INFECTION AND IMMUNITY, Apr. 2001, p. 2428–2434 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.4.2428–2434.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 4
Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC REGINA J. TANZER,1 DAVID LONGBOTTOM,2
AND
THOMAS P. HATCH1*
Department of Molecular Sciences, University of Tennessee Center for Health Sciences, Memphis, Tennessee 38163,1 and Moredun Research Institute, Pentlands Science Park, Penicuik, Midlothian EH26 OPZ, United Kingdom2 Received 6 October 2000/Returned for modification 1 December 2000/Accepted 4 January 2001
The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins. The cell envelope structure of Chlamydia is similar to that of other gram-negative bacteria, with an outer membrane (OM) containing lipopolysaccharide, a periplasm, and an inner membrane. However, two envelope features are unique to chlamydiae: an apparent lack of or deficiency in peptidoglycan and the presence of disulfide-bond-cross-linked proteins in the OM and the periplasm (reviewed in reference 15). Nevertheless, the infectious elementary body (EB) form of chlamydiae, but not the dividing reticulate body (RB) form, is osmotically stable, and chlamydiae are sensitive to -lactams and D-cycloserine. The sensitivity of chlamydiae to peptidoglycan synthesis-inhibiting drugs in the possible absence of peptidoglycan has been termed the chlamydial anomaly by Moulder (29). The scope of the anomaly has been expanded by the recent sequencing of the genomes of several chlamydial strains (18, 35, 38), revealing the presence of what has been thought to be all of the genes required for peptidoglycan synthesis (7). Ghuysen and Goffin (11) proposed a solution to the anomaly, suggesting that chlamydiae use their peptidoglycan genes to synthesize a glycanless wall polymer whose synthesis is penicillin sensitive. Three key points of their proposal are as follows: (i) the predicted amino acid sequence of the three chlamydial penicillinbinding proteins suggests that they are capable of carrying out cross-linking transpeptidase reactions but are incapable of transglycosylating N-acetylglucosamine-N-acetylmuramic acid disaccharides into a glycan polymer; (ii) potential chlamydial N-acetylmuramoyl-L-alanine amidases cleave cross-linked peptidyl polymers from the disaccharide subunits; and (iii) the disaccharide subunits, as part of lipid II, serve strictly as carriers and therefore do not accumulate in stoichiometric amounts. Ghuysen and Goffin (11) further speculated that the
glycanless polymer may be covalently linked to lipoproteins in the inner membrane or OM or to a highly disulfide-bondcross-linked protein structure in the periplasm. A disulfidecross-linked periplasmic structure was proposed by Everett and Hatch (10) to consist of cysteine-rich proteins (CRPs) that are encoded by a bicistronic operon and that are made only late in the chlamydial developmental cycle (1, 17, 22, 31). The CRPs consist of a 60-kDa doublet, which is the product of posttranslational processing of the omcB gene product (2), and a 12- to 15-kDa lipoprotein (9), which is a product of omcA and is speculated to be anchored to the OM by its lipid moiety (10). The CRPs are located in the sodium lauryl sarcosine (Sarkosyl)-insoluble fraction, called the Sarkosyl chlamydial OM complex (COMC), which consists of integral OM proteins and the highly disulfide-cross-linked periplasmic CRPs (10, 15). The predominant protein in COMCs is the major OM protein (MOMP). The MOMP lacks a homolog in other bacteria but functions, like many other gram-negative OM proteins, as a porin (4, 40, 41). It is present throughout the chlamydial developmental cycle and is disulfide cross-linked in EBs but not in logarithmically dividing RBs (16, 17, 33). A third class of proteins, variously referred to as polymorphic OM proteins (POMPs) and polymorphic membrane proteins, was identified in the Sarkosyl COMC of ovine abortion strains of Chlamydia psittaci by Cevenini et al. (6) and others (12, 13, 27, 37). Genes encoding 9 and 21 potential POMPs are present in the genomes of Chlamydia trachomatis and Chlamydia pneumoniae, respectively (18, 35, 38). The similarity of predicted POMP amino acid sequences across chlamydial species is low, ranging from about 10 to 60%. Similarities among POMPs within a chlamydial strain are almost equally low, with the notable exception of the ovine enzootic abortion (OEA) strain C. psittaci S26/3 POMPs 90A and 90B, which are identical, and POMPs 91A and 91B, which are 89% similar to each other and 86 and 87% similar, respectively, to POMPs 90A and 90B (26). The POMPs share no homology with other bacterial pro-
* Corresponding author. Mailing address: Department of Molecular Sciences, University of Tennessee Center for Health Sciences, 858 Madison Ave., Memphis, TN 38163. Phone: (901) 448-4664. Fax: (901) 448-8462. E-mail:
[email protected]. 2428
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teins but do share common features among themselves, including multiple GGA (I, L, and V) and FXXN repeats, large size (90 to 187 kDa), and carboxy-terminal phenylalanine residues (14, 26, 27, 38). The last property and predicted secondary structures suggest that POMPs are located in the OM, and immunoelectron and immunofluorescence microscopy studies indicate that one or more of the ovine POMPs and at least one C. pneumoniae POMP are surface exposed (19, 24, 25). However, it is not clear that all POMP genes are expressed as proteins and that all POMPs are located in the OM. For example, 5 of the 21 C. pneumoniae POMP genes contain frameshift mutations and 1 is truncated to encode a 56-kDa protein, and 1 of the predicted POMPs of OEA C. psittaci (POMP 98A; GenBank accession number U722499), C. trachomatis D, and C. pneumoniae lacks a signal sequence (14, 18, 38). The function of the POMPs is unknown. The paralogous nature of the proteins suggests that the more distantly related POMPs may have distinct functions (14), and animals that were immunized with insoluble detergent extracts of C. psittaci stains, which likely contained POMPs, were protected against challenge with infectious organisms (3, 39). The purpose of the present study was to determine the cellular location, disulfide-cross-linked nature, and developmental stage of synthesis of POMPs in C. psittaci avian strain 6BC. MATERIALS AND METHODS Purification of EBs and RBs. L cells were infected with C. psittaci 6BC. RBs or EBs were harvested at 15, 24, 28, and 48 h postinfection and purified by density centrifugation in a Beckman SW28 rotor for 30 min at 80,000 ⫻ g with a three-step gradient of 29, 34, and 40% Hypaque-76 (Nycomed Inc., Princeton, N.J.) in Dulbecco phosphate-buffered saline (GIBCO, Grand Island, N.Y.) containing 0.5 mM MgCl2 and 1.0 mM CaCl2 (PBS). The 48-h harvest was treated with the nonionic detergent Nonidet P-40 (0.25%; Sigma Chemical Co., St. Louis, Mo.) in PBS for 5 min at room temperature before purification to eliminate osmotically fragile RBs (17). RBs were collected at the 29%–34% interface; EBs were collected at the 34%–40% interface. Trypsin digestion. To determine the surface exposure of proteins, purified EBs were incubated with trypsin (60 g/ml; type III from bovine pancreas; Sigma) in PBS for 30 min at 37°C. Trypsin was inactivated by incubation with trypsin inhibitor (120 g/ml; type II-O from chicken egg white; Sigma) for 5 min at room temperature. The samples were pelleted by centrifugation in a microcentrifuge at 9,000 ⫻ g for 10 min and washed once with PBS containing trypsin inhibitor. Labeling of EBs. To label OM proteins, 25 Ci of 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazarine ([125I]TID; Amersham, Arlington Heights, Ill.) was added to gradient-purified EBs harvested from 2 ⫻ 108 infected cells in 200 l of PBS. The mixture was incubated for 1 h on ice in a dimly lit room before the [125I]TID was activated by exposure to a long-wave UV lamp for 1 h (10). Proteins in EBs and detergent-insoluble complexes were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and labeled proteins were detected by phosphorimaging of the dried gel or an immunoblot of the gel. Attempts were made to surface label EBs by reaction with 50 g of Nhydroxysulfosuccinimide-biotin (sulfo-NHS-biotin; Pierce, Rockford, Ill.) per ml in PBS (pH 8.0) for 30 min at room temperature. The reaction was quenched by the addition of an equal volume of 10 mM glycine in PBS (pH 7.4). EBs were pelleted by centrifugation at 9,000 ⫻ g for 10 min and washed once with glycinePBS. Proteins were fractionated by SDS-PAGE and electrophoretically transferred to an Immobilon polyvinylidene difluoride membrane (0.45-m pore size; Millipore Corp., Bedford, Mass.). Sulfo-NHS-biotin-reactive proteins were detected on the membrane with streptavidin-horseradish peroxidase and color development with 4-chloro-1-naphthol and hydrogen peroxide. Preparation of detergent-insoluble complexes. Purified RBs and EBs were extracted at 37°C for 30 min with either 2.0% SDS or 2.0% Sarkosyl (Sigma) in PBS. The samples were subjected to sonication for 30 s, pelleted by centrifugation at 14,500 ⫻ g for 20 min at 4°C, and washed once with the detergents to produce SDS-insoluble complexes and Sarkosyl COMCs. In one experiment,
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EBs were incubated for 30 min at 37°C in PBS containing 20 mM dithiothreitol (DTT) and 5% -mercaptoethanol (ME) and pelleted by centrifugation at 9,000 ⫻ g for 10 min before extraction with detergents. SDS-PAGE and immunoblotting. All samples were suspended in Laemmli solubilization buffer, with or without 5% ME and 10 mM DTT, heated to 90°C, and fractionated by SDS-PAGE on 7.5 to 15% gradient gels (21). Prestained protein standards were purchased from Bio-Rad, Hercules, Calif. (low range, catalog no. 161-0305; high range, catalog no. 161-0309; and broad range, catalog no. 161-0318). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes, and the membranes were reacted with an affinity-purified polyclonal antibody raised in sheep against the carboxyl-terminal portion of POMP 90A of OEA C. psittaci strain S26/3 (24–26). Mass spectrometry. Sarkosyl COMCs were fractionated by SDS-PAGE, and proteins were detected by zinc sulfate staining (5). Proteins in gel slices were treated with trypsin, and peptides were extracted and analyzed by matrix-assisted laser desorption ionization (MALDI)–time of flight mass spectrometry as described by Shevchenko et al. (36).
RESULTS Identification of OM proteins in C. psittaci 6BC. The protein profiles of whole EBs and the Sarkosyl COMC fraction of EBs of C. psittaci 6BC were examined by SDS-PAGE and immunoblotting (Fig. 1). Following harvesting and purification, EBs were treated with [125I]TID, a photoactivatable lipophilic reagent that specifically labels the portions of OM proteins that are embedded within the membrane. TID does not label periplasmic proteins or proteins in the inner membrane because it cannot enter or pass through the hydrophilic periplasm (10). Following treatment with [125I]TID and before the preparation of Sarkosyl COMCs, half of the EB preparations were treated with trypsin to test whether proteins were surface exposed. Prominent proteins that were noted by Coomassie brilliant blue staining of the COMC preparations (Fig. 1A, lane 3) included the MOMP, an unidentified 48-kDa protein just above the MOMP (protein 7), the CRP 60-kDa doublet, and a cluster of proteins migrating with apparent molecular weights of about 90,000 (protein 6), 96,000 (protein 5), 98,000 (protein 4), and 105,000, a band which was resolved to proteins 2 and 3 (band 2/3) by the analysis of EBs treated with trypsin (see below). An additional protein with a relative molecular weight of about 110,000 (protein 1) was barely visible with the Coomassie stain. The relative molecular weights were estimated from the protein standards on the right side of Fig. 1A. The large sizes of proteins 1 to 6 suggest that they may be members of the POMP family previously identified in C. trachomatis serovar L2, C. pneumoniae, and ovine strains of C. psittaci (6, 12, 13, 19, 26, 27, 30, 37). All of the putative POMPs were also found in whole EBs and were labeled with [125I]TID (Fig. 1B, lanes 1 and 3). The latter observation confirms that they are integral OM proteins. The MOMP and protein 7 were also labeled with [125I]TID, whereas the periplasmically located CRPs were not. Preliminary MALDI mass spectrometric analysis of a gel slice containing protein 7 was consistent with the presence of the MOMP in the band (data not shown); however, the current lack of a C. psittaci 6BC genomic database did not allow the identification of any other proteins that might be present in the gel slice or confirmation that the high-molecular-weight proteins are POMPs. Treatment of whole EBs with trypsin resulted in a reduction in the intensity of protein band 2/3, the disappearance of pro-
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FIG. 1. SDS-PAGE and immunoblot analysis of Sarkosyl COMCs of EBs labeled with [125I]TID. EBs were harvested and purified at 48 h postinfection and treated with [125I]TID. Where indicated, EBs were treated with trypsin before the preparation of Sarkosyl COMCs. All samples were heated in the presence of 10 mM DTT and 5% ME before electrophoresis. (A) Coomassie brilliant blue-stained gel, with molecular weights (MW, in thousands) of prestained protein standards at each side. (B) Phosphorimage of the dried gel. (C) Immunoblot of a duplicate gel reacted with monospecific sheep antiserum raised against C. psittaci ovine strain POMP 90A. (D) Phosphorimage of the immunoblot. Lanes: 1, EBs; 2, EBs treated with trypsin; 3, Sarkosyl (Sark) COMCs prepared from EBs; 4, Sarkosyl COMCs prepared from trypsin-treated EBs. The positions of putative POMPs (positions 1 to 6), an unknown protein (position 7), the CRPs, and the MOMP are indicated at the right side of each panel. T1, T2, and T3 indicate tryptic fragments. The assignment of TID-labeled tryptic fragments to specific immunoreactive proteins was determined by superimposition of the phosphorimage in panel D on the immunoblot in panel C.
tein 5, and the appearance of two trypsin fragments, T2 and T3, as noted in the Coomassie-stained gel (Fig. 1A, compare lane 1 with lane 2 and lane 3 with lane 4) and the phosphorimage of the gel (Fig. 1B). These observations suggest that at least two putative POMPs are surface exposed, protein 5 and one of two comigrating proteins in band 2/3 (we arbitrarily designated protein 3 as the trypsin-sensitive protein). The insensitivity of the MOMP and proteins 1, 2, 4, 6, and 7 to trypsin does not necessarily reflect a lack of surface exposure but rather may simply reflect the lack of an exposed trypsin-sensitive site. Attempts were made to identify surface-exposed proteins on EBs by reaction with the membrane-impermeable cross-linking reagent sulfo-NHS-biotin. A large proportion of the Coomassie-stained EB proteins, including the CRPs, were labeled with this reagent (data not shown), suggesting that this hydrophilic compound was capable of penetrating the periplasm of C. psittaci EBs. EBs and Sarkosyl COMCs, fractionated on the same gel (Fig. 1A and B), were analyzed by immunoblotting with a monospecific sheep polyclonal antibody raised against the Cterminal half of ovine C. psittaci POMP 90A. This antibody
reacts with a cluster of ovine strain proteins in COMCs consisting of the identical proteins POMP 90A and POMP 90B and highly homologous proteins, POMP 91A and POMP 91B (24–26). The antibody reacted strongly with proteins 4 and 5 and weakly with protein 6 as well as with trypsin fragments T1 and T2 (Fig. 1C). Fragment T2 also reacted with [125I]TID (Fig. 1B and D, lanes 2 and 4) and may represent an OMembedded peptide derived from protein 5 (Fig. 1C, lanes 2 and 4). In contrast, the antibody-reactive fragment T1 (Fig. 1C, lanes 2 and 4) was not labeled with [125I]TID (Fig. 1B and D), nor was it apparent on the Coomassie-stained gel (Fig. 1A). The failure to detect T1 by Coomassie staining and [125I]TID labeling suggests that it is a minor peptide, possibly a rare product of partial trypsin digestion. The third trypsin fragment, T3, failed to react with the antiserum; thus, it may be a cleavage product of protein 3. The properties of the putative POMPs of C. psittaci 6BC are summarized in Table 1. Disulfide bonds in POMPs. All POMP genes in C. trachomatis and C. pneumoniae and the six ovine C. psittaci POMP genes identified in DNA sequence databases are predicted to
POMPs OF C. PSITTACI 6BC
VOL. 69, 2001 TABLE 1. Properties of proteins in the Sarokosyl COMCs of C. psittaci 6BCa Protein(s)
[125I]TID labeling
Trypsin sensitivity
Reaction with antibodyb
1 2 3 4 5 6 7 MOMP CRPs
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺
⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫹ ⫹ ⫾ ⫺ ⫺ ⫺
a b
⫹, positive results; ⫺, negative result; ⫾, weak reaction. Anti-POMP 90A (C-terminal half).
encode proteins that contain cysteine residues. To investigate whether the putative POMPs of 6BC are interpeptide crosslinked by disulfide bonds, the effects of reducing agents on the ability of POMPs in detergent-insoluble complexes to enter polyacrylamide gels were examined. The protein profiles of the Sarkosyl COMCs and the SDS-insoluble complexes electro-
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phoresed in the presence of reducing agents were identical, consisting of the MOMP and likely degradation products of the MOMP, protein 7, the CRPs, and the putative high-molecular-weight POMPs (Fig. 2A, lanes 2 and 4). Because the strong anionic detergent SDS dissolves both the inner membrane and the OM of bacteria, the presence of these proteins in the SDS-insoluble fraction suggests that they are part of one or more insoluble supramolecular complexes. When the insoluble complexes were electrophoresed in the absence of DTT and ME, CRPs, the MOMP, and protein 7 failed to enter the gel (Fig. 2B, lanes 2 and 4), confirming that these proteins are highly interpeptide disulfide cross-linked. In contrast, diffusely Coomassie-stained bands were noted in the 98-kDa putative POMP region of the gel, in the absence of reducing agents (Fig. 2B, lanes 2 and 4). Immunoblot analysis confirmed that at least some of the diffusely stained bands cross-reacted with antibody raised against ovine POMP 90A (Fig. 2D, lanes 2 and 4). These results suggest that at least some of the C. psittaci 6BC POMPs, although part of a supramolecular complex, are not extensively interpeptide disulfide cross-linked. When Sarkosyl COMCs were prepared from EBs treated
FIG. 2. SDS-PAGE and immunoblot analysis of detergent-insoluble fractions of EBs. EBs were harvested and purified at 48 h postinfection. Where indicated, EBs were treated with 20 mM DTT and 5% ME before the preparation of Sarkosyl COMCs and SDS-insoluble complexes. All preparations were suspended in Laemmli buffer (21) with (A and C) and without (B and D) the addition of reducing agents (10 mM DTT and 5% ME), heated to 90°C, fractionated by SDS-PAGE, and analyzed by Coomassie staining (A and B) or immunoblotting with anti-POMP 90A (C and D). Lanes: 1, EBs; 2, Sarkosyl (Sark) COMC; 3, Sarkosyl COMC prepared from reduced EBs; 4, SDS-insoluble complex; 5, SDS-insoluble complex prepared from reduced EBs. The intensively stained band below the MOMP at approximately 40 kDa (lanes 1 to 3) is a degradation product of the MOMP. MW, molecular weight standards, in thousands.
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FIG. 3. SDS-PAGE (A) and immunoblot analysis (B) of EB and RB proteins during the developmental cycle. Electrophoresis was carried out under reducing conditions, and the blot was reacted with anti-POMP 90A. Lanes: 1, 15-h RBs; 2, 24-h RBs; 3, 28-h RBs; 4, 48-h EBs; 5 to 8, Sarkosyl COMCs of the samples shown in lanes 1–4. Prestained protein standards (MW, in thousands) were run at the sides of the panels. The positions of putative POMPs (positions 1 to 6), the CRPs, and the MOMP are indicated.
with reducing agents, the CRPs, but not the MOMP and protein 7, were rendered completely soluble (Fig. 2A, lane 3). This observation, which confirms the previous observations of Everett and Hatch (10), suggests that the CRPs are located in the periplasm and are found in the Sarkosyl COMC fraction only because of their extensive disulfide cross-linked nature, not because they are integral OM proteins. The intensity of the putative POMP bands was decreased in Sarkosyl COMCs prepared from reduced EBs (Fig. 2A, lane 3). The observations that the putative POMPs were labeled with [125I]TID and that some had exposed trypsin-sensitive sites (Fig. 1) support an OM rather than a periplasmic location for the POMPs. Therefore, the effect of reducing agents on the POMPs in COMCs suggests that disulfide bonds play a role in the maintenance of the POMPs in OMs. When EBs were reduced with DTT and M⌭ and then treated with SDS, no proteins were found in the insoluble fraction (Fig. 2A, lane 5), suggesting that the maintenance of all proteins in the SDS-insoluble complex is dependent upon disulfide bonds. Stage-specific expression of POMPs. To determine the developmental stage specificity of the putative POMPs, whole
chlamydiae and Sarkosyl COMCs were analyzed by SDSPAGE and immunoblotting at 15, 24, 28, and 48 h postinfection (Fig. 3). The 15- to 28-h harvests consisted of densitygradient-purified RBs, and the 48-h preparations were purified, Nonidet P-40-treated EBs (to eliminate osmotically fragile RBs in the EB fraction). The material loaded on the gels was adjusted so that the amount of the MOMP, which is present throughout the cycle, was approximately the same in all preparations. Under these conditions of analysis, the putative POMPs and the late-stage-specific CRPs were not detected in either whole RBs or COMC preparations at 15 h but were detected in increasing amounts as the infection progressed to completion between 28 and 48 h postinfection. These observations indicate that the six putative POMPs of C. psittaci 6BC are late-stage specific. DISCUSSION We identified three POMPs in C. psittaci 6BC that crossreact with antiserum prepared against the POMP 90 family of OEA C. psittaci S26/3. It is likely that three additional proteins (proteins 1 to 3) in C. psittaci 6BC are POMPs on the basis of
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their large sizes, location in the OM, and the lack of predicted non-POMP OM proteins of similar sizes in the C. trachomatis and C. pneumoniae genome databases (18, 35, 38). Two of the six putative POMPs have a surface-exposed, trypsin-sensitive site. The precise role of disulfide bonds in maintaining C. psittaci POMPs in the OM is not clear. In contrast to the MOMP, protein 7, and the CRPs, some proportion if not all of the POMPs were capable of migrating in gels at approximately the same rates in the presence and absence of reducing agents, suggesting that they are not interpeptide cross-linked. On the other hand, the POMPs were released from the SDS-insoluble complexes and partially solubilized from the Sarkosyl COMCs when the complexes were prepared from EBs treated with reducing agents. It is possible that intrapeptide disulfide bonds play some role in maintaining POMPs in detergent-insoluble complexes, perhaps by allowing a noncovalent association of POMPs with themselves or with a cross-linked MOMP in the OM. Alternatively, they may associate with supramolecular structures in the periplasm, such as the glycanless wall polymer proposed by Ghuysen and Goffin (11) or the disulfide-crosslinked CRP complex proposed by Everett and Hatch (10). The ability of the MOMP to form oligomeric complexes in SDS has been shown by Wyllie et al. (40) and McCafferty et al. (28) Immunoelectron microscopic studies by Longbottom et al. (24, 25) indicated that one or more members of the POMP 90 family are exposed on the surfaces of 24-h RBs and 48-h EBs of ovine C. psittaci S26/3 24 and 48 h postinfection. This observation is consistent with our finding that C. psittaci 6BC POMPs were found in gradient-purified RBs at 24 and 28 h postinfection and in EBs. However, we failed to detect POMPs and the CRP doublet proteins when we examined middlestage, logarithmically dividing RBs, suggesting that C. psittaci 6BC POMPs, like the CRPs, are late-stage specific and therefore are not required for RB growth and cell division. In a preliminary study, Lindquist and Stephens (23) reported that transcripts orthologous to the nine C. trachomatis D POMP genes could be detected in C. trachomatis L2 by reverse transcription-PCR between 10 and 48 h postinfection, suggesting that C. trachomatis POMPs are made early in the growth cycle. However, the POMP transcripts were not quantified, nor were their levels compared with the levels of other chlamydial gene transcripts; thus, the possibility that C. trachomatis POMP genes are relatively more highly expressed during the late phase of the developmental cycle cannot be excluded. The numbers of POMP genes in the genomes of C. psittaci 6BC and other C. psittaci strains are not known. Six POMP genes have been identified in the genome of C. psittaci S26/3; however, by analogy to C. trachomatis and C. pneumoniae, additional POMP genes are likely to be present in the genomes of C. psittaci strains. Our finding of six POMPs in the OM of C. psittaci 6BC is higher than the numbers found thus far in C. trachomatis (two) and C. pneumoniae (four) (19, 30). It is possible that many more POMP genes are expressed as proteins in chlamydiae growing in tissue cultures but are present in amounts not readily detected by conventional staining and labeling techniques. Alternatively, some POMP genes may be expressed only under specific in vivo conditions, as speculated by Birkelund et al. (8). The C. pneumoniae POMP genes are particularly interesting in that six genes do not appear to encode full-length proteins
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and thus may represent a reservoir for recombination or mutation (14, 18). At least one example of mutation has already been identified: the open reading frame (Cpn 449/450) that encodes Pmp10, also referred to as OMP5, in strain CDC/ CWL-029/VR1310 contains a frameshift, as sequenced by Kalman et al. (18), but is expressed as a full-length POMP, as reported by Knudsen et al. (19) and Pedersen et al. (34). Pedersen et al. (34) speculated that the expression of the open reading frame was dependent on the addition or deletion of nucleotides from a poly(G) tract within the coding region, most likely at the genomic level. An interesting result of our study was the finding that sulfoNHS-biotin, which is useful for labeling proteins exposed on the surfaces of eukaryotic cells, appeared to penetrate the OM of chlamydial EBs, labeling a wide range of proteins that were not labeled with [125I]TID. The EB form of chlamydiae is impermeable to ATP, GTP, and amino acids that are taken up by specific transport mechanisms in RBs (17). Bavoil et al. (4) suggested that the impermeability of EBs may be a function of the MOMP porin, which they found in a liposome swelling assay to require the reduction of disulfide bonds and the blockage of sulfhydryl groups with iodoacetamide for activity. However, Wyllie et al. (40) found that an MOMP reconstituted into planar lipid bilayers maintained porin activity without the blockage of sulfhydryl residues and that treatment of the bilayers with oxidative reagents did not inhibit activity. It is possible, therefore, that the hydrophilic cross-linking reagent gained entry to the periplasm through the MOMP porin and that the impermeability of EBs is related to the inactivity of specific transport systems in the inner membrane rather than to exclusion by the OM. Another interesting result of our study was the discovery of 48-kDa protein 7, which is one of the interpeptide-disulfidecross-linked OM proteins found in EBs but which does not appear to be late-stage specific (Fig. 3). Mass spectroscopic analysis of a gel slice containing protein 7 revealed the presence of an MOMP in the band. It is possible that protein 7 is a posttranslationally modified form of MOMP, as has been proposed by Kuo et al. (20) for the MOMP of C. trachomatis L2. However, Kuo et al. (20) presented evidence that a “standard” MOMP, rather than a protein of higher molecular weight, is glycosylated, as is the case for protein 7. Alternatively, protein 7 may be an unknown OM protein which is contaminated by the highly abundant MOMP, the result of smearing during electrophoresis. In either case, it is interesting that Newhall et al. (32) found in multiple serovars of C. trachomatis a similar band that reacted on immunoblots with the sera of some patients infected with C. trachomatis. We are currently attempting to identify the homolog of protein 7 in a DNA-sequenced strain of C. trachomatis. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI-19570 from the National Institute of Allergy and Infectious Diseases and the Scottish Executive Rural Affairs Department. Mass spectrometric studies were carried out in the Stout Neuroscience Laboratory at the University of Tennessee, which is supported by Public Health Service grant RR 105222 and National Science Foundation grant DBI 9604633. REFERENCES 1. Allen, J. E., M. C. Cerrone, P. R. Beatty, and R. S. Stephens. 1990. Cysteinerich outer membrane proteins of Chlamydia trachomatis display compensa-
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