From the
Center for Microbial Pathogenesis and
Departments of Medicine and Genetics & Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3710, the ¶Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, the ||Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, and the **Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536
Received for publication, January 23, 2003 , and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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Two recent developments have spawned interest in the processes by which T. pallidum acquires trace metals. One is the availability of the genomic sequence of the spirochete (9), which reveals that many genes encoding well characterized iron-containing proteins (e.g. cytochromes, superoxide dismutase, and tricarboxylic acid metalloenzymes) are lacking. The finding that T. pallidum expresses at least two experimentally confirmed iron-binding proteins, superoxide reductase (neelaredoxin) and rubredoxin, (10, 11, 12), argues that the bacterium does require iron. The second impetus to study metal utilization by T. pallidum is provided by the discovery of the six gene tro (transport-related operon) (Fig. 1) (13). The first gene of the operon, troA, encodes the solute binding protein (SBP)1 component of an ATP-binding cassette transporter (13, 14, 15) belonging to a newly described cluster (cluster nine or C9) of transition metal permeases (5). In addition to encoding an ATPase (TroB), two cytoplasmic membrane permeases (TroC and TroD), and a DtxR-like metalloregulator (TroR), the operon also encodes the glycolytic enzyme phosphoglyceromutase (Gpm) (13). Because the T. pallidum Gpm has no requirement for metals (16), the physiological benefit of transcriptionally linking the spirochete's sole copy of this essential enzyme to the Tro transporter genes is unclear.
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The identity of the metal ligand(s) for the Tro transporter is a subject of controversy (5, 6). Our analysis of TroA purified from Escherichia coli showed that Zn2+ was present in a 1:1 molar ratio, whereas other metals were present in trace or undetectable quantities (17). On the assumption that the SBP determines specificity of the transporter (18, 19), we proposed that the permease mediates Zn2+ influx (15). On the other hand, Posey et al. (20), using recombinant TroR for mobility shift DNA binding and DNase I footprint assays, reported that only Mn2+ could promote the binding of TroR to the tro promoter, leading them and others to conclude that the permease transports Mn2+ (5, 6, 7, 21, 22, 23, 24). Conclusions from both studies are, however, essentially extrapolations from in vitro data. There are precedents for metalloproteins binding Zn2+ rather than their primary substrate when heterologously expressed in E. coli (25, 26). Similarly, in vitro and in vivo studies of metalloregulators have not always yielded identical profiles of metal-dependent DNA binding (3, 27, 28). We therefore devised an experimental strategy that would permit a more direct assessment of Tro permease function and regulation. Here we examined (i) metal binding by apo-TroA; (ii) the functional capacity of the transporter when expressed in surrogate hosts; (iii) the metal specificity of TroR-mediated transporter repression in E. coli; and (iv) the effect of TroR-mediated repression on Gpm expression. Our findings indicate that the permease transports Zn2+, Mn2+, and possibly iron but that Zn2+ is likely to be the primary activator of TroR under physiological conditions. We also found that expression of Gpm was not tightly linked to transcription of the tro transporter genes. This revised understanding of metal uptake and regulation by the Tro permease provides a foundation for further analysis of its role in treponemal metal metabolism and, by extension, syphilis pathogenesis.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and MediaAnimal protocols described in this work were approved by the University of Connecticut Health Center Animal Care Committee under the auspices of Animal Welfare Assurance A3471-01. T. pallidum subspecies pallidum (Nichols-Farmington strain) was propagated by intratesticular inoculation of adult New Zealand White rabbits and harvested as described previously (29). Unless otherwise indicated, E. coli strain TOP10F' (Invitrogen) was the recipient strain for recombinant constructs and was grown in LB medium with appropriate antibiotic supplementation. For experiments requiring media with reduced metal contents, either M9 medium (Becton Dickinson, Sparks, MD) or a specially formulated minimal medium (Hazlett-Radolf minimal medium (HRMM)) was utilized for the growth of E. coli. M9 medium contains 1x M9 salts, 0.2% casamino acids, and 0.4% glucose. HRMM contains 0.4% glucose, 0.2% casamino acids, 0.2% ATP, 0.2% ammonium sulfate, 1 mM magnesium sulfate, 9 µM FeSO4, and 1.67% PIPES, pH 6.5. Yersinia pestis strains were grown in the defined medium PMH2 (30).
Generation of TroA apo Proteins and Determination of Dissociation ConstantsThe plasmid (pHTa:TroA), which encodes an isopropyl--D-thiogalactopyranoside-inducible polyhistidine-tagged TroA protein has been described previously (17). This plasmid also served as the template for site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) with primers TroA H199E 5' and TroA H199E 3', which changed the H codon (CAT) of residue 199 to a E codon (GAA), generating pHTa:TroA(H199E). Sequence analysis confirmed that only the desired mutation had been introduced. Expression and purification of His-TroA and His-TroA(H199E) were performed as previously described (17). His tags were removed by digestion with TEV protease (Invitrogen) followed by a second round of nickel-nitrilotriacetic acid chromatography, which captures free His tag, uncleaved fusion protein, and the His-tagged TEV protease. To generate apo-proteins suitable for metal binding studies, endogenously bound metals were removed from the purified proteins by overnight dialysis against 0.1 M sodium acetate buffer (pH 4.6) containing 10 mM 1,10 o-phenanthroline (Sigma) (31). Restoration of metal binding capacity was achieved by sequential dialysis against 0.1 M sodium acetate (pH 4.6) and 0.1 M sodium acetate (pH 6.5), respectively. The apo-proteins were adjusted to between 25 and 35 µM and aliquoted as 1.1-ml portions into 8000 molecular weight cut-off dialysis bags (SPECTRA/POR, Spectrum Medical Industries, Inc, Los Angeles, CA). Thereafter, all protein manipulations employed acid-washed plastic containers and buffers treated with Chelex-100 (Bio-Rad). The dialysis bags containing apo-protein were transferred to 50-ml conical tubes containing 45 ml of 0.1 M sodium acetate (pH 6.5) with Mn2+ or Zn2+ acetate (0100 µM) and dialyzed overnight. Protein and dialysate samples were analyzed for Zn2+ by inductively coupled plasma emission spectroscopy or for Mn2+ by graphite furnace atomic absorption spectroscopy at the Mayo Clinic (Rochester, MN). The protein concentrations were confirmed by amino acid analysis. Metal-toproteins ratios (
) were plotted against the free metal (Me2+) concentrations (µM) and fitted to sigmoidal curves using the equation
= ((N[Me2+])/(Kd + [Me2+])) + 0.3, where N (Me2+-binding sites) = 1 (17). The correction factor of 0.3 was included to better fit the sigmoidal curves to the data; Kd values obtained without this correction did not differ significantly from those presented in this work.
Y. pestis znu and mntH Mutants and Complementation PlasmidsTo generate the Y. pestis znu knockout plasmid pSucZnu3.5, we recovered the wild type znu locus from a Sau3AI genomic library (32) using a znuA probe and subcloned a 9.4-kb BglII/Asp718 fragment into pWSK129, generating pZnu2, which served both as a complementation plasmid as well as an intermediate in the construction of pSucZnu3.5. pZnu2 was digested with EcoRI and ApaI, treated with Klenow, and self-ligated, generating pZnu3, which harbors the znu locus with a 1265-bp deletion covering approximately half of znuB, all of znuC, and the bi-directional znuCB/znuA promoter. The 7.5-kb znu XbaI fragment from pZnu3 was ligated into the suicide vector pSuc1 (33), generating pSucZnu3.5, which was electroporated into Y. pestis KIM6+ (34). Clones arising from double cross-over events were selected by successively screening for ampicillin and sucrose resistance. A
znu isolate (KIM62077+) whose genotype was confirmed by Southern blot was used in this work. To generate a mntH knockout plasmid, the 5' and 3' regions of the mntH gene were PCR-amplified from Y. pestis DNA using primer pairs YpMntH5'-SalI/YpMntH5'-SmaIR and YpMntH3'-SmaIF/YpMntH3'-SpeI, digested with SalI/SmaI and SmaI/SpeI, respectively, and cloned in tandem into SalI/SpeI-digested suicide vector pKNG101 (35), creating p
MntH. The resulting insert consists of the mntH gene, lacking 600 bp of coding sequence, flanked by 300 and 80 bp at the 5' and 3' ends, respectively. Plasmid p
MntH was used to transform Y. pestis KIM6 and the isogenic yfe mutant Y. pestis KIM62031.1 (34). Clones arising from double cross-over events were selected by successively screening for streptomycin and sucrose resistance. Plasmid pMntH-Op was generated by PCR amplifying the mntH gene with its native promoter from Y. pestis KIM6 DNA using primer pair YpMntH5'-SalI/YpMntH3'-XbaI; the resulting fragment was cloned into pWSK129 (36) in the opposite orientation of the vector-encoded lacZ gene.
Construction of Permease PlasmidsThe promoter-less troABCD genes were PCR-amplified from T. pallidum chromosomal DNA using the proofreading polymerase Ex-Taq (Panvera, Madison, WI), primers NoPro TroA 5' EcoRI and TroD 3' EcoRI and cloned into TOPO pCR2.1 (Invitrogen). Following sequence confirmation, the insert was moved into the EcoRI site of pBAD18 (37), and a plasmid harboring the treponemal genes in the same orientation as the pBAD promoter (pTroA-D) was identified. pTroA(H199E)-D was generated from pTroA-D as described above and confirmed by sequence analysis. The primer pairs used to PCR amplify mntH, yfeABCD, scaCBA, and adcCBA from E. coli K-12, Y. pestis KIM6, S. gordonii DL1, and Streptococcus pneumoniae TIGR 4 genomic DNAs, respectively, for cloning into pBAD18, are presented in the supplemental table, as are the resulting plasmids. The divergently transcribed znu genes of E. coli K12 were amplified using primers Ec ZnuA 3' KpnI and Ec ZnuB 3' XbaI, digested, and directly cloned into KpnI/XbaI-digested pWSK129, generating pEcZnu.
To clone the complete tro operon, a DNA fragment extending from 106 bp upstream of the troA start codon to the gpm stop codon was PCR-amplified from T. pallidum DNA using Ex-Taq polymerase (Panvera) and primers TroPro 5' EcoRI and Gpm 3' SacII. The amplicon was digested with EcoRI and SacII and cloned into the EcoRI/SacII sites of plasmids pWSK129 and pWSK29 (36) such that the tro operon was oriented oppositely to that of the vector-encoded lacZ promoter, creating pPro-Gpm and pPro-Gpm Amp, respectively. Transformants were plated on M9 agar containing 50 µg/ml kanamycin (pPro-Gpm) or 100 µg/ml ampicillin (pPro-GpmAmp), 0.1 mM isopropyl--D-thiogalactopyranoside, 20 µg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal) and grown overnight at 30 °C. The cloned insert was sequenced using primers listed in the supplemental materials. A variant, pNoPro-Gpm, which lacks the tro promoter, was similarly generated using primers NoPro TroA 5' EcoRI and Gpm 3' SacII.
Complementation of Y. pestis and E. coli Transporter Mutants Complementation analysis of Y. pestis strains was conducted in PMH2 broth (30) and chelator gradient plates as described previously (34); all manipulations were conducted in acid-washed glassware. For experiments with broth, Y. pestis strains cultivated on tryptose blood agar slants were suspended in PMH2 medium to an A620 of 0.1 and grown overnight. The following morning they were diluted to an A620 of 0.1 in fresh PMH2 containing the required antibiotics with or without 0.2% arabinose. Growth at 30 °C in a shaking water bath was spectrophotometrically monitored. Arabinose-containing chelator gradient plates containing 01 µM TPEN or 05 µM conalbumin were inoculated and incubated for 3 days at 30 °C, after which growth into the metaldepleted zones was measured. Complementation analysis of a Zn2+ uptake-deficient E. coli strain was conducted in HRMM broth. Overnight HRMM cultures of E. coli GR354 (znu/
zupT) (38) bearing either control or transporter plasmids were diluted into fresh medium containing appropriate antibiotics, 0 or 0.2% arabinose, and various concentrations of either the Zn2+ chelator TPEN or ZnCl2. The cultures were incubated at 37 °C with vigorous aeration, and growth was spectrophotometrically monitored for 12 h.
65Zn and 54Mn Transport AssaysWith minor variation, transport assays were conducted as previously described (39, 40). E. coli strain MM2115 (mntH) (40) and E. coli strain GR362, which lacks all known Zn2+ uptake and efflux transporters (38), were transformed with pBAD18 or pTroA-D. Overnight cultures were diluted into 0.4% fresh Chelex 100-treated tryptone, 0.25% NaCl broth, grown for 3 h, induced with 10 mM arabinose for 1530 min, and added to isotope-containing broth (100 nM 65Zn2+ (Perkin-Elmer, Boston, MA) or 50 nM 54Mn2+ (Perkin-Elmer)). Culture samples, taken at intervals from 30 s to 5 min, were collected on 0.45-µm filters, rinsed twice with 5 ml of ice-cold medium, and processed for scintillation counting as described previously (39). Channels 401945 were used to detect photons derived from
radiation of 54Mn, whereas all channels were used to detect photons derived from 65Zn radiation.
Metal Sensitivity AssaysOvernight cultures were diluted 1:100 into fresh HRMM and plated on HRMM, 1% agarose plates containing 0.1 mg/ml ampicillin and either 0 or 0.2% L-arabinose. Sterile 6-mm paper disks (Becton Dickinson) were placed in the center of the air-dried plates and impregnated with 10 µl of 0.5 M ZnCl2, 1 M MnCl2, or 1 M FeSO4. Following overnight incubation at 37 °C, the zones of inhibition were measured in blinded fashion. The measurements from three to seven independent experiments were used to calculate the mean zones of inhibition. The differences in mean zone sizes were compared using the two-tailed Student's t test; p values ≤ 0.01 were considered significant. Although Fe2+ (FeSO4) was used in these assays, the lability of this ion under aerobic conditions likely allowed for oxidation to the Fe3+ ion.
Immunologic Reagents and Immunoblot AnalysisPolyclonal rat antiserum directed against TroA has been previously described (14). Antiserum was adsorbed with E. coli (pWSK129) lysates that had been electrophoresed on a 12.5% SDS-polyacrylamide gel and electrotransferred to nitrocellulose. A mouse monoclonal antibody directed against E. coli 70 was purchased from NeoClone Biotechnology (Madison, WI). To produce antisera directed against Gpm and TroR, the T. pallidum gpm and troR genes were amplified using primer pairs Gpm 5' NcoI/Gpm 3' EcoRI and TroR 5' NcoI/TroR EcoRI, digested with NcoI and EcoRI, and cloned into the expression vector pProExHTa (Invitrogen). The resulting plasmids (pHTa:Gpm and pHTa:TroR) encode isopropyl-
-D-thiogalactopyranoside-inducible polyhistidine-tagged proteins, which were purified from induced E. coli cell lysates by nickel-nitrilotriacetic acid chromatography and used to immunize 4-week-old male Sprague-Dawley rats as previously described (29). T. pallidum, E. coli, and Y. pestis lysates were resolved on 12.5% SDS-polyacrylamide gels and electrotransferred to nitrocellulose. The blots were incubated with 1:1000 to 1:5000 dilutions of the appropriate antiserum or anti-
70 monoclonal antibody and then probed with a 1:30,000 dilution of horseradish peroxidase-conjugated goat anti-rat (or anti-mouse) (Southern Biotechnology Associates, Birmingham, AL). The immunoblots were developed using the SuperSignal Femto chemiluminescence substrate (Pierce) followed by exposure to film (Amersham Biosciences). In the metal repression studies, the densitometric signals of immunoreactive bands were measured by an Alpha Innotech ChemiImager 4400 (Alpha Innotech Corporation, San Leandro, CA).
Detection of tro Transcripts by RT-PCRRNAs were isolated using Trizol (Invitrogen) and treated with RQ1 RNase-free DNase (Promega, Madison, WI) until the RNAs were free of contaminating DNA. RT-PCR reactions were performed with the Titan One-Step RT-PCR kit (Roche Applied Science) as previously described (10) using the RT-PCR primers listed in the supplemental table. For each primer set, three reactions were performed: (i) RT-PCR with RNA template, (ii) PCR with RNA template, and (iii) PCR with control template DNA. In reactions without RT, the Expand High Fidelity DNA polymerase (Roche Applied Science) was substituted for the RT/DNA polymerase enzyme mixture. Following the RT reaction (50 °C for 30 min), PCR was performed using the following parameters: 98 °C for 2 min followed by 30 cycles of 94 °C for 5 s; 63 °C for 10 s; and 68 °C for 45 s followed by a single terminal extension at 68 °C for 3 min. Densitometry was performed as above.
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RESULTS |
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X-ray crystal structures have been solved for two C9 SBPs, TroA of T. pallidum and PsaA of Streptococcus pneumoniae (15, 42). In TroA, Zn2+ is coordinated by residues His68, His133, His199, and Asp279; the corresponding residues in PsaA are His67, His139, Glu205, and Asp280. Alignment of amino acid sequences of C9 SBPs with experimentally confirmed metal ligands (see supplemental Fig. S1) showed that His68/67 and His133/139 are strictly conserved, residue 279/280 is either Asp or Glu, whereas residue 199/205 is either His or Glu. C9 SBPs with His at the 199/205 position tend to bind Zn2+, whereas those with Glu at this position predominantly bind Mn2+ or iron, suggesting that the identity of residue 199/205 could be a critical determinant of ligand specificity (15). We therefore mutated His199 to Glu in TroA and measured the affinities of the mutant apo-protein for Zn2+ and Mn2+. The mutation reduced the affinity of apo-TroA for Zn2+ by no more than 50% (Kd = 2.64 ± 0.8 µM) but, surprisingly, abrogated its capacity to bind Mn2+ (Fig. 2B).
Functional Analysis of the Heterologously Expressed Tro TransporterWe next asked whether the Tro permease could mediate uptake of Zn2+ and/or Mn2+. Because T. pallidum cannot be genetically manipulated, functional studies must be performed in a surrogate host. We therefore cloned the promoter-less troABCD genes downstream of the arabinose-inducible promoter of pBAD18 (37), creating pTroA-D (supplemental table). Immunoblot and RT-PCR analyses demonstrated that the four permease genes were expressed in E. coli in an arabinose-dependent manner (data not shown). Next, we sought to determine whether pTroA-D could complement growth defects conferred by metal transporter mutations. We initially chose to work with Y. pestis, which has been extensively utilized in previous metal transporter studies (34). In addition to a strain harboring a mutation in the Fe3+/Mn2+ transporter, Yfe, (34), we used allelic replacement to generate mutations in the Znu (Zn2+) (43) and MntH (Mn2+) (40, 44) transporters. The znu mutant and yfe/mntH double mutant displayed growth defects in metal-restricted broth medium that were corrected by the addition of Zn2+ and Mn2+, respectively; as reported previously for S. typhimurium and E. coli (44), mutation of mntH alone caused no discernible growth defect (data not shown). Expression of the Tro transporter, confirmed by immunoblot analysis for TroA (data not shown), failed to improve the growth of either the znu or yfe/mntH mutants, whereas expression of the homologous Znu or MntH permeases from plasmids pZnu2 and pMntH-Op (see the supplemental table) restored wild type growth (Fig. 3A). We also used an assay format in which bacterial strains are streaked onto solid medium containing increasing concentrations of iron or Zn2+ chelators (34). Growth of the yfe and znu mutants into the metal-depleted zone was enhanced by the plasmid-borne Y. pestis Yfe (pYfe3) (34) and Znu permeases but not by the Tro transporter (Fig. 3B). The lack of a manganese-specific chelator precluded a similar examination of Mn2+ uptake.
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Finally, we utilized a Zn2+ uptake-deficient strain of E. coli (38) to assess Zn2+-dependent complementation by the Tro permease. As with the znu mutant Y. pestis, growth of the znu/
zupT double mutant strain of E. coli in Zn2+-restricted broth medium was corrected by either the addition of Zn2+ or expression of the native Znu transporter (pEc Znu; supplemental table) but not by expression of the Tro genes (pTroA-D) (data not shown). Because E. coli Mn2+ uptake mutants have no discernable growth defect (39, 40), a similar analysis of Mn2+-dependent complementation was not performed.
To assess Tro-mediated metal transport more directly, we sought to detect uptake of isotopically labeled Zn2+ and Mn2+ in E. coli. Because potentially poor heterologous function could hinder detection of uptake, these assays were performed in strains deficient in metal transporters that were grown in medium depleted of metal to reduce internal stores. In a strain lacking all known Zn2+ uptake and efflux systems (38), the Tro permease reproducibly promoted modest uptake of 65Zn2+ (3-fold above background), confirming the functionality of the transporter; in parallel assays using a mntH mutant strain (40), we detected a similar degree of 54Mn2+ uptake by the Tro transporter (data not shown). Because complementation of a znu mutant E. coli by the homologous zinc transporter has been reported to be accompanied by an approximate 20-fold increase in 65Zn2+uptake (43), it seems likely that the 3-fold increase of 65Zn2+ uptake by the heterologously expressed Tro permease was not sufficient for complementation. Poor permease expression seemed a likely explanation for the low levels of metal uptake; indeed, immunoblot analysis revealed that TroA levels were
10- and 20-fold lower on a per cell basis in arabinose-induced E. coli (pTroA-D) and Y. pestis (pTroA-D), respectively, than in T. pallidum (data not shown).
Given the above evidence of poor heterologous function by the Tro transporter, we hypothesized that metal transport activity might be more readily discernible by assaying for enhanced growth inhibition during incubation with high concentrations of exogenous metals (22, 38, 40, 44, 45, 46). For these experiments, we chose to employ disk diffusion assays in which enhanced metal uptake is manifested within a lawn of bacteria as an increased zone of inhibition surrounding a metal-containing disk (44, 46). As controls, several previously characterized metal transporters, namely, Adc of S. pneumoniae (Zn2+) (47), MntH of E. coli (Mn2+) (40, 44), Sca of S. gordonii (Mn2+) (48), and Yfe (Fe3+/Mn2+) were cloned into pBAD18. The combined results for these experiments are presented in Fig. 4. Expression of the Tro and Adc permeases significantly enhanced Zn2+-dependent growth inhibition as compared with the pBAD18 vector or constructs expressing MntH or Yfe, neither of which appear to mediate 65Zn2+ uptake. Expression of a Tro transporter harboring the H199E TroA mutation also resulted in increased Zn2+-dependent growth inhibition, suggesting that the mutant SBP retained a near wild type conformation. Expression of Sca also enhanced Zn2+-dependent growth inhibition, consistent with Zn2+ inhibition of Sca-mediated Mn2+ uptake (48). Consistent with our binding data, Mn2+-dependent growth inhibition of transformants expressing the wild type, but not the H199E mutant, Tro transporter was comparable with that displayed by cells expressing MntH, Yfe, Sca, and Adc. Similarly, expression of the wild type, but not the mutant Tro permease, resulted in enhanced growth inhibition by iron, as did expression of MntH, Yfe, Sca, and Adc. These findings are in accord with the ability of the MntH and Yfe permeases to transport iron (34, 40, 44) and the ability of Fe3+ to partially rescue Adc mutants (47).
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Zn2+ Represses the tro Operon in Vivo at Much Lower Concentrations than Does Mn2+Gherardini and co-workers (20) reported that TroR is activated exclusively by Mn2+ in vitro. They also showed that expression of TroR could repress a tro promoter-lacZ reporter in E. coli, although they were unable to demonstrate metal dependence of TroR function in vivo. The Zn2+ binding and transport data reported above prompted us to re-examine the role of Zn2+ in TroR activation. We again used E. coli as a surrogate host for regulation studies. However, rather than expressing troR from an inducible strong promoter (20), we cloned the entire tro operon with its promoter into the very low copy number plasmid pWSK129 (36) generating pPro-Gpm. We reasoned that this construct would better mimic native TroR-tro promoter interactions and, at the same time, allow examination of gpm expression when the tro promoter is repressed.
Immunoblot and RT-PCR analyses confirmed that all of the tro genes were expressed by E. coli pPro-Gpm transformants (data not shown). Interestingly, unlike TroA and Gpm, which are readily detectable by immunoblot analysis, TroR could not be detected in either E. coli(pPro-Gpm) or T. pallidum by enhanced chemiluminescence (data not shown). Because transcriptional repressor proteins are typically found in very low abundance (23), these results supported the use of E. coli as a T. pallidum surrogate. As shown in Fig. 5A, Zn2+-mediated repression of TroA was apparent at 50 µM, whereas much higher concentrations of Mn2+ (> 2 mM) were required to effect a comparable repression. Iron concentrations up to 500 µM had no discernable effect on TroA expression (data not shown). By plotting the exogenous metal concentrations against the intensity of the TroA bands, we calculated mean EC50 values for Zn2+ and Mn2+ repression of TroA of 71 µM and 2.66 mM, respectively (data not shown). To increase the sensitivity of these assays, particularly for Mn2+, we next performed repression studies using strains of E. coli that had been engineered to have elevated levels of intracellular Zn2+ and Mn2+. In an E. coli strain defective for Zn2+ efflux (zntA/
zitB) (38), Zn2+-mediated repression of TroA was apparent at 10 µM and absolute at 50 µM Zn2+ (supplemental Fig. S2). In contrast, Mn2+-dependent repression in a MntH-overexpressing stain of E. coli was apparent at 50 µM Mn2+ and only 70% complete at 250 µM Mn2+ (supplemental Fig. S2), further suggesting that TroR is primarily Zn2+-responsive. Interestingly, repression of TroA was not accompanied by a commensurate decrease in Gpm (Fig. 5A). Densitometric analysis revealed that Gpm decreased by
50% under conditions that resulted in a greater than 90% decrease of TroA (data not shown). Separate immunoblot experiments using antibodies directed against the constitutively expressed
70 protein were used as loading controls (data not shown). To confirm that these protein data were indicative of changes in tro transcript levels, RT-PCR analysis was performed using primers specific for troA, the troR-gpm intergenic region, and the gpm coding region. Consistent with the above results, growth of E. coli (pPro-Gpm) in the presence of exogenous Zn2+ resulted in marked reductions of troA and troR-gpm transcripts (84 and 88% decrements, respectively; Fig. 5B). In contrast, gpm mRNA levels were reduced by 55%, whereas those of the vector-encoded kanamycin resistance gene were effectively unchanged (1.5% reduction). These results point to the existence of an autonomous gpm promoter. To confirm this, we preformed immunoblot analysis on E. coli bearing a variant of plasmid pPro-Gpm, dubbed pNoPro-Gpm, which lacks the tro promoter. E. coli (pNoPro-Gpm) contained readily detectable levels of Gpm, whereas TroA, as expected, was not detected (Fig. 5C). By performing a similar analysis with constructs lacking various amounts of sequence upstream of gpm, the autonomous gpm promoter activity was localized to 541 bp of DNA directly upstream of the gpm start codon (data not shown).
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DISCUSSION |
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As a platform for understanding the contribution of the Tro permease to transition metal homeostasis in T. pallidum, we surveyed the genome of the spirochete (9) for orthologs of known metal-dependent enzymes and metal transporters (Table I). As noted, T. pallidum lacks many iron-dependent proteins found in other bacteria; accordingly, the spirochete also lacks siderophore biosynthetic genes and dedicated iron uptake systems. Because Zn2+ is second in abundance only to iron within other bacteria, as well as within the obligate human host of the spirochete (41), the small number of iron metalloproteins in T. pallidum suggests that Zn2+ is likely the predominant transition metal in the bacterium. Indeed, a number of abundantly expressed putative Zn2+ metalloproteins participate in a variety of crucial cellular processes involving nucleic acid and protein synthesis/degradation, remodeling of peptidoglycan, and glycolysis (49, 50). Although the intimation that T. pallidum requires significant amounts of Mn2+ (20) has become widely accepted in the literature (5, 6, 7, 21, 22, 23, 24), our survey of the genome lends little support to this notion. We were able to identify only four orthologs of known Mn2+-dependent enzymes, whereas a number of well characterized Mn2+-metalloproteins, such as PrpA and PrpB protein phosphatases, Pkn2/PknA type kinases, family II inorganic pyrophosphatase, sphingomylinase, phosphatidylserine synthase, Mn2+-fructose-1,6-bisphosphate phosphatase (GlpX), L-fucose isomerase, cellobiose-6-phosphate hydrolase, arginase/agmatinase, pyruvate carboxylase, glutamine synthetase, Mn2+-RNaseH, SpoT, superoxide dismutase, catalase, and Mn2+-peroxidase (7) are notably absent. Unlike the phylogenetically distinct Mn2+-dependent enzymes of several Bacillus species, the treponemal Gpm has recently been demonstrated to have no requirement for Mn2+ (16). Significantly, T. pallidum does not encode orthologs of either of the Mn2+ transporters MntH or the P-type ATPase, MntA.
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The equal affinity of TroA for both Zn2+ and Mn2+ (Kd = 1 µM) suggests that the Tro permease could transport multiple metals. Notably, these affinities are similar to those of several other C9 permeases measured in whole cell radioisotope uptake studies (39, 48, 51). To extend these in vitro binding studies, we heterologously expressed the Tro transporter in the surrogate hosts Y. pestis and E. coli. Despite the inability of the Tro permease to complement the growth defects of Y. pestis and E. coli transporter mutants, likely because of poor heterologous expression and/or function, we were clearly able to discern transport activity in E. coli by assaying for 65Zn2+ and 54Mn2+ uptake and increased growth inhibition by Zn2+, Mn2+, and iron. Thus, the in vitro binding experiments and the in vivo functional analyses yield internally consistent data indicating that the Tro permease plays an important role in fulfilling the requirements of the spirochete for Zn2+, Mn2+, and possibly iron. Interestingly, among the C9 permeases, the capacity for transporting multiple metals appears to be a more common feature than is generally appreciated. In the only other analysis of multiple metal binding by a purified C9 SBP, the streptococcal MtsA protein was found to bind Zn2+, Fe3+, and Cu2+, whereas the intact permease transported both Fe3+ and Zn2+ (52). The Y. pestis Yfe and S. parasanguis Fim permeases transported both Fe3+ and Mn2+ (34, 53). Mutation of the S. pneumoniae Adc permease imparts a growth defect that is corrected primarily by Zn2+, and to lesser degrees by Cu2+ and Fe2+ (47). Finally, Zn2+ is a potent competitive inhibitor of Mn2+ uptake by the Sca, Sit, and Mnt ATP-binding cassette permeases (39, 48, 51). Because Zn2+ and iron can be bound with coordination numbers of either 4, 5, or 6, whereas Mn2+ can be coordinated through five or six binding interactions (54, 55), it is not surprising that many of the C9 SBPs, all of which have residues consistent with either a pentavalent or hexacoordinate ligand binding site, would be able to bind multiple metals.
Examination of the local architectures of the ligand-binding sites of TroA and PsaA revealed by the crystal structures (15, 42) enables us to explain the counter-intuitive observation that TroA (H199E) failed to bind manganese. Among the C9 SBPs containing His199, this amino acid is flanked on one or both sides by a bulky (Phe or Gln) or a negatively charged (Asp) residue (supplemental Fig. S1). In contrast, the flanking residues of the Glu199 subgroup of C9 SBPs are small (Gly, Ser, or Cys), thereby affording the carboxylate moiety the mobility to form bidentate interactions with the bound metal. The residues flanking His199 likely constrain and orient the N-H tautomer of His199, thereby promoting a favorable interaction of the N
2 atom with the coordinated metal (54). We hypothesize that by placing a Glu in this constraining environment, only a single carboxylate oxygen can interact with the bound metal. Moreover, the spatial proximity of residue 199 to His133 (15) would allow the remaining carboxylate oxygen of Glu199 to interact with the N
2 atom of His133, thereby preventing His133 from binding the coordinated metal. If so, the H199E mutation would actually impart a tetrahedral coordination geometry that is not conducive for Mn2+ binding but is frequently utilized in Zn2+-binding proteins (54, 55). The observation that TroA(H199E) binds Zn2+ but not Mn2+ suggests that the wild type TroA sequence has been maintained through a selective pressure favoring multiple metal binding capacity.
By cloning the entire tro operon into a very low copy number plasmid, we were able to observe TroR-mediated repression of the tro genes in vivo in response to physiologically relevant concentrations of Zn2+, whereas supraphysiological (>2 mM) concentrations of Mn2+ were required to effect a similar down-regulation. The concentrations of Zn2+ required to activate TroR (70 µM) are close to those (
10 µM) that activate the E. coli Zn2+-dependent transcriptional repressor, Zur (23). Whether the slightly higher levels of Zn2+ required to activate TroR represents a true "evolutionary tuning" of TroR to a higher intracellular Zn2+ content or merely an effect of the copy number (approximately seven) (36) of the plasmid pPro-Gpm remains to be seen. In stark contrast, the levels of Mn2+ required to activate TroR (
2 mM) are 2000-fold greater than those (
10 µM) that activate the E. coli Mn2+-dependent repressor MntR (56). Thus, we conclude that Zn2+ is the native co-repressor of the tro genes, in contrast to Posey et al. (20), who reported that TroR purified from inclusion bodies and renatured was exclusively activated by Mn2+ in vitro. The observation that both the E. coli and Bacillus subtilus Zur proteins, when purified from inclusion bodies and renatured, are activated by Zn2+ and Mn2+ in vitro, whereas neither of these Zn2+-dependent repressors responds to Mn2+ in vivo (27, 28) lends a cautionary note to results obtained with denatured/renatured repressor proteins used for in vitro assays. Based on the premise that TroR is activated by Mn2+ (20), it was suggested that repression of the tro operon was likely to occur during infection of the central nervous system. However, as a sexually transmitted pathogen, T. pallidum is frequently exposed to semen, which contains very high levels of Zn2+ (
1 to 2 mM) (57, 58, 59). Based on our findings, we suggest that Zn2+-mediated repression during exposure to semen is a more plausible scenario. As spirochetes disseminate from the site of inoculation and encounter much lower levels of Zn2+, such as those present in serum and cerebrospinal fluid (
15 and
2 µM, respectively) (60, 61), repression of the Tro permease would be alleviated.
The detection of troR-gpm co-transcripts by RT-PCR prompted the notion that Gpm expression was exclusively dependent upon transcription from the tro promoter (13). Gherardini and co-workers (16, 20) further suggested that TroR-mediated repression of Gpm within the central nervous system would decrease treponemal metabolism and thereby contribute to the latency of syphilitic infection. Because T. pallidum can only generate ATP by glycolysis, such a genetic arrangement seems inherently problematic because TroR-mediated repression of Gpm would hinder generation of ATP and thus the ability of the bacterium to escape a potentially metal-toxic environment. Consistent with the notion that glycolysis must proceed during TroR-mediated repression, we observed that Gpm expression continues when activity from the tro promoter is abolished. We postulate, therefore, that the net expression of gpm cumulatively results from transcriptional read-through from the tro promoter as well as from an autonomous gpm-specific promoter. Precedent for this type of genetic arrangement is provided by the streptococcal C9 permeases Sca, Fim, Adc, and Psa, all of which contain a downstream gene unassociated with either the transporter or its repressor that is expressed from both the permease promoter and from an autonomous promoter (45, 47, 62, 63). This observation, however, does not explain the physiological relevance of this genetic arrangement. We can only speculate that the level of Gpm activity that occurs during repression of the Tro permease is sufficient to meet the metabolic needs of the spirochete. Regardless of its transcriptional control, the prevalence of an unrelated protein in multiple C9 permease operons suggests that its presence is not coincidental, implying some currently unknown physiological advantage.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI-10573 (to K. R. O. H.), AI-26756 (to J. D. R.), AI25098 (to R. D. P.), and GM61748 (to M. E. M.) and by funds from the Mayo Foundation (to F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains a supplemental table and supplemental figures.
Present address: Div. of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521.
To whom correspondence should be addressed: Center for Microbial Pathogenesis, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3710. Tel.: 860-679-8391; Fax: 860-679-8130; E-mail: KHazlett{at}up.uchc.edu.
1 The abbreviations used are: SBP, solute binding protein; HRMM, Hazlett-Radolf minimal medium; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); RT, reverse transcription; TPEN, N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine.
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