Table IIITryptophan synthase activity in lysates of CY15077 trpBA transformants
      Enzyme sourceSpecific activity
      1 unit is the amount of enzyme that catalyses the disappearance (α reaction) or appearance (β reaction) of 0.1 μmol of indole in 20 min at 37 °C.
      β reactionα reactionαβ reaction
      units mg−1
      Vector aloneNDA
      NDA, no detectable activity.
      NDANDA
      L21.06 ± 0.02
      Specific activity ±1 S.D.
      NDANDA
      A0.08 ± 0.16NDANDA
      I3.43 ± 1.35NDANDA
      Purified Salmonella trpsynthase43.212.122.4
      3-a 1 unit is the amount of enzyme that catalyses the disappearance (α reaction) or appearance (β reaction) of 0.1 μmol of indole in 20 min at 37 °C.
      3-b NDA, no detectable activity.
      3-c Specific activity ±1 S.D.

       C. trachomatis Tryptophan Synthase Activity in Vivo

      It is clear from the complementation and in vitro activity studies that the trpB genes of C. trachomatis serovars L2, A, and I encode functional enzymes that require the presence of full-length TrpA for detectable activity. It has been previously reported that growth of most human serovars of C. trachomatis are tryptophan-dependent (
      • Jones M.L.
      • Gaston J.S.
      • Pearce J.H.
      ,
      • Coles A.M.
      • Reynolds D.J.
      • Harper A.
      • Devitt A.
      • Pearce J.H.
      ,
      • Morrison R.P.
      ). To determine whether C. trachomatis tryptophan synthase could function in vivo, HeLa cell infections were carried out under tryptophan-free conditions, and chlamydial growth was assessed after supplementation of the media with potential tryptophan precursors. It has been proposed that anthranilate may serve as a precursor for tryptophan biosynthesis in C. trachomatis(
      • Stephens R.S.
      ). However, the decrease in recoverable IFU for serovar L2 grown in tryptophan-free media did not change after supplementation with anthranilate (Fig. 6). Thus, anthranilate cannot by used by C. trachomatis for tryptophan synthesis. In addition, kynurenine, another tryptophan degradation product, was also unable to rescue C. trachomatis growth in tryptophan-efficient medium (data not shown).
      Figure thumbnail gr6
      Figure 6Effect of anthranilate on the growth ofC. trachomatis serovar L2 under tryptophan-free conditions. HeLa cells were infected with serovar L2 EBs at an m.o.i. of 3–5 IFU cell−1 in MEM, 10% dialyzed FCS containing 10 mg liter−1 tryptophan (+TRP), lacking tryptophan (−TRP), or lacking tryptophan but supplemented with increasing concentrations of anthranilate (AN). After 48 h, infected cells and culture supernatants were collected and used to infect a second HeLa cell monolayer for enumeration of recoverable IFU. Data are presented as the mean IFU (log10) of triplicate determinations from three separate experiments. Error bars have been omitted for clarity. The S.E. of any determination never exceeded 0.5 log10.
      Results from the in vitro enzyme assays indicated thatC. trachomatis TrpB could utilize indole for the synthesis of tryptophan. As shown in Fig. 7, the level of recoverable IFU under tryptophan-replete conditions varied depending upon the serovar. However, growth of all serovars was inhibited by the removal of tryptophan from the cell culture medium. In the presence of 100 μm indole, growth of all the genital (D-K, L1-L3) serovars recovered to tryptophan-replete levels or better, whereas there was no effect of indole supplementation on the growth of the ocular serovars (A-C, Ba). To confirm that indole was being utilized by C. trachomatis and was not converted into a tryptophan precursor by the host cell, HeLa cell infections were carried out in the presence of radiolabeled indole. All C. trachomatis serovars grown in the presence of tryptophan failed to incorporate 14C-labeled indole, suggesting that there may be some regulation of tryptophan synthase by tryptophan levels in the cell. Under tryptophan-free conditions, serovars L2, D, and I were able to incorporate 14C-labeled indole, whereas serovar A and mock-infected HeLa cells showed no indole incorporation (Fig.8). Thus, it would appear that in vivo, C. trachomatis genital serovars are able to synthesize tryptophan directly from indole and that lack of a full-length TrpA results in the inability of ocular serovars to utilize indole.
      Figure thumbnail gr7
      Figure 7Effect of indole on the growth of 15C. trachomatis reference serovars under tryptophan-free conditions. HeLa cells were infected with EBs at an m.o.i. of 3–5 IFU cell−1 in MEM, 10% dialyzed FCS containing 10 mg liter−1 tryptophan (+TRP), lacking tryptophan (−TRP), or lacking tryptophan but supplemented with 100 μm indole (−TRP+IND). After 48–72 h, infected cells and culture supernatants were collected and used to infect a second HeLa cell monolayer for enumeration of recoverable IFU. Data are presented as the mean IFU (log10) of triplicate determinations from three separate experiments. Error bars have been omitted for clarity. The S.E. of any determination never exceeded 0.5 log10.
      Figure thumbnail gr8
      Figure 8Utilization of indole by C. trachomatis during HeLa cell infection under tryptophan-free conditions. HeLa cells were infected with C. trachomatis serovar L2, D, A, and I EBs at an m.o.i. of 3–5 IFU cell−1 or left uninfected (mock) in MEM, 10% dialyzed FCS supplemented with 10 μCi of 14C-indole and containing 10 mg liter−1 tryptophan (TRP+) or lacking tryptophan (TRP−). After 36–48 h of incubation at 37 C, the infected cells were lysed, and incorporated radioactivity was determined by scintillation counting.

      DISCUSSION

      Host response to chlamydial infection involves production of the protective cytokine IFN-γ, which induces the expression of indoleamine-2,3-dioxygenase and, thus, promotes tryptophan degradation in the host cells (
      • Beatty W.L.
      • Morrison R.P.
      • Byrne G.I.
      ,
      • Beatty W.L.
      • Belanger T.A.
      • Desai A.A.
      • Morrison R.P.
      • Byrne G.I.
      ,
      • Byrne G.I.
      • Lehmann L.K.
      • Landry G.J.
      ). Thus, the ability to synthesize tryptophan may be an important survival factor for C. trachomatis during the course of infection, allowing the intracellular bacteria to persist in the presence of IFN-γ-induced tryptophan limitation. This study was undertaken to determine the extent of heterogeneity in the C. trachomatis trpregion and to determine whether trpB and trpA, respectively, encoding the β and α subunits of tryptophan synthase, were expressed as functional enzymes.
      Both trpB and trpA were expressed inC. trachomatis as shown by RT-PCR analysis of transcripts from infected HeLa cells. Results indicate that trpB andtrpA can be expressed as a single transcript, similar to what has been observed for the trp operon of other bacteria (
      • Crawford I.P.
      ,
      • Yanofsky C.
      ). C. trachomatis encodes a putative tryptophan repressor (trpR), which presents the possibility that transcription of the trp genes may be regulated by the tryptophan concentration in the host cell. High levels transcriptional repression of the trp operon by the TrpR-tryptophan repressor complex has been observed in many Gram-negative bacteria (
      • Rose J.K.
      • Squires C.L.
      • Yanofsky C.
      • Yang H.L.
      • Zubay G.
      ). However, results from the present study indicate that there must be a basal level of trp gene expression in C. trachomatis, as we could detect both TrpB and TrpA in purified EBs obtained from HeLa cells infected in tryptophan-replete medium.
      The C. trachomatis trpB gene sequences from the 15 reference serovars were nearly identical, with only four single nucleotide polymorphisms observed. All of the amino acids essential for activity, as identified in E. coli TrpB (His86, Lys87, Glu109, Arg148, Leu188, Cys230, Asp305, Phe306, Glu350) (
      • Miles E.W.
      ,
      • Fluri R.
      • Jackson L.E.
      • Lee W.E.
      • Crawford I.P.
      ,
      • Tanizawa K.
      • Miles E.W.
      ,
      • Miles E.W.
      • Kawasaki H.
      • Ahmed S.A.
      • Morita H.
      • Nagata S.
      ,
      • Higgins W.
      • Miles E.W.
      • Fairwell T.
      ) are conserved in the C. trachomatis proteins, suggesting that they should have enzymatic activity. This was indeed the case as shown by both genetic complementation studies and in vitro assays of β reaction activity in crude cell lysates. TrpB from serovars L2, A, and I was capable of catalyzing the β-replacement reaction of indole and serine to form tryptophan. However, our results suggested a unique property of the C. trachomatis TrpB compared with that characterized from other Gram-negative bacteria. Thus, C. trachomatis TrpB appeared to have an absolute requirement for TrpA for function; no β reaction activity was detectable in the absence of TrpA or in the presence of truncated TrpA from serovar A. In contrast,E. coli TrpB has been shown to have activity in the absence of TrpA, albeit at a lower level than observed in its presence (
      • Lane A.N.
      • Kirschner K.
      ,
      • Miles E.W.
      • McPhie P.
      ,
      • Crawford I.P.
      • Yanofsky C.
      ). Therefore, the requirement for TrpA activation of TrpB appears to be more stringent in the C. trachomatis enzyme than in that of E. coli or Salmonella.
      A larger number of polymorphisms were found in the nucleotide sequences of C. trachomatis trpA compared withtrpB; however, there was still greater than 98% identity among the sequences from the various serovars. Interestingly, TrpA from all serovars retained the invariant catalytic residues, Glu49 and Asp60, but had changed most of the other highly conserved amino acids (Phe22, Thr183, Gly211, Gly213, Gly234, Ser235) in the active site pocket. These residues have been shown by mutagenesis to be critical for TrpA activity (
      • Nagata S.
      • Hyde C.C.
      • Miles E.W.
      ,
      • Milton D.L.
      • Napier M.L.
      • Myers R.M.
      • Hardman J.K.
      ,
      • Allen M.K.
      • Yanofsky C.
      ,
      • Yanofsky C.
      • Helinski D.R.
      • Maling B.D.
      ,
      • Yanofsky C.
      • Horn V.
      ,
      • Lim W.K.
      • Sarkar S.K.
      • Hardman J.K.
      ) and are key residues involved in binding IGP in the Salmonella TrpBA crystal structure (
      • Miles E.W.
      ,
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      ,
      • Weyand M.
      • Schlichting I.
      ). Given the changes in these key amino acids, it is not surprising that we did not detect α reaction activity in the genetic complementation studies nor the in vitro activity assays of lysates from cells overexpressing the C. trachomatis tryptophan synthase.
      A polymorphic mutational “hot spot” was identified intrpA from the genital chlamydiae serovars at nt 530 and 532, resulting in three possible amino acids combinations at positions 177 and 178 in the translated protein. These amino acids lie in TrpA loop 6, a region identified in the Salmonella tryptophan synthase crystal structure as being highly flexible and important for subunit-subunit interactions between TrpB and TrpA, metabolite channeling, and substrate binding (
      • Miles E.W.
      ,
      • Miles E.W.
      ,
      • Hyde C.C.
      • Ahmed S.A.
      • Padlan E.A.
      • Miles E.W.
      • Davies D.R.
      ,
      • Ruvinov S.B.
      • Miles E.W.
      ,
      • Brzovic P.S.
      • Sawa Y.
      • Hyde C.C.
      • Miles E.W.
      • Dunn M.F.
      ,
      • Brzovic P.S.
      • Hyde C.C.
      • Miles E.W.
      • Dunn M.F.
      ,
      • Miles E.W.
      ,
      • Schneider T.R.
      • Gerhardt E.
      • Lee M.
      • Liang P.H.
      • Anderson K.S.
      • Schlichting I.
      ). It is possible that the sequence polymorphisms observed in the TrpA loop 6 region may affect interactions between the α and β subunits and thus influence TrpB activity. In total, the unusual primary structure of TrpA suggests that the main function of the C. trachomatis α-subunit could be to position TrpB in the appropriate or favorable conformation to efficiently carry out the β reaction. Detailed kinetic characterization of purified TrpB and TrpA from the different serovars will be required to determine this.
      The deletion and frameshift mutations in trpA from serovars A and C, originally identified by Shaw et al. (
      • Shaw A.C.
      • Christiansen G.
      • Roepstorff P.
      • Birkelund S.
      ), were also found in serovar Ba in the present study. Interestingly, these mutations appear to be predictive for the tissue tropism of the isolates. All of the ocular serovars had a 3-base deletion (nt 408–410) and a single nucleotide deletion (nt 528), resulting in a truncated TrpA, whereas none of the genital serovars did. Similarly, non-conservative point mutations also cluster the serovars into ocular and genital strains (i.e. at nt 499 and 511). We are currently investigating whether this correlation betweentrpA sequence and serovar tissue tropism holds true for clinical isolates.
      It has been postulated that C. trachomatis may be able to scavenge tryptophan degradation products such as anthranilate from the host cell for use as precursors in tryptophan biosynthesis (
      • Stephens R.S.
      ). Results from the present study clearly indicate that anthranilate could not rescue C. trachomatis growth in HeLa cells grown in tryptophan-free medium. This is not surprising given the absence of several key enzymes in the tryptophan biosynthesis pathway. AlthoughC. trachomatis encodes a phosphoribosyl anthranilate isomerase (trpF), it lacks the gene for anthranilate phosphoribosyltransferase required for conversion of anthranilate to phosphoribosyl anthranilate as well as the gene for IGP synthase (trpC), required for the conversion of 1-(o-carboxylphenylamino)-1-deoxyribulose-5-phosphate to IGP (see Fig. 1).
      Because of the lack of tools for genetic manipulation, it has not been possible to produce site-specific mutants in C. trachomatis. Despite this limitation, our results indicate that the tryptophan synthase detected in C. trachomatis RBs appears to function similarly to the recombinant enzymes expressed in E. coli. Thus, C. trachomatis growing within HeLa cells was able to utilize indole for growth in the absence of tryptophan. Only genital serovars could utilize indole, consistent with the observations thatin vitro the truncated TrpA (found in ocular serovars) could not enhance TrpB activity in the β reaction. Indole was used directly by C. trachomatis and was not processed by the HeLa cells into some other precursor molecule, as mock-infected cells exhibited no [14C]indole incorporation. In addition, C. trachomatis serovar A could not incorporate ]14C]indole, further confirming that its TrpB is unable to function in the absence of full-length TrpA.
      Taken together, our results indicate that tryptophan synthase encoded by C. trachomatis trpBA is functional for conversion of indole to tryptophan, which permits the growth of genital serovars under conditions of tryptophan starvation. In stark contrast, ocular serovars A, Ba, and C, with a mutation in trpA, resulting in the production of a truncated protein, are unable to utilize indole for growth in the absence of tryptophan. What might the clinical significance of these findings be? It is well known that ocular serovars rarely cause genital infections, and genital serovars are rarely associated with blinding trachoma (
      • Schachter J.
      ,
      • Schachter J.
      ,
      • Mabey D.
      • Bailey R.
      ). The molecular basis for this distinct tissue tropism has never been defined. To our knowledge the differing abilities to synthesize tryptophan is the first demonstration of a distinction in the biosynthetic capacity between the ocular and genital serovars.
      The unusual properties of the chlamydial tryptophan synthase raise the question as to what might be the true substrate in vivo. Our findings support the hypothesis that it is likely indole. Under normal physiological conditions, indole is not readily available as a metabolite in mammalian cells. Indole is, however, a major byproduct of tryptophan degradation in bacteria encoding the enzyme tryptophanase (
      • Yanofsky C.
      ). Common enteric bacteria such as E. coli andProteus sp. are known producers of indole (
      • Yanofsky C.
      ,
      • Kamath A.V.
      • Yanofsky C.
      ). The same two organisms are also part of the normal genital microflora and important urogenital pathogens (
      • Gibbs R.S.
      ,
      • Larsen B.
      • Monif G.R.
      ). Other indole-producing organisms (
      • Balows A.
      ) known to colonize the female genital tract (
      • Larsen B.
      • Monif G.R.
      ) includePeptostreptococcus asaccharolyticus,Fusobacterium species, Bacteroides species,Haemophilus influenza, and Weeksella virosa. This raises the intriguing possibility that C. trachomatisserovars that infect the genital tract may be able to use indole produced by other microflora, either endogenous or the result of infection, as a substrate to synthesize their own tryptophan for growth. Co-infection with indole-producing organisms may allow for the rescue of chlamydial organisms persisting in a non-replicating form in response to host IFN-γ and its subsequent effect on intracellular tryptophan levels. The ability to synthesize tryptophan from indole may be important for the persistence of C. trachomatis within the genital tract epithelium, with important consequences for disease transmission as well as for the inflammatory sequelae associated with chronic infection. Because the eye is normally a sterile niche, ocular serovars are less likely to encounter sources of indole during the course of infection, thereby eliminating the selective pressure to maintain a functional tryptophan synthase.

      Acknowledgements

      We thank Dr. Edith Wilson Miles for providing us with purified Salmonella tryptophan synthase and IGP and Dr. Charles Yanofsky for several E. coli mutants. We are also indebted to both for helpful advice.

      REFERENCES

        • Moulder J.W.
        Microbiol. Rev. 1991; 55: 143-190
        • Schachter J.
        Pathol. Immunopathol. Res. 1989; 8: 206-220
        • Schachter J.
        Stephens R.S. Chlamydia Intracellular Biology, Pathogenesis, and Immunity. American Society for Microbiology, Washington, D. C.1999: 139-170
        • Kuo C.C.
        • Jackson L.A.
        • Campbell L.A.
        • Grayston J.T.
        Clin. Microbiol. Rev. 1995; 8: 451-461
        • Campbell L.A.
        • Rosenfeld M.
        • Kuo C.C.
        Trends Microbiol. 2000; 8: 255-257
        • Kuo C.
        • Campbell L.A.
        Mol. Med. Today. 1998; 4: 426-430
        • Grayston J.T.
        • Kuo C.C.
        • Campbell L.A.
        • Wang S.P.
        • Jackson L.A.
        Cardiologia. 1997; 42: 1145-1151
        • Stephens R.
        Moulder J.W. Antigenic Variation of Chlamydia trachomatis. CRC Press, Inc., Boca Raton, FL1989
        • Mabey D.
        • Bailey R.
        Br. J. Ophthalmol. 1999; 83: 1261-1263
        • Belland R.J.
        • Scidmore M.A.
        • Crane D.D.
        • Hogan D.M.
        • Whitmire W.
        • McClarty G.
        • Caldwell H.D.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13984-13989
        • Brunham R.C.
        Stephens R.S. Chlamydia Intracellular Biology, Pathogenesis, and Immunity. American Society for Microbiology, Washington, D. C.1999: 211-238
        • Kim S.K.
        • DeMars R.
        Curr. Opin. Immunol. 2001; 13: 429-436
        • Loomis W.P.
        • Starnbach M.N.
        Curr. Opin. Microbiol. 2002; 5: 87-91
        • Beatty W.L.
        • Morrison R.P.
        • Byrne G.I.
        Microbiol. Rev. 1994; 58: 686-699
        • Beatty W.L.
        • Belanger T.A.
        • Desai A.A.
        • Morrison R.P.
        • Byrne G.I.
        Infect. Immun. 1994; 62: 3705-3711
        • Taylor M.W.
        • Feng G.S.
        FASEB J. 1991; 5: 2516-2522
        • Boehm U.
        • Klamp T.
        • Groot M.
        • Howard J.C.
        Annu. Rev. Immunol. 1997; 15: 749-795
        • Stephens R.S.
        • Kalman S.
        • Lammel C.
        • Fan J.
        • Marathe R.
        • Aravind L.
        • Mitchell W.
        • Olinger L.
        • Tatusov R.L.
        • Zhao Q.
        • Koonin E.V.
        • Davis R.W.
        Science. 1998; 282: 754-759
        • Read T.D.
        • Brunham R.C.
        • Shen C.
        • Gill S.R.
        • Heidelberg J.F.
        • White O.
        • Hickey E.K.
        • Peterson J.
        • Utterback T.
        • Berry K.
        • Bass S.
        • Linher K.
        • Weidman J.
        • Khouri H.
        • Craven B.
        • Bowman C.
        • Dodson R.
        • Gwinn M.
        • Nelson W.
        • DeBoy R.
        • Kolonay J.
        • McClarty G.
        • Salzberg S.L.
        • Eisen J.
        • Fraser C.M.
        Nucleic Acids Res. 2000; 28: 1397-1406
        • Kalman S.
        • Mitchell W.
        • Marathe R.
        • Lammel C.
        • Fan J.
        • Hyman R.W.
        • Olinger L.
        • Grimwood J.
        • Davis R.W.
        • Stephens R.S.
        Nat. Genet. 1999; 21: 385-389
        • Shirai M.
        • Hirakawa H.
        • Kimoto M.
        • Tabuchi M.
        • Kishi F.
        • Ouchi K.
        • Shiba T.
        • Ishii K.
        • Hattori M.
        • Kuhara S.
        • Nakazawa T.
        Nucleic Acids Res. 2000; 28: 2311-2314
        • Shaw A.C.
        • Christiansen G.
        • Roepstorff P.
        • Birkelund S.
        Microbes Infect. 2000; 2: 581-592
        • Miles E.W.
        Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172
        • Miles E.W.
        Subcell. Biochem. 1995; 24: 207-254
        • Miles E.W.
        Chem. Rec. 2001; 1: 140-151
        • Lane A.N.
        • Kirschner K.
        Eur. J. Biochem. 1983; 129: 571-582
        • Miles E.W.
        • McPhie P.
        J. Biol. Chem. 1974; 249: 2852-2857
        • Crawford I.P.
        • Yanofsky C.
        Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 1161-1170
        • Tipples G.
        • McClarty G.
        J. Bacteriol. 1991; 173: 4932-4940
        • Caldwell H.D.
        • Kromhout J.
        • Schachter J.
        Infect. Immun. 1981; 31: 1161-1176
        • Lacy M.J.
        • Voss E.W., Jr.
        J. Immunol. Methods. 1986; 87: 169-177
        • Smith O.H.
        • Yanofsky C.
        Methods Enzymol. 1962; 5: 794-806
        • Miles E.W.
        J. Biol. Chem. 1970; 245: 6016-6025
        • Crawford I.P.
        • Nichols B.P.
        • Yanofsky C.
        J. Mol. Biol. 1980; 142: 489-502
        • Fluri R.
        • Jackson L.E.
        • Lee W.E.
        • Crawford I.P.
        J. Biol. Chem. 1971; 246: 6620-6624
        • Tanizawa K.
        • Miles E.W.
        Biochemistry. 1983; 22: 3594-3603
        • Miles E.W.
        • Kawasaki H.
        • Ahmed S.A.
        • Morita H.
        • Nagata S.
        J. Biol. Chem. 1989; 264: 6280-6287
        • Higgins W.
        • Miles E.W.
        • Fairwell T.
        J. Biol. Chem. 1980; 255: 512-517
        • Hyde C.C.
        • Ahmed S.A.
        • Padlan E.A.
        • Miles E.W.
        • Davies D.R.
        J. Biol. Chem. 1988; 263: 17857-17871
        • Nichols B.P.
        • Yanofsky C.
        Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5244-5248
        • Nagata S.
        • Hyde C.C.
        • Miles E.W.
        J. Biol. Chem. 1989; 264: 6288-6296
        • Milton D.L.
        • Napier M.L.
        • Myers R.M.
        • Hardman J.K.
        J. Biol. Chem. 1986; 261: 16604-16615
        • Shirvanee L.
        • Horn V.
        • Yanofsky C.
        J. Biol. Chem. 1990; 265: 6624-6625
        • Allen M.K.
        • Yanofsky C.
        Genetics. 1963; 48: 1065-1083
        • Yanofsky C.
        • Helinski D.R.
        • Maling B.D.
        Cold Spring Harbor Symp. Quant. Biol. 1961; 26: 11-24
        • Yanofsky C.
        • Horn V.
        J. Biol. Chem. 1972; 247: 4494-4498
        • Weyand M.
        • Schlichting I.
        Biochemistry. 1999; 38: 16469-16480
        • Jones M.L.
        • Gaston J.S.
        • Pearce J.H.
        Microb. Pathog. 2001; 30: 299-309
        • Coles A.M.
        • Reynolds D.J.
        • Harper A.
        • Devitt A.
        • Pearce J.H.
        FEMS Microbiol. Lett. 1993; 106: 193-200
        • Morrison R.P.
        Infect. Immun. 2000; 68: 6038-6040
        • Stephens R.S.
        Stephens R.S. Chlamydia Intracellular Biology, Pathogenesis, and Immunity. American Society for Microbiology Press, Washington, D. C.1999: 9-28
        • Byrne G.I.
        • Lehmann L.K.
        • Landry G.J.
        Infect. Immun. 1986; 53: 347-351
        • Crawford I.P.
        Bacteriol. Rev. 1975; 39: 87-120
        • Yanofsky C.
        Bacteriol. Rev. 1960; 24: 221-245
        • Rose J.K.
        • Squires C.L.
        • Yanofsky C.
        • Yang H.L.
        • Zubay G.
        Nat. New Biol. 1973; 245: 133-137
        • Lim W.K.
        • Sarkar S.K.
        • Hardman J.K.
        J. Biol. Chem. 1991; 266: 20205-20212
        • Ruvinov S.B.
        • Miles E.W.
        FEBS Lett. 1992; 299: 197-200
        • Brzovic P.S.
        • Sawa Y.
        • Hyde C.C.
        • Miles E.W.
        • Dunn M.F.
        J. Biol. Chem. 1992; 267: 13028-13038
        • Brzovic P.S.
        • Hyde C.C.
        • Miles E.W.
        • Dunn M.F.
        Biochemistry. 1993; 32: 10404-10413
        • Miles E.W.
        J. Biol. Chem. 1991; 266: 10715-10718
        • Schneider T.R.
        • Gerhardt E.
        • Lee M.
        • Liang P.H.
        • Anderson K.S.
        • Schlichting I.
        Biochemistry. 1998; 37: 5394-5406
        • Yanofsky C.
        J. Bacteriol. 2000; 182: 1-8
        • Kamath A.V.
        • Yanofsky C.
        J. Biol. Chem. 1992; 267: 19978-19985
        • Gibbs R.S.
        Am. J. Obstet. Gynecol. 1987; 156: 491-495
        • Larsen B.
        • Monif G.R.
        Clin. Infect. Dis. 2001; 32: 69-77
        • Balows A.
        Balows A. Truper H.G. Dworkin M. Harder W. Schleifer K.-H. The Prokaryotes. Springer-Verlag New York Inc., New York1991: 1-4
        • Kunst F.
        • Ogasawara N.
        • Moszer I.
        • Albertini A.M.
        • Alloni G.
        • Azevedo V.
        • Bertero M.G.
        • Bessieres P.
        • Bolotin A.
        • Borchert S.
        • Borriss R.
        • Boursier L.
        • Brans A.
        • Braun M.
        • Brignell S.C.
        • Bron S.
        • Brouillet S.
        • Bruschi C.V.
        • Caldwell B.
        • Capuano V.
        • Carter N.M.
        • Choi S.K.
        • Codani J.J.
        • Connerton I.F.
        • Danchin A.
        • et al.
        Nature. 1997; 390: 249-256
        • Bult C.J.
        • White O.
        • Olsen G.J.
        • Zhou L.
        • Fleischmann R.D.
        • Sutton G.G.
        • Blake J.A.
        • FitzGerald L.M.
        • Clayton R.A.
        • Gocayne J.D.
        • Kerlavage A.R.
        • Dougherty B.A.
        • Tomb J.F.
        • Adams M.D.
        • Reich C.I.
        • Overbeek R.
        • Kirkness E.F.
        • Weinstock K.G.
        • Merrick J.M.
        • Glodek A.
        • Scott J.L.
        • Geoghagen N.S.
        • Venter J.C.
        Science. 1996; 273: 1058-1073