Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands1
Author for correspondence: Juke S. Lolkema. Tel: +31 50 3632155. Fax: +31 50 3632154. e-mail: j.s.lolkema{at}biol.rug.nl
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ABSTRACT |
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Keywords: TCA cycle intermediate, promoter fusion, divalent metal ioncitrate complex, membrane vesicles, exchange
Abbreviations: FCCP, carbonylcyanide p-trifluormethoxy-phenylhydrazone; MSMYE, minimal salts medium/0·05% yeast extract; RSO, right-side-out; TCA, tricarboxylic acid
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INTRODUCTION |
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Older studies have reported the ability of the TCA cycle intermediates cis-aconitate and isocitrate to induce and competitively inhibit the uptake of citrate in wild-type cells of B. subtilis, suggesting the presence of a specific transport system in this organism (McKillen et al., 1972 ). More-recent studies have identified a number of transporters for citrate in B. subtilis. CimH, mentioned above, catalyses symport of citrate and protons (Krom et al., 2001
). The secondary transporters CitM and CitH also transport citrate in symport with protons, but only in the presence of divalent metal ions (Boorsma et al., 1996
; Krom et al., 2000
). Uptake studies of the two transporters separately expressed in E. coli cells confirmed that the metal ioncitrate complex is the transported species and that the metal-ion specificity of the two transporters is complementary, i.e. CitM transports citrate in complex with Mg2+, Mn2+, Ni2+, Co2+ or Zn2+, and CitH recognizes citrate in complex with Ca2+, Ba2+ or Sr2+ (Krom et al., 2000
). Expression of citM is strictly regulated: gene activation depends on the action of the two-component regulatory pair CitSCitT, which senses the presence of citrate in the medium (Yamamoto et al., 2000
). Furthermore, gene expression is subject to catabolite repression (Warner et al., 2000
). The strict regulation of citM expression by medium components makes it likely that CitM is the main uptake system during growth of B. subtilis on citrate as the sole carbon source.
Here, we have studied the involvement of the Mg2+citrate transporter CitM in the growth of B. subtilis on TCA cycle intermediates. It follows that CitM is both necessary and sufficient for growth of the organism on citrate as the sole carbon source. In addition, it is shown that CitM supports the growth of B. subtilis on isocitrate which, like citrate, is shown to be an inducer of citM expression. Subsequently, transport studies using membrane vesicles and resting cells demonstrated that CitM transports the complex of isocitrate and divalent metal ions.
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METHODS |
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E. coli TOP10 was transformed with plasmid pWSKCitM, which contains the gene encoding the Mg2+citrate transporter CitM under the control of the lac promoter (Krom et al., 2000 ). Recombinant cells were grown in LB medium supplemented with 100 µg carbenicillin ml-1 and were induced with 0·1 mM IPTG at an OD660 value of 0·2, after which the cells were allowed to grow for an additional 2 h.
Preparation of membrane vesicles.
E. coli TOP10 cells expressing CitM were harvested at an OD660 value of 0·81·0 and right-side-out (RSO) membrane vesicles were prepared by the osmotic shock lysis procedure described by Kaback (1983) . The vesicles were resuspended in 50 mM potassium PIPES (pH 6·5), aliquoted in 0·5 ml samples, rapidly frozen in liquid nitrogen and, subsequently, stored at -80 °C. The protein concentration was determined with the Bio-Rad DC Protein Assay Kit.
Transport assays.
(i) 63Ni2+ uptake in whole cells.
Cells of B. subtilis were harvested by centrifugation, washed once and resuspended in 50 mM potassium PIPES (pH 6·5) and stored on ice until use. Transport activity was determined by the rapid-filtration method (Lolkema et al., 1994 ). Briefly, 98 µl of a cell suspension with a final OD660 value of 1 was incubated for 5 min at 30 °C. At time-point zero, 2 µl of a mixture of 63Ni2+ [12·66 mCi (mg Ni)-1, 468 MBq (mg Ni)-1; Amersham] and citrate or DL-isocitrate was added to the cell suspension, yielding a final concentration of 12·5 µM 63Ni2+ and 0·1252·5 mM citrate or 110 mM DL-isocitrate. Samples were taken at time points between 0 and 5 min. Uptake of 63Ni2+ was stopped by the addition of 2 ml of ice-cold 0·1 M LiCl to the suspension, immediately followed by filtration of the suspension through a 0·45 µm pore-size nitrocellulose filter. The filters were washed once with the same LiCl solution and submerged in scintillation fluid. The retained radioactivity was counted in a liquid scintillation counter. Uptake rates were determined from the linear part of each uptake curve.
(ii) [1,5-14C]citrate uptake in membrane vesicles of E. coli.
RSO membrane vesicles of E. coli TOP10 containing CitM were diluted in 50 mM potassium PIPES (pH 6·5) supplemented with 10 mM MgCl2 to a final membrane protein concentration of 50 or 100 µg ml-1 in a total assay volume of 100 µl. An electrochemical proton gradient was allowed to develop at 30 °C for 2 min after the addition of 10 mM potassium ascorbate and 100 µM phenazine methosulfate to the assay mix under a flow of water-saturated air with magnetic stirring, after which the uptake was initiated by the addition of [1,5-14C]citrate (114 mCi mmol-1, 4·218 GBq mmol-1; Amersham) to the assay mix to a final concentration of 4·5 µM. The uptake of [1,5-14C]citrate was quenched and the samples were treated as described above. Inhibitors were present at a concentration of 1 mM.
(iii) Exchange in membrane vesicles of E. coli.
RSO membrane vesicles were allowed to accumulate [1,5-14C]citrate or L-[U-14C]proline (260 mCi mmol-1, 9·62 GBq mmol-1; Amersham) as described above for 1·5 min. Subsequently, in exchange experiments, various substrates were added to the assay mixes at the indicated concentrations and the internalized label was followed over time. In efflux experiments, 10 µM of the protonophore carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) was added to the assay mixes, which completely dissipates the proton motive force. Samples were taken between 10 s and 3 min and treated as described earlier.
ß-Galactosidase assay.
ß-Galactosidase activity of the B. subtilis cells was determined at 28 °C by the method of Miller (1972) using ONPG as the substrate. Cells from 2 ml of a cell culture were harvested by centrifugation. The cell pellet was suspended in a buffer containing 100 mM Na-phosphate, 10 mM KCl, 1 mM MgSO4 and 1 mM DTT (pH 7·0) and the cells were lysed using the lysozyme treatment in the presence of 10 µM DNase. Specific ß-galactosidase activities are expressed as the o-nitrophenol released per min per cell density at 28 °C (Miller units). The values reported are means of two independent measurements. Background activities were measured in B. subtilis 168 and amounted to 0·30·5 Miller units.
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RESULTS |
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CitM is known to transport exclusively citrate complexed to divalent metal ions (Krom et al., 2000 ). The MSMYE medium contained 0·123 mM Mg2+, apparently enough to support growth on citrate, but citrate was present in the medium in large excess in this study (5 mM). Accordingly, growth of B. subtilis on citrate was considerably improved when an additional concentration of 1 mM MgCl2 was added to the medium (Fig. 1b
). Higher concentrations of citrate, up to 10 mM, did not further improve growth of the organism (not shown). Similarly, growth of B. subtilis on isocitrate was significantly improved in the presence of 1 mM MgCl2, especially in the case of D-isocitrate (Fig. 1c
, d
). At higher Mg2+ concentrations, some enhancement of growth of the organism was observed on cis-aconitate (Fig. 1e
; see Discussion). Apparently, growth of B. subtilis on these substrates had been limited by a low metal ion concentration in the medium. To exclude the possibility that the improved growth of B. subtilis in response to Mg2+ was due to the addition of MgCl2 itself, growth of the organism was followed in the presence of the poor growth substrate tricarballylate and the good growth substrate L-malate, which is utilized independently of CitM. In both cases, the addition of 1 mM MgCl2 to the medium did not affect the growth characteristics of B. subtilis (Fig. 1h
).
In summary, CitM supports growth of B. subtilis on citrate and D-isocitrate in the presence of Mg2+. No apparent involvement of CitM was observed during growth of the organism on succinate, fumarate and L-malate, while no growth of the organism was observed in the presence of cis-aconitate, 2-oxoglutarate, D-malate, oxaloacetate or tricarballylate.
Analysis of citM expression
Expression of citM by B. subtilis was determined qualitatively by growing B. subtilis CM002, a strain that contains the lacZ reporter gene fused behind the citM promoter region integrated into the chromosome (see Methods), on LB agar plates containing the chromogenic substrate X-Gal to monitor LacZ activity. Control plates scored negative. Supplementing the plates with different TCA cycle substrates revealed that besides citrate, DL-isocitrate, D-isocitrate and cis-aconitate were apparently able to induce citM expression in B. subtilis CM002 (not shown).
Induction of citM expression was followed over time during growth of B. subtilis CM002 on CSE minimal medium in the presence of citrate, DL-isocitrate and cis-aconitate. The citM promoter activity was measured quantitatively by measuring the ß-galactosidase activity of the cells during their growth. The cultures were inoculated with uninduced cells. In the presence of citrate, ß-galactosidase activity of the cultures increased to reach a steady-state value in the late-exponential phase of growth (Fig. 2a). The pre-steady-state period represents the time required for the ß-galactosidase expression level to equilibrate between the synthesis rate and the growth rate (Warner & Lolkema, 2002
). A similar time dependence for ß-galactosidase activity was observed when B. subtilis CM002 was grown in the presence of DL-isocitrate, but the level of expression appeared to be somewhat lower than that observed for citrate (Fig. 2b
). No induction of expression by cis-aconitate was observed during the exponential growth phase but, surprisingly, after 24 h, long after the cells had entered the stationary phase, induction was as high as that observed during growth of the organism on citrate or DL-isocitrate (Fig. 2c
). The differences in ß-galactosidase activity levels in the exponential growth phase between B. subtilis CM002 cultures grown on citrate or DL-isocitrate and on cis-aconitate could not be explained by different growth rates (Warner & Lolkema, 2002
), since these were not affected much by the different substrates. To exclude the possibility that the observed induction of ß-galactosidase activity by cis-aconitate might be the result of the slow conversion (hydration) of this TCA cycle intermediate into citrate or isocitrate during the course of bacterial growth, CSE minimal medium containing cis-aconitate was pre-incubated for 24 h at 37 °C under continuous shaking before inoculation. The induction pattern generated after this pre-treatment was unchanged (not shown).
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Inhibition of uptake does not prove that the inhibitor is transported, i.e. the inhibitor may bind to the transporter without being translocated. Heterologous exchange of labelled citrate and unlabelled substrates provides an assay to demonstrate translocation of the latter (Bandell & Lolkema, 1999 ). Membrane vesicles prepared from E. coli cells expressing CitM were allowed to accumulate [1,5-14C]citrate driven by the proton motive force until a plateau was reached. Addition of FCCP, a protonophore that dissipates the proton motive force, to the assay mix resulted in very rapid efflux of the label from the lumen of the vesicles. In fact, all of the label was released within 10 s of the addition of FCCP (Fig. 3a
). When instead of FCCP 1 mM of unlabelled citrate was added to the assay mix, the release of the label was also fast, but 1020% of the label was still inside the membranes at t=10 s. Release of the label under these conditions is the result of homologous exchange during which the transporter transports unlabelled citrate into the vesicle in exchange for the exit of labelled citrate. To rule out the possibility that the addition of 1 mM of citrate to the assay mix would dissipate the proton motive force, i.e. would mimic the effect of FCCP, the effect of 1 mM of citrate on the accumulation level of L-[U-14C]proline in the RSO vesicles was measured as a control (Fig. 3b
). No significant release of L-[U-14C]proline was observed under these conditions, while treatment of the assay mix with FCCP resulted in the loss of the label in 3 min, suggesting that the addition of 1 mM of citrate to the assay mix did not affect the magnitude of the proton motive force significantly.
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DISCUSSION |
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Citrate transporters are found in many bacterial species; these transporters allow bacteria to utilize citrate by its degradation via the TCA cycle or via one of the citrate-fermentation pathways (Bott, 1997 ). However, E. coli is an exception to this rule although it contains all of the enzymes necessary for citrate metabolism, it cannot utilize citrate because it lacks a functional citrate-transport system (Bott, 1997
). In contrast to citrate, few reports are available on the utilization of isocitrate by bacteria and data on transporters with specificity for isocitrate are similarly scarce. It has been argued that transporters for citrate may also transport isocitrate (Kay, 1978
), but this is definitely not generally true. For instance, the citrate transporters of the 2-hydroxycarboxylate transporter family (CimH of B. subtilis, CitP of lactic acid bacteria and CitS of Klebsiella pneumoniae) do not recognize isocitrate (van der Rest et al., 1992
; Bandell et al., 1997
; Krom et al., 2001
). However, a tricarboxylate transport system has been described in Pseudomonas fluorescens that is induced by citrate and transports citrate and D-isocitrate. A second system in this organism is induced by tricarballylate and transports citrate, cis-aconitate and tricarballylate (Kay, 1978
). Salmonella typhimurium has been reported to be able to grow on citrate, cis-aconitate and isocitrate by using a thus-far unique uptake system, which involves a periplasmic binding protein that specifically binds citrate, isocitrate and L-erythro-2-fluorocitrate (Somers et al., 1981
; Sweet et al., 1984
; Widenhorn et al., 1988
). In this study, we have shown that B. subtilis can grow on isocitrate as a sole carbon source and that the Mg2+citrate transporter CitM is responsible for the uptake from the medium by three criteria. (i) The B. subtilis CitM-deficient strain CITMd lost the ability to grow on isocitrate completely (Fig. 1c
, d
), as was also observed for growth on citrate. (ii) Like citrate, isocitrate appeared to be an inducer of citM expression, on solid and in liquid media. (iii) Heterologous exchange experiments demonstrated that CitM transports isocitrate. Similar to citrate, isocitrate is taken up in complex with a divalent metal ion.
The results obtained with cis-aconitate are confusing; this confusion is likely to be caused by the presence of impurities in commercially available cis-aconitate preparations. Growth of B. subtilis in MSMYE medium resulted in complete lysis of the cells after 24 h incubation. When grown in the presence of cis-aconitate, we did not see additional growth of the organism, but the cells did not lyse and were still viable after 24 h incubation (not shown). Moreover, some B. subtilis growth enhancement was observed when 1 mM Mg2+ was added to the medium in addition to cis-aconitate, strongly suggesting the involvement of CitM in the uptake of this TCA cycle intermediate (Fig. 1e). The citM-expression studies showed induction of CitM by cis-aconitate, but only after prolonged incubation of B. subtilis in the stationary phase. Unfortunately, the exchange studies revealed the presence of a contaminant in cis-aconitate (
5%) that was also a substrate of CitM and was, therefore, most likely to be citrate or isocitrate. No evidence was obtained that cis-aconitate itself is a substrate of CitM. We tentatively conclude that the growth effects and induction pattern observed when B. subtilis is grown in the presence of cis-aconitate, and previous claims made in the literature (McKillen et al., 1972
), must be ascribed to impurities in the cis-aconitate used; hence, cis-aconitate is not a growth substrate of B. subtilis.
In conclusion, B. subtilis is capable of growing on the TCA cycle intermediates citrate and isocitrate, mediated by CitM, on succinate and fumarate, mediated by DctP (Asai et al., 2000 ), and on L-malate. A consequence of the involvement of CitM in B. subtilis growth on citrate and isocitrate is that optimal growth of the organism requires higher concentrations of Mg2+ to be present than normally required (compare Fig. 1b
, d
, h
). No growth of B. subtilis was detected when cis-aconitate, 2-oxoglutarate, D-malate, oxaloacetate or tricarballylate was the sole carbon source. The lack of growth of B. subtilis on 2-oxoglutarate that we observed is in contradiction with a report claiming growth of this organism on 2-oxoglutarate mediated by a low-affinity, inducible transport system with a Km of 6·7 mM (Fournier et al., 1972
). Analysis of the B. subtilis genome (Kunst et al., 1997
) provides no clue as to the identity of such a 2-oxoglutarate transporter.
CitM has been shown to take up citrate in complex with Mg2+, Zn2+, Mn2+, Co2+ or Ni2+. The improved growth of B. subtilis on isocitrate upon the addition of extra Mg2+ (Fig. 1c, d
) to the growth medium and the 63Ni2+ uptake experiments (Fig. 5
) indicated that isocitrate is also transported in complex with Mg2+ and Ni2+. Further experiments showed that the uptake of [1,5-14C]citrate in the presence of 10 mM of Mg2+, Zn2+, Mn2+, Co2+ or Ni2+ could, in all cases, be significantly inhibited by the presence of 1 mM of isocitrate in the medium (not shown). These experiments suggest that the metal ion specificity in the complexes that are transported by CitM is the same for isocitrate and citrate, but it cannot be excluded that complexes with other divalent metal ions are not substrates for CitM. Citrate appears to be a stronger chelator of divalent metal ions than isocitrate. For instance, the complex formation constants for Mn2+isocitrate and Mn2+citrate (2·55 and 3·54 mM, respectively) (Martell & Smith, 1977
) indicate a 10-fold lower affinity of isocitrate for Mn2+ than citrate. In our experiments, about seven times more DL-isocitrate than citrate was required to inhibit the 63Ni2+ uptake activity in whole cells of B. subtilis CITMd by 50%. Four times less of the naturally occurring isomer D-isocitrate was needed in whole cells of B. subtilis than L-isocitrate, indicating that D-isocitrate is a better chelator than L-isocitrate (not shown). Consequently, optimal growth of B. subtilis on D-isocitrate requires relatively high concentrations of divalent metal ions in the medium.
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ACKNOWLEDGEMENTS |
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Received 31 May 2002;
revised 9 July 2002;
accepted 12 July 2002.