Division of Infectious Diseases, School of Public Health, 140 Earl Warren Hall, University of California, Berkeley, CA 94720, USA1
Francis I. Proctor Foundation, University of California, San Francisco, CA 94143, USA2
Author for correspondence: Richard S. Stephens. Tel: +1 510 643 9900. Fax: +1 510 643 1537. e-mail: rss{at}uclink4.berkeley.edu
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ABSTRACT |
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Keywords: major outer-membrane protein, porin, 2-oxoglutarate
Abbreviations: MOMP, major outer-membrane protein; TCA cycle, tricarboxylic acid cycle
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INTRODUCTION |
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Previous experimental results have supported the energy parasite hypothesis that suggests that chlamydial growth depends on the acquisition of ATP and other high-energy metabolites from the host (Moulder, 1991 ). Interestingly, the genome sequence has revealed that Chlamydia may have the potential to produce oxidative and substrate-level ATP. Chlamydiae have the enzymic machinery for the EmbdenMeyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle (Kalman et al., 1999
; Stephens et al., 1998
). However, according to predictions based upon the genome sequence, the TCA cycle is incomplete in Chlamydia and is apparently missing three enzymes: citrate synthase, aconitase and isocitrate dehydrogenase (Kalman et al., 1999
; Stephens et al., 1998
). The implication of the missing TCA cycle enzymes is that glutamate and 2-oxoglutarate are obtained from the host cell as carbon and energy substrates. If 2-oxoglutarate is available from the host cell, then the cycle may be initiated with 2-oxoglutarate as an initial substrate and ending with the production of oxaloacetate. Chlamydiae encode an orthologue of a dicarboxylate transporter (SodTi). This orthologue is most similar to a transporter in spinach chloroplast envelopes that transports 2-oxoglutarate into the chloroplast (Weber et al., 1995
). Therefore, in Chlamydia this translocator could transport 2-oxoglutarate through the inner membrane, allowing for net carbon entry into the TCA pathway. Thus, it is predicted that chlamydiae obtain 2-oxoglutarate from the host cell. It has recently been shown in vitro that chlamydiae utilize glucose as the major source of carbon but that 2-oxoglutarate also serves to support chlamydial viability and growth (Iliffe-Lee & McClarty, 2000
).
Chlamydia has a major outer-membrane protein (MOMP) that functions as a general porin (Bavoil et al., 1984 ; Jones et al., 2000
; Wyllie et al., 1998
). The genome has also revealed a number of predicted outer-membrane proteins that have not been previously identified in Chlamydia. We recently used the genome analysis to aid in the discovery of a putative porin (PorB). PorB is localized in the outer-membrane complex of Chlamydia and is surface accessible (Kubo & Stephens, 2000
). This protein has porin activity when tested in a liposome-swelling assay but is significantly less efficient in diffusion of polysaccharide and amino acid solutes than MOMP (Kubo & Stephens, 2000
). Relative to the preponderance of MOMP in chlamydial outer membranes, PorB is much less abundant (Kubo & Stephens, 2000
). Differences in general pore-forming activity, as well as differences in the amount present in the chlamydial outer membrane, suggest a unique role for each of the porins. The presence of PorB in small amounts is difficult to understand unless PorB has a role as a substrate-specific porin that is efficient in the selective transport of particular classes of molecules. RT-PCR analysis and cell staining at various time points indicated that this protein is expressed throughout the developmental cycle (Kubo & Stephens, 2000
). Thus PorB expression is not differentially regulated and is likely not a secondary general porin that serves an analogous purpose as MOMP, being upregulated under different growth conditions.
We sought to determine if PorB is a substrate-specific porin that preferentially facilitates the translocation of certain classes of substrates into the periplasmic space. Our approach was to use the information derived from the chlamydial genome to aid in the identification of metabolites that are predicted to be obtained from the host and determine if these metabolites enter through PorB and/or MOMP.
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METHODS |
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Expression and purification of PorB and MOMP.
PorB was cloned and expressed in the E. coli TOP10 (Invitrogen), as previously described (Kubo & Stephens, 2000 ). The protein was isolated from E. coli outer membranes using octylglucoside by previously described methods (Kubo & Stephens, 2000
). Briefly, E. coli containing the PorB gene was grown until the cultures reached an OD600 of 0·6 and arabinose was added to induce the expression of PorB. PorB was cloned with a C-terminal His tag and was purified by nickel column chromatography using the His Bind Purification system (Novagen) after removing the octylglucoside by dialysis using PBS and then 1x BIND buffer (Novagen).
The synthetic gene encoding MOMP (ompA) was constructed in E. coli HMS 174 (DE3) and has been previously described (Jones et al., 2000 ). E. coli HMS 174 (DE3) without the plasmid was used as a control strain. The outer membranes of E. coli expressing MOMP and E. coli not expressing MOMP were isolated using the method of Osborn & Munson (1974)
with a few previously described modifications (Jones et al., 2000
). Purified protein and outer membranes for use in the liposome-swelling assay were quantitated according to the Lowry method.
Liposome-swelling assay.
This assay was performed according to the method of Nikaido & Rosenberg (1983) with the following modifications: (1) liposomes were made by mixing 5·0 µmol phosphatidylcholine and 0·02 µmol dicetyl phosphate with PorB or outer-membrane proteins to OD400 readings between 0·4 and 0·7 and (2) the liposome drying time was longer than 2 min (5 min), but at a lower temperature of 37 °C. Liposomes were made with dextran T-40 (15% dextran T-40 in 5 mM Tris/HCl, pH 7·5) inside. Since stachyose is impermeable to the porins, it was used as a control to determine the iso-osmotic concentration of other solutes. The concentration of stachyose that produced no swelling or shrinking of the proteoliposomes was determined to be the iso-osmotic concentration. The swelling rates were determined as d(1/OD400)/dt from the optical-density changes between 10 and 20 s (Nikaido & Rosenberg, 1983
).
Liposome-swelling assay for testing anions.
Liposomes were made according to the method described above with a few modifications. The following were added to phosphatidylcholine and dicetyl phosphate dried with PorB (6 µg): 4 mM NAD+, 12 mM stachyose, 1 mM imidazole-NAD buffer (pH 6·0). The test solution consisted of 1 mM imidazole-NAD (pH 6·0), 1 mM sodium-NAD and 6 mM disodium salt of the anion to be tested (2-oxoglutarate, succinate, oxaloacetate, malate or citrate). Control liposomes without protein were used to determine the isotonic concentration of the test solutions.
Enzyme-linked liposome-swelling assay.
Liposomes were made as described above with addition of 50 mM potassium phosphate, 2·5 mM NAD+, 0·2 mM thiamin pyrophosphate, 1·0 mM magnesium chloride, 0·13 mM coenzyme A, 2·6 mM cysteine and 5·0 units 2-oxoglutarate dehydrogenase. Various concentrations of 2-oxoglutarate (0·0011 mM) were used as test solutes. Liposomes containing PorB (6 µg) and control liposome without protein were made with the reaction mixture, washed through a Sephadex column (S-300) equilibrated with reaction mixture without 2-oxoglutarate dehydrogenase, and placed inside a cuvette. 2-Oxoglutarate was added to the reaction and mixed. The formation of NADH was measured by the increase in A340.
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RESULTS |
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To determine if PorB had specificity for any molecule(s), we studied the genome sequence to determine if the inferred biology of Chlamydia could provide an idea of which molecules Chlamydia might need to obtain from the host. This analysis provided a list of orthologues of transporters that are important in the translocation of solutes across the inner membrane, including amino acid, polysaccharide, oligopeptide and dicarboxylate transporters (Stephens et al., 1998 ). Previous analysis of MOMP porin activity showed that amino acids, mono-and disaccharides and oligopeptides enter efficiently through MOMP (Jones et al., 2000
). However, PorB did not allow for the efficient entry of either amino acids or polysaccharides (Kubo & Stephens, 2000
). The presence of an orthologue to an inner-membrane dicarboxylate transporter, and the fact that Chlamydia spp. appear to have a truncated TCA cycle, suggest that chlamydiae may require exogenous 2-oxoglutarate from the host cell. Therefore, we tested the hypothesis that dicarboxylates could enter through the chlamydial outer membrane by measuring 2-oxoglutarate diffusion through the two known porins, PorB and MOMP.
The liposome-swelling assay with PorB and MOMP showed that the diffusion of 2-oxoglutarate was more efficient through PorB than MOMP (Fig. 1). Several controls were employed to support the PorB-based diffusion data. No diffusion of 2-oxoglutarate was seen using liposomes without protein or when using liposomes containing another chlamydial outer-membrane protein (Omp85) that is a conserved bacterial protein (data not shown). Chlamydial Omp85 was used as a control protein; it was cloned, expressed in E. coli and purified by the same method used to purify PorB. Lysates of E. coli not expressing PorB were treated the same way as E. coli expressing PorB and subjected to nickel column chromatography. The column eluate was used as a control in all the assays to verify that no E. coli contaminants were responsible for the porin activity observed. The presence of the His tag on PorB did not affect its ability to function as a porin but it is possible that the His tag could unexpectedly influence the diffusion of some of the substrates. However, the activity of PorB was dose-response dependent as increasing amounts of PorB added to liposomes resulted in increased swelling rates (data not shown).
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DISCUSSION |
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MOMP functions as a general porin that allows for the diffusion of various types of molecules within a size limit through the outer membrane, and those necessary for Chlamydia will be transported through the inner membrane by transporters. PorB is present in much smaller amounts than MOMP (Kubo & Stephens, 2000 ) and likely has a more specific role than MOMP to allow for particular classes of molecules to be transported at a faster rate, much like substrate-specific porins found in other bacteria (Koebnik et al., 2000
). Since chlamydiae rely on the host for much of the nutrients they need to grow, it is not surprising that they have substrate-specific porins for the efficient transport of essential nutrients. PorB may be present to ensure that chlamydiae acquire 2-oxoglutarate to provide necessary metabolic intermediates and energy. Gene transcription and protein analyses have shown that porB is not developmentally regulated (Kubo & Stephens, 2000
) and other genes such as transporters for glutamate (gltT) and glucose (uhpC) also are not developmentally regulated (Iliffe-Lee & McClarty, 2000
). Thus Chlamydia is unlike most other bacteria that respond to environmental change by altering gene expression of porins and transporters. This may be because chlamydiae only grow in a singular environment within a host-cell vacuole; however, it is unknown how in vitro cultures may differ from in vivo infection in terms of the availability of carbon sources for chlamydial growth.
After diffusion through the outer membrane, there are many inner-membrane transporters that actively transport molecules into the cytoplasm of Chlamydia. The presence of a gene encoding a dicarboxylate transporter orthologue (SodTi) in Chlamydia suggests that 2-oxoglutarate is transported across the inner membrane. The SodTi transporter in chloroplasts functions as a translocator that brings 2-oxoglutarate into the stroma in exchange for malate (Weber et al., 1995 ). Therefore, the SodTi orthologue in Chlamydia may function in much the same way by transporting 2-oxoglutarate through the inner membrane into the chlamydial cytosol. Once 2-oxoglutarate is transported through the inner membrane, it can then be used in the chlamydial TCA loop since chlamydiae apparently do not have the capability of producing 2-oxoglutarate and it has recently been shown that chlamydiae may utilize 2-oxoglutarate as a carbon source to support growth (Iliffe-Lee & McClarty, 2000
).
PorB is the only porin in bacteria to show efficient diffusion for select dicarboxylates and is the second porin found in Chlamydia. PorB has a different function from MOMP and PorB can distinguish between similar molecules. However, the substrate selectivity determined for PorB is not biologically unique since a peroxisome porin also has specificity for dicarboxylates (Reumann et al., 1998 ). Peroxisomes are found in all but the most primitive eukaryotic cells and, like Chlamydia, they have access to intracellular substrates. Like PorB, the peroxisome porin does not form a general diffusion pore, but has particular properties analogous to specific and inducible porins that have been characterized in some Gram-negative bacteria (Koebnik et al., 2000
; Luckey & Nikaido, 1980
). Although PorB and the peroxisome porin have analogous functions, sequence comparisons as well as secondary structure comparisons did not reveal significant similarity.
Investigation of the porin function of MOMP and PorB has allowed for a general understanding of the permeability of the outer membrane of Chlamydia and its relevance to the unique metabolic machinery as inferred by the genome sequence analysis. MOMP facilitates the efficient diffusion of polysaccharides (i.e. glucose) and amino acids including glutamate (Jones et al., 2000 ) that are then transported across the inner membrane by UhpC and GltT, respectively (Stephens et al., 1998
). In contrast, PorB facilitates the efficient diffusion of dicarboxylates (i.e. 2-oxoglutarate) that are transported across the inner membrane by SodTi. Thus, PorB has an important role in providing TCA cycle intermediates that also produce cofactors such as NADH for the respiratory chain (McClarty, 1999
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 April 2001;
revised 26 June 2001;
accepted 23 July 2001.