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A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelop of E coli
Biochimie (1990) 72, 385-395 (~) Soci~t6fran~aisede biochimie et biologic molrculaire/Elsevier, Paris
385
A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli JM Bolla*, A Bernadac,
C Lazdunski, JM Pages
Centre de Biochimie et de Biologie Moldculaire, CNRS, 31 chemin Joseph-Aiguier, BP 71, 13402 Marseille Cedex 9, France
(Received 26 April 1990; accepted 28 June 1990)
Summary - Induciblehybrid genes encodingtwo large domains, a periplasmicdomain consistingof the PhoS sequence and an outer membrane
domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesizedhybrid polypeptidesare correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivitytoward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogoldlabellingon ultrathin cryosections.Insertion of a hydrophobicsequence betweenPhoS and OmpF improvesthe trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimerie conformationwhich is recognizedby specificantibodies. Moreover, the deletion of the C-terminalregion impairs the functionalinsertion of the OmpF domain; this indicatesthat the last membrane-spanningregion of OmpF is necessaryfor the correct folding and orientation of the protein in the outer membrane. outer membrane / export / immunolocalization/ colicin receptor
Introduction
O m p F is one of the m a j o r proteins of the o u t e r membrane of E coli. It is assembled as a functional trimer at the cell surface, and strongly associated to lipopolysaccharide [1, 2]. This porin has been shown to ta~,llltOtt~ t l t ~ ~ l l t l y O l 3 1 ! l i : l l I llyUl oplllllt; I I I U I I ~ I ~ U I ~ 111 the cell [3]. In addition, O m p F acts as a specific receptor for colicins and phages [1, 2]. The polypeptide synthesized in a precursor form in the cytosol is translocated across the cytoplasmic membrane where the leader peptidase cleaves its signal peptide [4, 5, 6, 7]. The later steps in export, ie, transfer to outer membrane, trimerization and insertion, are dependent on lipid synthesis suggesting a lipopolysaccharide role (LPS) [8]. The possibility of a specific sequence acting as an outer membrane address during the sorting process and its identification in the mature polypeptide is a crucial and open question. In fact, various intermediate steps are required before the complete integration and stability of a functiona~ protein in the membrane occurs [9, 10]. Thus, one cannot exclude the hypothesis that several domains of the molecule exposed at the surface during various conformational changes, as reported for O m p A [11, 12], act sequentially during the integration pathway. The exis-
*Correspondence and reprints
tence of a sorting signal common to several outer membrane proteins has been proposed [13]. However, at present, no evidence has been reported concerning the existence of a topogenic sequence of this kind, ensuring the localization of these proteins [14, 15]. Henning et al suggested the possible role of a region located between residues 154 to i80 of OmpA in the sorting mechanism. Amino acid substitutions in this region impaired incorporation in the outer membrane but not the translocation across the cytoplasmic membrane, indicating a strategic region for assembly in the mature sequence of O m p A [16]. In the case of FhuA, 2 regions may be required for correct, stable assembly: the C-terminal region is essential for the final folding of the protein in the membrane [17]; moreover, Coulton et al recently proposed that some information present in the sequence between residues 88 to 180 was a prerequisite for stable association with the outer membrane [18]. Recently, some evidence was obtained concerning the role of the C-terminal region of PhoE during the final assembly of the protein. The last membrane-spanning domain is required for correct outer membrane localization of the PhoE protein [19]. In order to obtain information about later steps in O m p F assembly, we have constructed hybrid genes comprising the quasi-complete sequence of PhoS, the
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periplasmic p h o s p h a t e binding protein [20], fused to various lengths of O m p F sequence. T h e presence of the total p r o m o t e r region o f p h o S gene m a d e overproduction of the chimeric p r o t e m possible under phosphate starvation. This kind of hybrid gene has been successfully used for the expression and purification of the P h o S - m h G R F ( G r o w t h - h o r m o n e Releasing Factor) protein, the presence of the PhoS signal sequence ensuring export of the hybrid to the periplasm [21]. However, the G R F region, consisting of a small peptide (44 a m i n o acid residues), represents only a few percent of the hybrid. In the case of P h o S - O m p F hybrids, we constructed larger proteins containing 2 putative domains, a periplasmic domain due to PhoS and a m e m b r a n e region involving the O m p F part, of an equivalent size. It has previously been dem~ntrated that the exchange of signal sequence between periplasmic and outer m e m b r a n e proteins did not alter the final location of the molecule [17, 22]. It was thus postulated that the mature sequence of the protein contained information ensuring the ultimate cellular location [23]. In our constructs, the presence of quasi total sequence of the 2 proteins m a y confer a dual address to these hybrids. T h e questions that arised after the processing step are firstly, what is the information on the polypeptide chain which will govern the final localization of the molecule and secondly, what is the effect of a large periplasmic domain (PhoS) on the putative trimerization of the O m p F domain? T h e analyses have been carried out using monoclonal antibodies directed against cell surface epitopes of O m p F [24, 25] and by testing the sensitivity toward colicins A and N of E coli B ompF, after expression of the various hybrid proteins.
Materials and Methods
medium (TGLP), cell samples were removed and total cellular proteins were analysed by SDS-PAGE. Pulse and pulse chase experiments Thirty rain and 90 min after induction, samples (5 x 108cells) were labelled for 1 rain at 37oC with [14C]-Iabelledamino acids (10 ttCi. ml-I) and chased for 20 min in the presence of unlabelled amino acids. For pulse chase experiments, 15 min and I h 15 after induction, samples (5 × l0 s cells) were labelled with [35S]-methionine(150 IxCi/ ml). An excess of unlabelled metbionine was added after a 20 s pulse and samples were subsequently removed during the chase at 0, 20, 40 s, 1, 2, 5, 10 and 30 mm. Each sample was immediately frozen in liquid nitrogen in the presence of chloramphenicol (50 ltg/ml). Solubilization and immunoprecipitations These were carried out as previously described [10]. Briefly, samples were resuspended and homogenized in Tris/EDTA/SDS buffer (50 mM Tris-HC! pH 8.0, 1 mM EDTA, I% SDS). An equivalent volume of this material was then solubilized at 56O C or 96~C. A 15fold excess of immnnoprecipitation buffer (I0 mM Tris-HCI pH 7.4, 150 mM NaCI, 5 mM EDTA) containing Triton X-100 (1% final concentration) was added. Incubation with the various antibodies was then carried out according to [10]. SDS- PAGE and immunoblotting SDS-PAGE (10% acrylamide), protein staining and fluorography were carried out as previously described [10]. The immunoblotting was carried out according to [21]. Colicin sensitivity The coUcin assay by survival test was previously described [28]. Briefly, after 15 rain of induction the cells (1 ml of suspension at DOa~0nm= 0.5) were added to various concentrations of colicins diluted from 10 to l0 Tin 10 mM sodium phosphate pH 7.4, Triton X-100 (0.1%) buffer for 20 min at 37oC. The cellular suspension was then diluted by 1 vol of fresh medium. The percentage of surviving cells was monitored by the ratio of optical densities at 600 nm from culture treated with colicins to untreated culture after 2 h at 37oC. lmmunolabelling of proteins on ultrathin frozen sections
Chemicals [35S]-Methionine was purchased from Amersham. [14C].labelled amino acids were purchased from the Commissariat/~l'i~nergieAtomique. The restriction enzymes, T4 DNA ligase and Klenow fragment of DNA polymerase I were purchased from Boehringer Manheim. Trypsin (TPCK) and trypsin inhibitor were purchased from Sigma Chemical Inc. Bacterial strain, plasmids and medium E coli B strain BZB 1107 (ompF: Tn5, Km R) from Dr Eisei~ has been described previously [25]. Piasmids pSN5182 and pLG361 have also been described previously [17, 26]. Tris-glucose medium, supplemented with required nutrients and phosphate, according to Morita et al [26], was used. Tetracyclin (10 Itg/ml) and Kanamycin (50 p,g/ ml) were used for selection of transformants and for ensuring the maintenance of strains and plasmids. Expression of hybrid genes Derepression of the pho regulon was carried out as previously described [27]. At various times after transfer to low phosphate
The detailed procedure for fixation, sectioning, immunolabelling and staining has been previously described [29]. Protease accessibifity experiments After 50 min of induction, cells were labelled for 10 min with [z4C]amino acids (5 IxCI/ ml) and chased for 10 rain with non radioactive amino acids (1% final concentration). After centrifugation at 4oC, cell pellets were resuspended in ice cold Tris 100 mM pH 8 containing either (10 raM) MgCI2 or (5 raM) EDTA [30]. Trypsin (100 I~g/ml final concentration) was added to half of each sample and incubation was carried out for 30 rain in ice. Trypsin inhibitor (500 Ixg/mi final concentration) was then added and samples were precipitated with trichloro acetic acid for 15 rain in ice. Precipitates were solubilized at 96oC for 10 rain in preheated sample buffer before immunoprecipitation. Antibodies Polyclonal antisera directed against PhoS and OmpF have been previously described as have monoclonal antibodies directed against native OmpF [24-26].
Assembly of PhoS-OmpF hybrid proteins in E coli
Results
Construction of a series of phoS-OmpF hybrid genes The plasmid pSN5182 [26] carrying the phoSgene with its own promoter was used as a vector. Different fragments of the ompFgene were cloned into the single restriction enzyme cleavage site for Sspl present in pSN5182. This blunt-ended site is in frame and located 10 codons upstream of the TAA stop codon of phoS gene. The orapF carrying plasmid pLG361 [17] was digested with restriction enzymes, and the relevant fragments were purified as described in Materials and Methods. After ligation, transformants were selected for tetracyclin resistance and the corresponding plasmids were purified. In order to check the correct orientation of the inserted fragment, digestion by appropriate enzymes was carried out. The pAB5 plasmid was constructed by exchanging the small Bglll-EcoRl fragment present in pAB3 for the corresponding one purified from pAB4. Transformants were selected for the presence of both the Pstl and HincII sites in the hybrid gene. Figure 1 shows the proteins encoded by the different constructed hybrid genes. The fusion proteins had all but 10 of the residues of PhoS protein fused to various polypeptide regions derived from OmpF. In the case of pAB3 and pAB5, the fusion takes place between residue 311 of mature PhoS and the 5th
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Fig 1. Structure of PhoS-OmpF hybrid proteins. Stippled areas, prePhoS protein; black areas, OmpF signal sequence; open areas, mature OmpF protein. Numbers above PhoS, amino acids position within the PhoS protein; numbers below OmpF, amino acids position within the OmpF protein. The number of residues of each protein is indicated for its mature sequence, a.a.: aminoacids.
387
amino acid residue of the OmpF signal sequence and they are only different in COOH-terminal region (fig 1). The former construct comprises the OmpF sequence with the last 29 amino acid residues deleted, while the latter contains the complete sequence of OmpF. pAB4 was constructed by digesting with Klenow fragment of DNA polymerase I the PstI extremities of the ompF fragment from pLG361, resulting in an in-frame deletion of the first amino acid of mature OmpF. The BZB 1107 strain was transformed by the 3 plasmids. This ompF E coli B strain was selected to allow immunodeteetion of trimerie forms using several monoclonal antibodies as probes.
Synthesis and processing of the PhoS-OmpF hybrid proteins At various times after growth under phosphate limitation conditions, aliquots of cultures were removed, and total cellular proteins analysed by S D S - P A G E and immunoblotting. The respective apparent molecular weight of the hybrids were in accordance with D N ~ analysis of the different fusion genes and the putative size of their products (fig 1). The polyclonal antisera directed against either PhoS [26] or OmpF [24] proteins were used for immunodetection because hybrid proteins containing the quasicomplete sequence (311 amino acid residues compared to 321) of PhoS and almost 80% of the OmpF sequence, harbour antigenic determinants recognized by these 2 polyclonal antibodies (fig 2). BZB and BZB pLG361 cells were used as controls in order to compare the different fusion gene products to the background. At 90 rain of induction, a product was significantly recognized by the 2 antisera (fig 2). The migration of the immun0-recognized proteins was consistent with the calculated molecular weight of hybrid molecules. At a later time after transfer to phosphate-limiting medium, several other bands were detected by the 2 antisera. Among them, a product was observed migrating above the mature form. A stable accumulation of the precursor form has been previously shown during the hyperproduction of PhoS [27]. Kinetics analyses suggested that a jamming of export sites occured when the synthesis of PhoS reached a high production level [27]. Such a process could also induce the accumulation of hybrid precursors. In addition, some products with lower apparent molecular weights were immunorecognized indicating a degradation of the hybrid. During normal export, in most cases cleavage of the signal peptide is a good indication that correct translocation of precursor forms across the cytoplasmic membrane has occured, although exceptions to this case have been reported [5]. The efficiency of processing
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was thus analysed with the various hybrids at 15 and 75 min after induction (fig 3). During the early growth period in phosphate minimal medium, only a weak signal corresponding to the precursor form of hybrids was detected in the pulse, the large majority of immunoprecipitated material corresponding to mature form. By 75 rain after induction, a large amount of a product migrating slower than mature form was accumulated. This polypeptide, related to precursor and appar,~ntly stable during the chase, could reflect jamming of the export machinery as previously reported during the hyperproduction of PhoS [27].
Assembly of the chimeric proteins
A B
Porin trimers are very stable structures not dissociated even in the presence of SDS, at temperatures below 75°C [9]. It was thus interesting to investigate the ability of the various chimeric proteins to assume such a conformation in order to determine whether the information comprised in the OmpF part could be expressed. To this end, 2 different experimental approaches were used. The kinetics of trimerization were studied and immunological analyses using various monoclonal antibodies as conformational probes were carded out. The results concerning the rate of trimerization after 1 h 15 of induction are presented in figure 4. No material migrating at high molecular weight was detected after a long chase time with PAB3 (fig 4A), even when
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Assemblyof PhoS-OmpFhybrid proteins in E coli
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the gel was overexposed. In contrast with PAB4 and PAB5, products showing a slow migration corresponding to hybrid oligomers were detected (figs 4B and 4C). At an early time of induction, the same pattern was obtained for the 3 hybrids but precursor forms were not accumulated (data not shown). In addition, the presence of oligomers was not related to overproduction because the high molecular weight material was detected as early as 15 rain after induction (data not shown). These results suggested that the COOH-terminal region of OmpF was necessary to promote efficient oligomerization of hybrids because PAB3 was not able to form oligomers. Only 1 major band corresponding to putative trimeric form was obtained with PAB4 in contrast to PAB5 which presented several high molecular weight products. Immunological analyses were performed using monoclonal antibodies previously described to rule out the hypothesis that some aggregation process could promote the presence of such high molecular weight products (fig 5). PAB3 hybrid protein which was recognized by polyclonal antisera directed either against OmpF or against PhoS, was not immunoprecipitated by the various monoclonal antibodies (MoF) specific to OmpF. The antigenic sites recognized by MoF 18, 19 and 20 have been recently located at the COOH-terminal region of OmpF (Fourel et al, unpublished results), moreover the epitope recognized by MoF18 has been detected on the monomeric surface [31]. Failure to
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(bottom) were labelled with [14C]-amino acids after 90 min of induction. Cell samples were subsequently solubilized in TES buffer at 56°C, immunoprecipitated and loaded on 10% polyacrylamide gels after denaturation in sample buffer. Lanes 1 and 2 correspond to anti-PhoS and anti-OmpF polyclonal antisera respectively; lanes 3 to 10 correspond to MoF1, MoF3, MoF4, MoF7, MoF18, MoF19, MoF20 and MoF21 monoclonal antibodies respectively. Monoclonal antibodies were previously described [24, 25].
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JM Bolla et al
detect PAB3 could thus be related to a lack of epitope removed by the deletion of the last 30 residues of OmpF. In addition, the absence of signal with other MoF suggests that PAB3 is either unfit to trimerize or only able to organize very unstable trimers that are dissociated under the experimental conditions used. Some of the MoF [ 1, 3, 4, 7] have previously been described and recognize cell surface-epitopes present only on the trimeric conformation of OmpF [24]. In the case of PAB5 a significant signal was observed with these antibodies indicating that some chimeric molecules haboured the respective epitopes. These results suggest that 2 conformational populations of processed PAB5 molecules were detected in the cell. The ratio of the chimeric proteins immunoprecipitated with MoF to the material immunoprecipitated with the polyclonal represents the percentage of hybrid porin which contains the respective native antigenic determinants. 48% of the PAB5 molecules (with regard to MoF1, 4 and 7 to polyclonal antiserum ratio) are then able to reach a native conformation as checked by such immunological tools. The remaining population which cannot express the native epitopes, may be accumulated and mis-assembled during overproduction of hybrids. The percentage of trypsin-sensitive PAB5 molecule roughly correlated with the immunological analysis carded out with MoF.
Protease accessibility of hybrid proteins It has recently been reported that alterations of membrane proteins, such as insertion or deletion, can promote orotease suscentihilitv [32_ 331 In nrd~r tn invp~-
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bility of the 3 hybrids (PAB3, PAB4 and PAB5) was studied. To avoid the problem due to the distribution of methionine residues (absent in PhoS mature sequence), the experiment was carried out after [14C]amino acids labelling. In the presence of Mg 2+ ions, hybrid proteins were resistant to trypsin degradation (fig 6). In the presence of EDTA, as previously reported [34], the periplasmic domain of OmpA was cleaved generating the 24 kDa protected fragment (fig 6 left). Under these conditions, various patterns of sensitivity were obtained with the hybrids. PAB3 was almost completely sensitive to trypsin digestion (fig 6A) and no protected fragment was immunodetected with both antisera, indicating an extensive degradation of the 2 domains. This indicated that the OmpF region of PAB3 presented higher susceptibility to trypsin, suggesting a conformation different from native OmpF. With PAB4, only a few percent of hybrid molecules are accessible to protease, the majority being resistant to degradation (fig 6B). In the case of PAB5, half ot the hybrids (46%) were degraded and a resulting fragment was immunoprecipi-
rated by the antiserum directed against OmpF (fig 6C). This fragment, migrating with an apparent molecular weight of 40 kDa, was not recognized by the anti-PhoS serum~ indicating a degradation of the PhoS part. The high sensitivity of this domain was completely different from the trypsin resistance of native periplasmic PhoS. It could be compared to the extensive susceptibility of the membrane associated prePhoS previously reported [35] and thus suggested a labile unfolded conformation of PhoS exposing a large number of trypsic sites. It was concluded that oniy some of the PAB5 hybrids could generate an OmpF plotected fragment.
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Assemblyof PhoS-OmpF hybrid proteins in E coil
Colicin sensitivities of ompF E coil B expressing the hybrid proteins OmpF has been described a; functioning as (part of) the receptor for certain t'/hages and is required for the efficient killing action of a number of colicins [ 1,2]. For colicin N, OmpF is involved both in the binding step
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and in the translocation of the bacteriocin across the outer membrane [36, 37]. In the case of colicin A, the receptor structure is mere complex comprising BtuB, OmpF and LPS [28, 38]. The sensitivity of BZB 1107 (ompF E coil B) was tested 15 min after transfer to phosphate- limiting medium. Cells of ompF E coli B free of plasmid or transformed with pLG301, carrying the ompF gene, were used as negative and positive controls respectively (fig 7), tinder these conditiens, the same significant colicin A sensitivity was obtained when the synthesis of PAB3 and PAB4 was induced, PAB5 being 10 times more sensitive in the assay. With colicin N, a sensitivity was observed only when PAB4 or PAB5 were produced. Thus, PAB3 presenting neither stable trimeric forms under solubilisatio.~ conditions used, nor antigenic sites related to native trimers, can nevertheless restore partial coliein A uptake. This may indicate that a few molecules could insert into the outer membrane and act as an efficient receptor for colicin A, whereas no colicin N receptor activity is observed. The difference in sensitivity observed with PAB3, 4 and 5 compared to wild type Or~pF may reflect the transmembrane conformation of such hybrids containing a large periplasmic domain. However, the production of PAB4 and 5, both containing the COOH-terminal region, restored only partial sensitivity toward colicin N. This suggested that the membrane orientation of OmpF in the chimeric proteins could be modified by the PhoS region, and as a consequence could modify the affinity of colicin N for its receptor site.
lmmunocytolocalization of the chimeric proteins
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Fig 7. Sensitivity toward colicins A and N. The strains BZB 1107 (D) or carrying pLG361 (e), pAB3 (B), pAB4 (A) or pAB5 (O) were induced for 15 min in TGLP medium. Aliquots of each culture were then incubated with dilutions of colicin A (A), or colicin N (B) for 20 min. After dilution in fresh TGHP medium, the cell suspensions were grown for 2 h at 37°C and the percentage of survivals checked as described in Materialsand Method~, was plotted as a function of colicin concentration.
Tl~,e sensitivity towards colicin A suggested that chimeric proteins were at least partly localized in the outer membrane. In addition, oligomeric forms which exhibited native cell surface exposed epitopes were detected with PAB5. The synthesis of hybrid proteins may induce jamming of the export machinery and consequently abortive export and miscompartmentalization of newly synthesized molecules [39]. Such a phenomenon was shown to cause accumulation of cytoplasmic and membrane-associated precursor forms and sometimes cytoplasmic aggregation of overproduced proteins [14, 40], Subseque,ntly, during classical fractionati6n procedures, this aggregated material can be artifactually distributed in a membrane compartment. As previously described [14, 39, 40],.immunolabelling of frozen thin sections of E coil can m most cases permit the localization of proteins and prevent, after fractionation experiments, incor_-ect conclusions being derived from aggregation, etc. In order to investigate the location of various products, immunogoid labellings were performed after 1 h 30 of induction (fig 8). The
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monoclonal antibody MoFI8 was selected instead of polyclonal antiserum to avoid a cross reaction with PhoE which is induced duri-g culture in low phosphate medium. No labelling was observed in control cells and in cells containing PAB3 which do not contain the specific epitope. In contrast, immunogold labelling associated to the outer membrane was clearly detected during the expression of PAB4 and PAB5 (fig 8). Electron-dense areas were observed in cytoplasm. Although these areas were significantly labelled with the antiserum directed against PhoS (fig 8), they were not recognized with the monodonal antibody MoF 18
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(compare fig 8A to 8B). These results suggest a microconcentration of hybrids in discrete cytoplasmic regions where molecular aggregation probably occured. These accumulated products probably correlate with the precursor form immunodetected at long periods of incubation (figs 2, 3) and with non native form (fig 5). These aggregated precursor molecules which did not present the native epitope, are not recognized by MoF 18. During overproduction of PhoS similar results were reported with a cytoplasmic labelling corresponding to accumulation of pre-PhoS due to jamming of the export machinery [27].
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Fig 8. Localisation of PhoS-OmpF hybrids by immunolabelling. Cell samples of E coli BZB 1107 (a), or carrying pAB3 (b), pAB4 (c) or pAB5 (d) were removed after 1 h 30 of culture in TGLP medium. Ultrathin frozen sections were prepared and incubated with either an anti-PhoS serum (A) or a monoclonal antibody directed against OmpF (B). Labelling was performed with gold-coated protein A.
Assembly of PhoS-OmpF hybrid proteins in E coli
Discussion
We have presented in this study the construction of several PhoS-OmpF hybrid proteins. Two large domains corresponding to PhoS and OmpF containing information for periplasmic and outer membrane localization respectively were present in these hybrids. The question of the efficiency of export, of the cellular distribution and of the topology of these proteins must then be considered. Previous studies demonstrated that prePhoS protein fused to mhGRF was efficiently synthesized and processed and that the hybrid PhoS-GRF was localized in the periplasmic space [21, 41]. This suggested that PhoS domain protein could promote efficient export of fused polypeptide. It was also shown that the signal sequence and part of the mature sequence of a periplasmic protein (/3-1actamase) was able to direct an outer membrane protein PhoE to its correct compartment [22, 34]. During expression of the various PhoS-OmpF hybrid proteins in an ompF E coli B strain, we analysed their export by assaying the kinetics of signal sequence cleavage (fig 3). In order to study the subcellular localization of these proteins, we used 2 reliable experimental approaches: immunogold labelling and trypsin accessibility. In a second step, we probed the ability of these hybrids to exhibit OmpF properties. Their conformational and functional characteristics were checked by immunological analyses of the trimerization pattern and by their ability to restore colicin sensitivity. The hybrid PAB5 contained the quasi complete sequence of PhoS fused to the preO-mpF sequence deleted from the positively charged amino-terminal part ( - 22 to - 17). Oligomeric products related to trimeric hybrid forming about 40-50% of mature PAB5 molecules were detected in the cells, and cellsurface exposed epitopes corresponding to the native OmpF trimers were immunorecognized on the chimeric protein. The sensitivity toward trypsin in the presence of EDTA indicated that among the 46% of the sensitive hybrids corresponding to correctly localized molecules, the PhoS part of PAB5 was accessible and degraded, while the OmpF domain was resistant as previously reported for the native porin [42]. These results suggest that this PhoS domain cannot reach the native conformation exhibited by the wild type periplasmic phosphate binding protein when anchored in the outer membrane via the OmpF part. This could explain the failure of outer membrane labelling with the antiserum directed against PhoS. Although no evidence was obtained about the conformation of the PhoS domain from hybrids into the membrane, it is possible that the artificial association of PhoS polypeptide with the outer membrane
393
modifies the antigenic sites by steric hindrance or association with outer membrane components. Alternatively, the deletion of last COOH region of PhoS could alter the antigenic properties of the molecule. In the light of the trypsin resistance of periplasmic PhoS [36], it must be concluded that the PhoS domain is not properly folded in hybrid molecules. It is likely that the translocation competent conformation of PhoS (partially unfolded) cannot return to the native conformation due to the continuous association with a membrane even after translocation. With periplasmie proteins, the dissociation from the inner membrane and final folding constitutes a distinct step in the export pathway as demonstrated with fl-lactamase [43] and maltose binding protein [44]. The efficiently assembled PAB5 molecules restore the sensitivity of OmpF cells toward colicins A and N to almost the same level as cells harbouring the ompF gene. When the COOH-terminal region (311-340 residues) was deleted generating PAB3 chimeric protein, the hybrid was unfit to promote stable oligomerization and to present native epitopes. It-exhibited larger protease sensitivity as a consequence of its abortive assembly. In addition, it could only restore the colicin A sensitivity while resistance to colicin N was observed. These results indicate that the COOH-terminal region of OmpF is necessary to promote stable assembly of the protein in the outer membrane. In the case of PAB4, the PhoS region was directly fused to the 2nd residue of OmpF mature sequence. This last chimera presented oligomeric forms different from PAB5 but was able to restore colicin A and N sen151tlVltl~5.
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molecules were susceptible to protease degradation. A significant immunogold labelling was nevertheless observed on the outer membrane with a monoclona! antibody directed against a cell surface-exposed monomeric epitope of OmpF. Thus, the hydrophobic segment of OmpF signal peptide inserted between the 2 domains could stabilize the outer membrane oligomers or enhance the membrane insertion but did not significantly modify the receptor function of OmpF domains and the exposure of antigenic determinants. Results obtained with PAB3 compared to those obtained with PAB4 and PAB5 indicate that the information required for the correct and stable insertion of hybrids is contained in the COOH-terminal region of OmpF. This sequence corresponds in fact to the last membrane spanning peptide proposed in the models described in [45] and [46]. These results, in addition to a recent report [19] describing similar properties of the carboxy-terminal region of PhoE strongly suggest that the last part of porin sequences are necessary during the oligomerization process in order to promote a correct assembly within
394
JM Bolla et al
the outer membrane. Hikita et al have recently observed that the carboxy-terminal region of OmpF and OmpC can be exchanged without alteration in porin assembly [47]. Thus, the role of the last putative membrane spanning region could be a common strategic element during the stable assembly of porins. The insertion of the hydrophobic part of OmpF signal peptide into the mature sequence of OmpC has recently been reported to impair the assembly of the chimenc porin in the outer membrane without altering its export [48]. In our case, the same sequence located between 2 separate domains, PhoS and OmpF, improved the insertion of hybrids. Thus, this location, which ensures a clean separation between the OmpF and PhoS regions in PAB5 could therefore facilitate independent folding of complete OmpF. This process may be perturbed by neighbouring PhoS polypeptide in the other constructs.
Acknowledgments We thank D Cavard, JL Eisel~, M Knibiehler, B Holland and Shinagawa for their generous gift of strains, plasmids, colicins and antiserum, and lmmunotech for carrying out the preparation of monoclonal antibodies. We gratefully acknowledge the helpful discussions with SP Howard and D Cavard, the help of M Bidaud in computer analysis of DNA and M Payan in secretarial support. This work was supported by the Centre National de la Recherche Scientifique, the lnstitut de la Sant~ et de la Recherche Mrdicale (CRE No 86.1020) and the Fondation pour la Recherche Mrdicale.
References 1 Lugtenberg B, Van Alphen L (1983) Molecular architecture and functioning of the outer membrane of Escherichm co!i and other gram-negative bacteria. Biochim Biophys Acta 737, 51-115 2 Nikaido H, Vaara M (i987) Outer membrane. In: Escherichia eoli and Salmonella typhimurium, Cellular and Molecular Biology (Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE, eds) ASM Publications, Washington, 1, 7-22 3 Hancock REW (1987) Role of porins in outer membrane permeability. J Bacteriol 169, 929-933 4 Lee C, Beckwith J (1986) Co-translational and post-translational proteins translocation in procaryotic systems. Ann Rev Cell Biol 2, 315-336 5 Randall LL, Hardy SJS, Thom JR (1987) Export of protein: a biochemical view. Ann Rev Microbio141, 507-541 6 Baker K, Mackman N, Holland IB (1987) Genetics and biochemistry of the assembly of proteins into the outer membrane of E coil Prog Biophys Mol Bio149, 89-115 7 Saier MH, Werner PK, Muller M 0989) Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotie process. Microbiol Rev 53,333-366 8 Bolla JM, Lazdunski C, Pages JM (1988) The assembly of the major outer membrane protein OmpF of Escherichia coil depends on lipid synthesis. E M B O J 7, 3595-3599 9 Reid J, Fang H, Gehring K, Klebba PE, Nikaido H (1988) Targeting of porin to the outer membrane of Escherichia coil J Biol Chem 263, 7753-7759 10 Pages JM, Bolla JM (1988) Assembly of the OmpF porin in E~cherichia coli B. Eur J Biochem 176, 655-660
11 Freudl R, Schwartz H, Strerhof YD, Gamon K, Hindennach I, Henning U (1086) An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem 261, 11355-11361 12 Geller BL, Green HM (1989) Translocationof Pro-OmpA across inner membrane vesicules of Escherichia coli occurs in two consecutive energetically distinct steps. J Biol Chem 264, 16465-16469 13 Benson SA, Bremer E, Silhavy TJ (1984) Intragenic regions required for Lamb export. Proc Nail Acad Sci USA 81, 3830-3834 14 Freudl R, Schwartz H, Klose M, Mowa RN, Henning U (1985) The nature of information, required for export and sorting present within the outer membrane protein OmpA of Escherichia coli K-12. E M B O J 4, 3593-3598 15 Bosch D, Leunissen J, Verbakel J, de Jong M, van Erp H, Tommassen J (1986) Periplasmie accumulation of truncated forms of outer membrane PhoE proteins of Escherichia coli K12. J Mol Biol 189,449-455 16 Klose M, Schwarz H, Mac Intyre S, Freudl R, Eschbach ML, Henning U (1988) Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide. J Biol Chem 263, 13291- 13296 17 Jackson HE, Pratt JE, Stocker NG, Holland IB (1985) An inner membrane protein N-terminal signal sequence is able to promote efficient Iocalisation of an outer membrane protein in Escherichia coil E M B O J 4, 2377-2383 18 Coulton, JW, Reid GK, Campana A (1988) Export of hybrid proteins FhuA-LacZ and FhuA-PhoA to the cell envelope of Escherichia coli K-12. J Bacteriol 170, 2267-2275 19 Bosch D, Scholten H, Verhagen C, Tommassen J (1989) The role of the earboxy-terminal membrane-spanningfragment in the biogenesis of Escherichia coli K-12 outer membrane protein PhoE. Mol & Gen Genet 216, 144-148 20 Magota K, Otsuji N, Miki T, Horiuchi T, Tsunasawa S, Kondo J, Sakiyama F, Amemura M, Morita T, Shinagawa H, Nakata A (1984) Nueleotide sequence of the phoSgene, the structural gene for the Phosphate-binding protein of Escherichia coli. J Bacteriol 157, 909-917 21 Anba J, Baty D, Lloubrs R, Pagrs JM, Joseph-Liauzun E, Shire D, Roskam W, Lazdunski C (1987) Expression vector promotinQ the gvnl'h~glgand ~vn,'sr~c~f*h. h. . . . . . . . . . . *k k. . . . . . releasing factor in Escherichia coli. Gene 53, 219-229 22 Tommassen J, van Tol H, Lugtenberg B (1983) The ultimate localization of an outer membrane protein of Escherichia coli K12 is not determined by the signal sequence. E M B O J 2, 1275-1279 23 Jackson ME, Pratt JM, Holland IB (1986) Intermediates in the assembly of the TonA polypeptide into the outer membrane of Escherichia coli K-12. J Mol Biol 189, 477-486 24 Pages JM, Pagrs C, Bernadac A, Princ6 P (1988) Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12. Mol lmmuno125, 555-563 25 Lupi N, Bourgois A, Bernadac A, Laboucari6 S, Pagrs JM (1989) Immunological analysis of porin polymorphism in Escherichia coli B and K-12. Mol lmmuno126, 1027-1036 26 Morita T, Amemura M, Makino K, Shinagawa H, Magota K, Otsuji N, Nakata A (1983) Hyperproduction of phosphatebinding protein, PhoS, and pre-PhoS protein in E coli carrying a cloned phoSgene. Eur J Biochem 130,427-435 27 Pagrs JM, Anba J, Bernadac A, Shinagawa H, Nakata A, Lazdunski C (1984) Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coll. Eur J Biochem 143,499-505 28 Cavard D, Lazdunski C (1981) Involvement of BtuB and OmpF proteins in binding and uptake of colicin A. FEMS Microbiol Left 12, 311-316 29 Anba J, Bernadac A, Pagrs JM, Lazdunski C (1984) The periseptal annulus in Escherichia coil Biol Cell 50, 2"13-278 --
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Assembly of PhoS-OmpF hybrid proteins in E coli 30 Bosch D, Voorhout W, Tommassen J (1988) Export and localization of N-terminally truncated derivatives of Escherichia coil K-12 outer membrane protein PhoE. J Biol Chern 263, 9952-9957 31 Pagrs JM, Bolla JM, Bernadac A, Fourel D (1990) Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli. Biochimie 72, 169-176 32 Freudl R, Mac lntyre S, Degen M, Henning U (1986b) Cell surface exposure of the outer membrane protein OmpA of Escherichin coli K-12. J Moi Biol 188,491-494 33 Morona R, Tommassen J, Henning U (1985) Demonstration of a bacteriophage receptor site on the Escherichia coli K-12 outer membrane protein OmpC by the use of protease. E u r J Biochem 150, 161-169 34 Tommassen J, Lugtenberg B (1984) Amino terminus of outer membrane PhoE protein: localization by use of a bla-phoE hybrid gene. J Bacteriol 157, 327-329 35 Anba J, Pagrs JM, Lazdunski C (1986) Mode of transfer of the phosphate-binding protein through the cytoplasmic membrane in Escherichia coli. F E M S Microbiol Lett 34, 215-219 36 Tommassen J, Pugsley AP, Korteland J, Lugtenberg B (1984) Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K-12. Mol & Gen Genet 197, 503-508 37 Pugsley AP (1984) The ins and outs of colicins. Microbiol Sci 1, 168-176 & 203-205 38 Chai TJ, Wu V, Foulds J (1982) Colicin A receptor: role of two Escherichia coli outer membrane proteins (OmpF protein and btuB gene product) and lipopolysaccharide. J Bacteriol 151, 983-988 39 Tommassen J (1986) Fallacies of E coli cell fractionations and
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consequences thereof for protein export models. Microbiol Pathogenesis I, 225-228 40 Tommassen J, Lennissen J, van Damme-Jongsten M. Overdium P (1985) Failure of E coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm. E M B O J 4, 1041- 1047 41 Anba J, B~,rnadacA, Lazdunski C, Pages J (1988) Improving the stability of a foreign protein in the periplasmic space of Escherichin coli. Biochimie 70, 727-733 42 Rosenbusch JP (1974) Characterization of the major envelope protein from Escherichia coil J Biol Chem 249. 8019-8029 43 Minsky A, Summers RG, Knowles JR (1986) Secretion ofl3-1actamase into the periplasm of E coli: evidence for a distinct release step associated with a conformational change. Proc NatlAcad Sci USA 83, 4180-4184 44 Thom JR, Randall LL (1988) Role of the leader peptide of Maltose-Binding-Protein in two steps of the export process. J Bacteriol 170, 5654-5661 45 Benson SA, Occi JLL, Sampson BA (1988) Mutations that alter the pore function of OmpF porin of Escherichia coli K-12. J Mol Bio1203,961-970
46 Carlton P, Rosenbusch JP (1985) Folding patterns of porin and bacteriorhodopsin. E M B O J 4, 1593-1597 47 Hikita L, Satake Y, Yamada H, Mizuno T, Mizushima S (1989) Structural and functional characterization of the OtnpF and OmpC porins of the Escherichia coli outer membrane: studies involving chimeric proteins. Res Microbiol 140, 177-190 48 Shinkai A, Yamada H, Mizuno T, Miznshima S (1989) Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E coli. J Biochem 106, 323-330
385
A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli JM Bolla*, A Bernadac,
C Lazdunski, JM Pages
Centre de Biochimie et de Biologie Moldculaire, CNRS, 31 chemin Joseph-Aiguier, BP 71, 13402 Marseille Cedex 9, France
(Received 26 April 1990; accepted 28 June 1990)
Summary - Induciblehybrid genes encodingtwo large domains, a periplasmicdomain consistingof the PhoS sequence and an outer membrane
domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesizedhybrid polypeptidesare correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivitytoward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogoldlabellingon ultrathin cryosections.Insertion of a hydrophobicsequence betweenPhoS and OmpF improvesthe trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimerie conformationwhich is recognizedby specificantibodies. Moreover, the deletion of the C-terminalregion impairs the functionalinsertion of the OmpF domain; this indicatesthat the last membrane-spanningregion of OmpF is necessaryfor the correct folding and orientation of the protein in the outer membrane. outer membrane / export / immunolocalization/ colicin receptor
Introduction
O m p F is one of the m a j o r proteins of the o u t e r membrane of E coli. It is assembled as a functional trimer at the cell surface, and strongly associated to lipopolysaccharide [1, 2]. This porin has been shown to ta~,llltOtt~ t l t ~ ~ l l t l y O l 3 1 ! l i : l l I llyUl oplllllt; I I I U I I ~ I ~ U I ~ 111 the cell [3]. In addition, O m p F acts as a specific receptor for colicins and phages [1, 2]. The polypeptide synthesized in a precursor form in the cytosol is translocated across the cytoplasmic membrane where the leader peptidase cleaves its signal peptide [4, 5, 6, 7]. The later steps in export, ie, transfer to outer membrane, trimerization and insertion, are dependent on lipid synthesis suggesting a lipopolysaccharide role (LPS) [8]. The possibility of a specific sequence acting as an outer membrane address during the sorting process and its identification in the mature polypeptide is a crucial and open question. In fact, various intermediate steps are required before the complete integration and stability of a functiona~ protein in the membrane occurs [9, 10]. Thus, one cannot exclude the hypothesis that several domains of the molecule exposed at the surface during various conformational changes, as reported for O m p A [11, 12], act sequentially during the integration pathway. The exis-
*Correspondence and reprints
tence of a sorting signal common to several outer membrane proteins has been proposed [13]. However, at present, no evidence has been reported concerning the existence of a topogenic sequence of this kind, ensuring the localization of these proteins [14, 15]. Henning et al suggested the possible role of a region located between residues 154 to i80 of OmpA in the sorting mechanism. Amino acid substitutions in this region impaired incorporation in the outer membrane but not the translocation across the cytoplasmic membrane, indicating a strategic region for assembly in the mature sequence of O m p A [16]. In the case of FhuA, 2 regions may be required for correct, stable assembly: the C-terminal region is essential for the final folding of the protein in the membrane [17]; moreover, Coulton et al recently proposed that some information present in the sequence between residues 88 to 180 was a prerequisite for stable association with the outer membrane [18]. Recently, some evidence was obtained concerning the role of the C-terminal region of PhoE during the final assembly of the protein. The last membrane-spanning domain is required for correct outer membrane localization of the PhoE protein [19]. In order to obtain information about later steps in O m p F assembly, we have constructed hybrid genes comprising the quasi-complete sequence of PhoS, the
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JM Bolla et al
periplasmic p h o s p h a t e binding protein [20], fused to various lengths of O m p F sequence. T h e presence of the total p r o m o t e r region o f p h o S gene m a d e overproduction of the chimeric p r o t e m possible under phosphate starvation. This kind of hybrid gene has been successfully used for the expression and purification of the P h o S - m h G R F ( G r o w t h - h o r m o n e Releasing Factor) protein, the presence of the PhoS signal sequence ensuring export of the hybrid to the periplasm [21]. However, the G R F region, consisting of a small peptide (44 a m i n o acid residues), represents only a few percent of the hybrid. In the case of P h o S - O m p F hybrids, we constructed larger proteins containing 2 putative domains, a periplasmic domain due to PhoS and a m e m b r a n e region involving the O m p F part, of an equivalent size. It has previously been dem~ntrated that the exchange of signal sequence between periplasmic and outer m e m b r a n e proteins did not alter the final location of the molecule [17, 22]. It was thus postulated that the mature sequence of the protein contained information ensuring the ultimate cellular location [23]. In our constructs, the presence of quasi total sequence of the 2 proteins m a y confer a dual address to these hybrids. T h e questions that arised after the processing step are firstly, what is the information on the polypeptide chain which will govern the final localization of the molecule and secondly, what is the effect of a large periplasmic domain (PhoS) on the putative trimerization of the O m p F domain? T h e analyses have been carried out using monoclonal antibodies directed against cell surface epitopes of O m p F [24, 25] and by testing the sensitivity toward colicins A and N of E coli B ompF, after expression of the various hybrid proteins.
Materials and Methods
medium (TGLP), cell samples were removed and total cellular proteins were analysed by SDS-PAGE. Pulse and pulse chase experiments Thirty rain and 90 min after induction, samples (5 x 108cells) were labelled for 1 rain at 37oC with [14C]-Iabelledamino acids (10 ttCi. ml-I) and chased for 20 min in the presence of unlabelled amino acids. For pulse chase experiments, 15 min and I h 15 after induction, samples (5 × l0 s cells) were labelled with [35S]-methionine(150 IxCi/ ml). An excess of unlabelled metbionine was added after a 20 s pulse and samples were subsequently removed during the chase at 0, 20, 40 s, 1, 2, 5, 10 and 30 mm. Each sample was immediately frozen in liquid nitrogen in the presence of chloramphenicol (50 ltg/ml). Solubilization and immunoprecipitations These were carried out as previously described [10]. Briefly, samples were resuspended and homogenized in Tris/EDTA/SDS buffer (50 mM Tris-HC! pH 8.0, 1 mM EDTA, I% SDS). An equivalent volume of this material was then solubilized at 56O C or 96~C. A 15fold excess of immnnoprecipitation buffer (I0 mM Tris-HCI pH 7.4, 150 mM NaCI, 5 mM EDTA) containing Triton X-100 (1% final concentration) was added. Incubation with the various antibodies was then carried out according to [10]. SDS- PAGE and immunoblotting SDS-PAGE (10% acrylamide), protein staining and fluorography were carried out as previously described [10]. The immunoblotting was carried out according to [21]. Colicin sensitivity The coUcin assay by survival test was previously described [28]. Briefly, after 15 rain of induction the cells (1 ml of suspension at DOa~0nm= 0.5) were added to various concentrations of colicins diluted from 10 to l0 Tin 10 mM sodium phosphate pH 7.4, Triton X-100 (0.1%) buffer for 20 min at 37oC. The cellular suspension was then diluted by 1 vol of fresh medium. The percentage of surviving cells was monitored by the ratio of optical densities at 600 nm from culture treated with colicins to untreated culture after 2 h at 37oC. lmmunolabelling of proteins on ultrathin frozen sections
Chemicals [35S]-Methionine was purchased from Amersham. [14C].labelled amino acids were purchased from the Commissariat/~l'i~nergieAtomique. The restriction enzymes, T4 DNA ligase and Klenow fragment of DNA polymerase I were purchased from Boehringer Manheim. Trypsin (TPCK) and trypsin inhibitor were purchased from Sigma Chemical Inc. Bacterial strain, plasmids and medium E coli B strain BZB 1107 (ompF: Tn5, Km R) from Dr Eisei~ has been described previously [25]. Piasmids pSN5182 and pLG361 have also been described previously [17, 26]. Tris-glucose medium, supplemented with required nutrients and phosphate, according to Morita et al [26], was used. Tetracyclin (10 Itg/ml) and Kanamycin (50 p,g/ ml) were used for selection of transformants and for ensuring the maintenance of strains and plasmids. Expression of hybrid genes Derepression of the pho regulon was carried out as previously described [27]. At various times after transfer to low phosphate
The detailed procedure for fixation, sectioning, immunolabelling and staining has been previously described [29]. Protease accessibifity experiments After 50 min of induction, cells were labelled for 10 min with [z4C]amino acids (5 IxCI/ ml) and chased for 10 rain with non radioactive amino acids (1% final concentration). After centrifugation at 4oC, cell pellets were resuspended in ice cold Tris 100 mM pH 8 containing either (10 raM) MgCI2 or (5 raM) EDTA [30]. Trypsin (100 I~g/ml final concentration) was added to half of each sample and incubation was carried out for 30 rain in ice. Trypsin inhibitor (500 Ixg/mi final concentration) was then added and samples were precipitated with trichloro acetic acid for 15 rain in ice. Precipitates were solubilized at 96oC for 10 rain in preheated sample buffer before immunoprecipitation. Antibodies Polyclonal antisera directed against PhoS and OmpF have been previously described as have monoclonal antibodies directed against native OmpF [24-26].
Assembly of PhoS-OmpF hybrid proteins in E coli
Results
Construction of a series of phoS-OmpF hybrid genes The plasmid pSN5182 [26] carrying the phoSgene with its own promoter was used as a vector. Different fragments of the ompFgene were cloned into the single restriction enzyme cleavage site for Sspl present in pSN5182. This blunt-ended site is in frame and located 10 codons upstream of the TAA stop codon of phoS gene. The orapF carrying plasmid pLG361 [17] was digested with restriction enzymes, and the relevant fragments were purified as described in Materials and Methods. After ligation, transformants were selected for tetracyclin resistance and the corresponding plasmids were purified. In order to check the correct orientation of the inserted fragment, digestion by appropriate enzymes was carried out. The pAB5 plasmid was constructed by exchanging the small Bglll-EcoRl fragment present in pAB3 for the corresponding one purified from pAB4. Transformants were selected for the presence of both the Pstl and HincII sites in the hybrid gene. Figure 1 shows the proteins encoded by the different constructed hybrid genes. The fusion proteins had all but 10 of the residues of PhoS protein fused to various polypeptide regions derived from OmpF. In the case of pAB3 and pAB5, the fusion takes place between residue 311 of mature PhoS and the 5th
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387
amino acid residue of the OmpF signal sequence and they are only different in COOH-terminal region (fig 1). The former construct comprises the OmpF sequence with the last 29 amino acid residues deleted, while the latter contains the complete sequence of OmpF. pAB4 was constructed by digesting with Klenow fragment of DNA polymerase I the PstI extremities of the ompF fragment from pLG361, resulting in an in-frame deletion of the first amino acid of mature OmpF. The BZB 1107 strain was transformed by the 3 plasmids. This ompF E coli B strain was selected to allow immunodeteetion of trimerie forms using several monoclonal antibodies as probes.
Synthesis and processing of the PhoS-OmpF hybrid proteins At various times after growth under phosphate limitation conditions, aliquots of cultures were removed, and total cellular proteins analysed by S D S - P A G E and immunoblotting. The respective apparent molecular weight of the hybrids were in accordance with D N ~ analysis of the different fusion genes and the putative size of their products (fig 1). The polyclonal antisera directed against either PhoS [26] or OmpF [24] proteins were used for immunodetection because hybrid proteins containing the quasicomplete sequence (311 amino acid residues compared to 321) of PhoS and almost 80% of the OmpF sequence, harbour antigenic determinants recognized by these 2 polyclonal antibodies (fig 2). BZB and BZB pLG361 cells were used as controls in order to compare the different fusion gene products to the background. At 90 rain of induction, a product was significantly recognized by the 2 antisera (fig 2). The migration of the immun0-recognized proteins was consistent with the calculated molecular weight of hybrid molecules. At a later time after transfer to phosphate-limiting medium, several other bands were detected by the 2 antisera. Among them, a product was observed migrating above the mature form. A stable accumulation of the precursor form has been previously shown during the hyperproduction of PhoS [27]. Kinetics analyses suggested that a jamming of export sites occured when the synthesis of PhoS reached a high production level [27]. Such a process could also induce the accumulation of hybrid precursors. In addition, some products with lower apparent molecular weights were immunorecognized indicating a degradation of the hybrid. During normal export, in most cases cleavage of the signal peptide is a good indication that correct translocation of precursor forms across the cytoplasmic membrane has occured, although exceptions to this case have been reported [5]. The efficiency of processing
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was thus analysed with the various hybrids at 15 and 75 min after induction (fig 3). During the early growth period in phosphate minimal medium, only a weak signal corresponding to the precursor form of hybrids was detected in the pulse, the large majority of immunoprecipitated material corresponding to mature form. By 75 rain after induction, a large amount of a product migrating slower than mature form was accumulated. This polypeptide, related to precursor and appar,~ntly stable during the chase, could reflect jamming of the export machinery as previously reported during the hyperproduction of PhoS [27].
Assembly of the chimeric proteins
A B
Porin trimers are very stable structures not dissociated even in the presence of SDS, at temperatures below 75°C [9]. It was thus interesting to investigate the ability of the various chimeric proteins to assume such a conformation in order to determine whether the information comprised in the OmpF part could be expressed. To this end, 2 different experimental approaches were used. The kinetics of trimerization were studied and immunological analyses using various monoclonal antibodies as conformational probes were carded out. The results concerning the rate of trimerization after 1 h 15 of induction are presented in figure 4. No material migrating at high molecular weight was detected after a long chase time with PAB3 (fig 4A), even when
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C ....... Fig 2. Expression of the hybrid proteins in ompF E coli strain. Samples were removed at 0, 1 h 30 and 3 h of induction and total cell proteins were analysed by SDS-PAGE. BZB and pLG correspond respectively to BZB 1107 and BZB 1107 carrying pLG 361 after 3 h of culture in TGLP medium. (A): Coomassie blue staining of total cellular proteins. After electrotransfer to nitrocellulose, proteins were detected with antisera directed against OmpF (B) and PhoS (C). The triangle and dot indicate the migration of OmpF and PhoS respectively. Only the relevant part of the gel and blots are shown. Mr standard: (a) 94 kDa, (b) 67 kDa, (c) 43 kDa.
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Fig 3. Maturation of the various PhoS-OmpF hybrid proteins. At 15 min (A, C, E) or 1 h 15 (B, D, F) after transfer to TGLP medium, the cells BZB 1107 carrying pAB3 (A, B), pAB4 (C, D) or pAB5 (E, F) were pulse labelled for 20 s (lane 0) with [35S]-methionineand chased with unlabelled methionine for 20, 40 s, 1, 2, 5, 10 and 30 rain (lane 1 to 7 respectively). Immunoprecipitations with an anti-PhoS serum were then carried out and samples were analysed by SDS-PAGE after solubilization at 96oC. The migration of precursor (p) and mature form (m) of each hybrid are indicated.
Assemblyof PhoS-OmpFhybrid proteins in E coli
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the gel was overexposed. In contrast with PAB4 and PAB5, products showing a slow migration corresponding to hybrid oligomers were detected (figs 4B and 4C). At an early time of induction, the same pattern was obtained for the 3 hybrids but precursor forms were not accumulated (data not shown). In addition, the presence of oligomers was not related to overproduction because the high molecular weight material was detected as early as 15 rain after induction (data not shown). These results suggested that the COOH-terminal region of OmpF was necessary to promote efficient oligomerization of hybrids because PAB3 was not able to form oligomers. Only 1 major band corresponding to putative trimeric form was obtained with PAB4 in contrast to PAB5 which presented several high molecular weight products. Immunological analyses were performed using monoclonal antibodies previously described to rule out the hypothesis that some aggregation process could promote the presence of such high molecular weight products (fig 5). PAB3 hybrid protein which was recognized by polyclonal antisera directed either against OmpF or against PhoS, was not immunoprecipitated by the various monoclonal antibodies (MoF) specific to OmpF. The antigenic sites recognized by MoF 18, 19 and 20 have been recently located at the COOH-terminal region of OmpF (Fourel et al, unpublished results), moreover the epitope recognized by MoF18 has been detected on the monomeric surface [31]. Failure to
i
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Fig 5. Culture of BZB 1107 carrying pAB5 (top) or pAB3 Fig 4. Assembly of PhoS-OmpF hybrids. At 1 h 15 after transfer to TGLP medium, the cells BcZB 1107 carrying pAB3 (A), pAB4 (B) or pAB5 (C) were pulse labelled and chased as described in figure 4. Samples were solubilized at 56oC and immunoprecipitations were carried out with an anti-PhoS serum. The immunoprecipitates were then heated at 56oC before application. Migration of trimers (t0 and monomers ( ( ) are indicated.
(bottom) were labelled with [14C]-amino acids after 90 min of induction. Cell samples were subsequently solubilized in TES buffer at 56°C, immunoprecipitated and loaded on 10% polyacrylamide gels after denaturation in sample buffer. Lanes 1 and 2 correspond to anti-PhoS and anti-OmpF polyclonal antisera respectively; lanes 3 to 10 correspond to MoF1, MoF3, MoF4, MoF7, MoF18, MoF19, MoF20 and MoF21 monoclonal antibodies respectively. Monoclonal antibodies were previously described [24, 25].
3Q0
JM Bolla et al
detect PAB3 could thus be related to a lack of epitope removed by the deletion of the last 30 residues of OmpF. In addition, the absence of signal with other MoF suggests that PAB3 is either unfit to trimerize or only able to organize very unstable trimers that are dissociated under the experimental conditions used. Some of the MoF [ 1, 3, 4, 7] have previously been described and recognize cell surface-epitopes present only on the trimeric conformation of OmpF [24]. In the case of PAB5 a significant signal was observed with these antibodies indicating that some chimeric molecules haboured the respective epitopes. These results suggest that 2 conformational populations of processed PAB5 molecules were detected in the cell. The ratio of the chimeric proteins immunoprecipitated with MoF to the material immunoprecipitated with the polyclonal represents the percentage of hybrid porin which contains the respective native antigenic determinants. 48% of the PAB5 molecules (with regard to MoF1, 4 and 7 to polyclonal antiserum ratio) are then able to reach a native conformation as checked by such immunological tools. The remaining population which cannot express the native epitopes, may be accumulated and mis-assembled during overproduction of hybrids. The percentage of trypsin-sensitive PAB5 molecule roughly correlated with the immunological analysis carded out with MoF.
Protease accessibility of hybrid proteins It has recently been reported that alterations of membrane proteins, such as insertion or deletion, can promote orotease suscentihilitv [32_ 331 In nrd~r tn invp~-
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bility of the 3 hybrids (PAB3, PAB4 and PAB5) was studied. To avoid the problem due to the distribution of methionine residues (absent in PhoS mature sequence), the experiment was carried out after [14C]amino acids labelling. In the presence of Mg 2+ ions, hybrid proteins were resistant to trypsin degradation (fig 6). In the presence of EDTA, as previously reported [34], the periplasmic domain of OmpA was cleaved generating the 24 kDa protected fragment (fig 6 left). Under these conditions, various patterns of sensitivity were obtained with the hybrids. PAB3 was almost completely sensitive to trypsin digestion (fig 6A) and no protected fragment was immunodetected with both antisera, indicating an extensive degradation of the 2 domains. This indicated that the OmpF region of PAB3 presented higher susceptibility to trypsin, suggesting a conformation different from native OmpF. With PAB4, only a few percent of hybrid molecules are accessible to protease, the majority being resistant to degradation (fig 6B). In the case of PAB5, half ot the hybrids (46%) were degraded and a resulting fragment was immunoprecipi-
rated by the antiserum directed against OmpF (fig 6C). This fragment, migrating with an apparent molecular weight of 40 kDa, was not recognized by the anti-PhoS serum~ indicating a degradation of the PhoS part. The high sensitivity of this domain was completely different from the trypsin resistance of native periplasmic PhoS. It could be compared to the extensive susceptibility of the membrane associated prePhoS previously reported [35] and thus suggested a labile unfolded conformation of PhoS exposing a large number of trypsic sites. It was concluded that oniy some of the PAB5 hybrids could generate an OmpF plotected fragment.
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Fig 6. Trypsin accessibility experiments. After 50 min of culture in TGLP medium, cells of BZB 1107 carrying pAB3 (A), pAB4 (B) or pAB5 (C) were labelled with [n4C]-amino acids and treated with trypsin in the presence of Mg2÷ ions or EDTA as indicated. After 30 min of incubation and then TCA precipitation, immunoprecipitations were carried out with anti-OmpA, anti-OmpF or anti-PhoS sera, as indicated. The positions of the OmpA protein (10, OmpA protected fragment (~), migration of the respective fusion proteins (o), as well as of PAB5 protected fragment (*) are shown.
Assemblyof PhoS-OmpF hybrid proteins in E coil
Colicin sensitivities of ompF E coil B expressing the hybrid proteins OmpF has been described a; functioning as (part of) the receptor for certain t'/hages and is required for the efficient killing action of a number of colicins [ 1,2]. For colicin N, OmpF is involved both in the binding step
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39
and in the translocation of the bacteriocin across the outer membrane [36, 37]. In the case of colicin A, the receptor structure is mere complex comprising BtuB, OmpF and LPS [28, 38]. The sensitivity of BZB 1107 (ompF E coil B) was tested 15 min after transfer to phosphate- limiting medium. Cells of ompF E coli B free of plasmid or transformed with pLG301, carrying the ompF gene, were used as negative and positive controls respectively (fig 7), tinder these conditiens, the same significant colicin A sensitivity was obtained when the synthesis of PAB3 and PAB4 was induced, PAB5 being 10 times more sensitive in the assay. With colicin N, a sensitivity was observed only when PAB4 or PAB5 were produced. Thus, PAB3 presenting neither stable trimeric forms under solubilisatio.~ conditions used, nor antigenic sites related to native trimers, can nevertheless restore partial coliein A uptake. This may indicate that a few molecules could insert into the outer membrane and act as an efficient receptor for colicin A, whereas no colicin N receptor activity is observed. The difference in sensitivity observed with PAB3, 4 and 5 compared to wild type Or~pF may reflect the transmembrane conformation of such hybrids containing a large periplasmic domain. However, the production of PAB4 and 5, both containing the COOH-terminal region, restored only partial sensitivity toward colicin N. This suggested that the membrane orientation of OmpF in the chimeric proteins could be modified by the PhoS region, and as a consequence could modify the affinity of colicin N for its receptor site.
lmmunocytolocalization of the chimeric proteins
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, 106
, to, -~. to, -4 t0-s eolicin N (mg/ml)
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Fig 7. Sensitivity toward colicins A and N. The strains BZB 1107 (D) or carrying pLG361 (e), pAB3 (B), pAB4 (A) or pAB5 (O) were induced for 15 min in TGLP medium. Aliquots of each culture were then incubated with dilutions of colicin A (A), or colicin N (B) for 20 min. After dilution in fresh TGHP medium, the cell suspensions were grown for 2 h at 37°C and the percentage of survivals checked as described in Materialsand Method~, was plotted as a function of colicin concentration.
Tl~,e sensitivity towards colicin A suggested that chimeric proteins were at least partly localized in the outer membrane. In addition, oligomeric forms which exhibited native cell surface exposed epitopes were detected with PAB5. The synthesis of hybrid proteins may induce jamming of the export machinery and consequently abortive export and miscompartmentalization of newly synthesized molecules [39]. Such a phenomenon was shown to cause accumulation of cytoplasmic and membrane-associated precursor forms and sometimes cytoplasmic aggregation of overproduced proteins [14, 40], Subseque,ntly, during classical fractionati6n procedures, this aggregated material can be artifactually distributed in a membrane compartment. As previously described [14, 39, 40],.immunolabelling of frozen thin sections of E coil can m most cases permit the localization of proteins and prevent, after fractionation experiments, incor_-ect conclusions being derived from aggregation, etc. In order to investigate the location of various products, immunogoid labellings were performed after 1 h 30 of induction (fig 8). The
392
J M B o l l a et al
monoclonal antibody MoFI8 was selected instead of polyclonal antiserum to avoid a cross reaction with PhoE which is induced duri-g culture in low phosphate medium. No labelling was observed in control cells and in cells containing PAB3 which do not contain the specific epitope. In contrast, immunogold labelling associated to the outer membrane was clearly detected during the expression of PAB4 and PAB5 (fig 8). Electron-dense areas were observed in cytoplasm. Although these areas were significantly labelled with the antiserum directed against PhoS (fig 8), they were not recognized with the monodonal antibody MoF 18
A
(compare fig 8A to 8B). These results suggest a microconcentration of hybrids in discrete cytoplasmic regions where molecular aggregation probably occured. These accumulated products probably correlate with the precursor form immunodetected at long periods of incubation (figs 2, 3) and with non native form (fig 5). These aggregated precursor molecules which did not present the native epitope, are not recognized by MoF 18. During overproduction of PhoS similar results were reported with a cytoplasmic labelling corresponding to accumulation of pre-PhoS due to jamming of the export machinery [27].
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Fig 8. Localisation of PhoS-OmpF hybrids by immunolabelling. Cell samples of E coli BZB 1107 (a), or carrying pAB3 (b), pAB4 (c) or pAB5 (d) were removed after 1 h 30 of culture in TGLP medium. Ultrathin frozen sections were prepared and incubated with either an anti-PhoS serum (A) or a monoclonal antibody directed against OmpF (B). Labelling was performed with gold-coated protein A.
Assembly of PhoS-OmpF hybrid proteins in E coli
Discussion
We have presented in this study the construction of several PhoS-OmpF hybrid proteins. Two large domains corresponding to PhoS and OmpF containing information for periplasmic and outer membrane localization respectively were present in these hybrids. The question of the efficiency of export, of the cellular distribution and of the topology of these proteins must then be considered. Previous studies demonstrated that prePhoS protein fused to mhGRF was efficiently synthesized and processed and that the hybrid PhoS-GRF was localized in the periplasmic space [21, 41]. This suggested that PhoS domain protein could promote efficient export of fused polypeptide. It was also shown that the signal sequence and part of the mature sequence of a periplasmic protein (/3-1actamase) was able to direct an outer membrane protein PhoE to its correct compartment [22, 34]. During expression of the various PhoS-OmpF hybrid proteins in an ompF E coli B strain, we analysed their export by assaying the kinetics of signal sequence cleavage (fig 3). In order to study the subcellular localization of these proteins, we used 2 reliable experimental approaches: immunogold labelling and trypsin accessibility. In a second step, we probed the ability of these hybrids to exhibit OmpF properties. Their conformational and functional characteristics were checked by immunological analyses of the trimerization pattern and by their ability to restore colicin sensitivity. The hybrid PAB5 contained the quasi complete sequence of PhoS fused to the preO-mpF sequence deleted from the positively charged amino-terminal part ( - 22 to - 17). Oligomeric products related to trimeric hybrid forming about 40-50% of mature PAB5 molecules were detected in the cells, and cellsurface exposed epitopes corresponding to the native OmpF trimers were immunorecognized on the chimeric protein. The sensitivity toward trypsin in the presence of EDTA indicated that among the 46% of the sensitive hybrids corresponding to correctly localized molecules, the PhoS part of PAB5 was accessible and degraded, while the OmpF domain was resistant as previously reported for the native porin [42]. These results suggest that this PhoS domain cannot reach the native conformation exhibited by the wild type periplasmic phosphate binding protein when anchored in the outer membrane via the OmpF part. This could explain the failure of outer membrane labelling with the antiserum directed against PhoS. Although no evidence was obtained about the conformation of the PhoS domain from hybrids into the membrane, it is possible that the artificial association of PhoS polypeptide with the outer membrane
393
modifies the antigenic sites by steric hindrance or association with outer membrane components. Alternatively, the deletion of last COOH region of PhoS could alter the antigenic properties of the molecule. In the light of the trypsin resistance of periplasmic PhoS [36], it must be concluded that the PhoS domain is not properly folded in hybrid molecules. It is likely that the translocation competent conformation of PhoS (partially unfolded) cannot return to the native conformation due to the continuous association with a membrane even after translocation. With periplasmie proteins, the dissociation from the inner membrane and final folding constitutes a distinct step in the export pathway as demonstrated with fl-lactamase [43] and maltose binding protein [44]. The efficiently assembled PAB5 molecules restore the sensitivity of OmpF cells toward colicins A and N to almost the same level as cells harbouring the ompF gene. When the COOH-terminal region (311-340 residues) was deleted generating PAB3 chimeric protein, the hybrid was unfit to promote stable oligomerization and to present native epitopes. It-exhibited larger protease sensitivity as a consequence of its abortive assembly. In addition, it could only restore the colicin A sensitivity while resistance to colicin N was observed. These results indicate that the COOH-terminal region of OmpF is necessary to promote stable assembly of the protein in the outer membrane. In the case of PAB4, the PhoS region was directly fused to the 2nd residue of OmpF mature sequence. This last chimera presented oligomeric forms different from PAB5 but was able to restore colicin A and N sen151tlVltl~5.
l"luwt;:;V~;l,
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molecules were susceptible to protease degradation. A significant immunogold labelling was nevertheless observed on the outer membrane with a monoclona! antibody directed against a cell surface-exposed monomeric epitope of OmpF. Thus, the hydrophobic segment of OmpF signal peptide inserted between the 2 domains could stabilize the outer membrane oligomers or enhance the membrane insertion but did not significantly modify the receptor function of OmpF domains and the exposure of antigenic determinants. Results obtained with PAB3 compared to those obtained with PAB4 and PAB5 indicate that the information required for the correct and stable insertion of hybrids is contained in the COOH-terminal region of OmpF. This sequence corresponds in fact to the last membrane spanning peptide proposed in the models described in [45] and [46]. These results, in addition to a recent report [19] describing similar properties of the carboxy-terminal region of PhoE strongly suggest that the last part of porin sequences are necessary during the oligomerization process in order to promote a correct assembly within
394
JM Bolla et al
the outer membrane. Hikita et al have recently observed that the carboxy-terminal region of OmpF and OmpC can be exchanged without alteration in porin assembly [47]. Thus, the role of the last putative membrane spanning region could be a common strategic element during the stable assembly of porins. The insertion of the hydrophobic part of OmpF signal peptide into the mature sequence of OmpC has recently been reported to impair the assembly of the chimenc porin in the outer membrane without altering its export [48]. In our case, the same sequence located between 2 separate domains, PhoS and OmpF, improved the insertion of hybrids. Thus, this location, which ensures a clean separation between the OmpF and PhoS regions in PAB5 could therefore facilitate independent folding of complete OmpF. This process may be perturbed by neighbouring PhoS polypeptide in the other constructs.
Acknowledgments We thank D Cavard, JL Eisel~, M Knibiehler, B Holland and Shinagawa for their generous gift of strains, plasmids, colicins and antiserum, and lmmunotech for carrying out the preparation of monoclonal antibodies. We gratefully acknowledge the helpful discussions with SP Howard and D Cavard, the help of M Bidaud in computer analysis of DNA and M Payan in secretarial support. This work was supported by the Centre National de la Recherche Scientifique, the lnstitut de la Sant~ et de la Recherche Mrdicale (CRE No 86.1020) and the Fondation pour la Recherche Mrdicale.
References 1 Lugtenberg B, Van Alphen L (1983) Molecular architecture and functioning of the outer membrane of Escherichm co!i and other gram-negative bacteria. Biochim Biophys Acta 737, 51-115 2 Nikaido H, Vaara M (i987) Outer membrane. In: Escherichia eoli and Salmonella typhimurium, Cellular and Molecular Biology (Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE, eds) ASM Publications, Washington, 1, 7-22 3 Hancock REW (1987) Role of porins in outer membrane permeability. J Bacteriol 169, 929-933 4 Lee C, Beckwith J (1986) Co-translational and post-translational proteins translocation in procaryotic systems. Ann Rev Cell Biol 2, 315-336 5 Randall LL, Hardy SJS, Thom JR (1987) Export of protein: a biochemical view. Ann Rev Microbio141, 507-541 6 Baker K, Mackman N, Holland IB (1987) Genetics and biochemistry of the assembly of proteins into the outer membrane of E coil Prog Biophys Mol Bio149, 89-115 7 Saier MH, Werner PK, Muller M 0989) Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotie process. Microbiol Rev 53,333-366 8 Bolla JM, Lazdunski C, Pages JM (1988) The assembly of the major outer membrane protein OmpF of Escherichia coil depends on lipid synthesis. E M B O J 7, 3595-3599 9 Reid J, Fang H, Gehring K, Klebba PE, Nikaido H (1988) Targeting of porin to the outer membrane of Escherichia coil J Biol Chem 263, 7753-7759 10 Pages JM, Bolla JM (1988) Assembly of the OmpF porin in E~cherichia coli B. Eur J Biochem 176, 655-660
11 Freudl R, Schwartz H, Strerhof YD, Gamon K, Hindennach I, Henning U (1086) An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem 261, 11355-11361 12 Geller BL, Green HM (1989) Translocationof Pro-OmpA across inner membrane vesicules of Escherichia coli occurs in two consecutive energetically distinct steps. J Biol Chem 264, 16465-16469 13 Benson SA, Bremer E, Silhavy TJ (1984) Intragenic regions required for Lamb export. Proc Nail Acad Sci USA 81, 3830-3834 14 Freudl R, Schwartz H, Klose M, Mowa RN, Henning U (1985) The nature of information, required for export and sorting present within the outer membrane protein OmpA of Escherichia coli K-12. E M B O J 4, 3593-3598 15 Bosch D, Leunissen J, Verbakel J, de Jong M, van Erp H, Tommassen J (1986) Periplasmie accumulation of truncated forms of outer membrane PhoE proteins of Escherichia coli K12. J Mol Biol 189,449-455 16 Klose M, Schwarz H, Mac Intyre S, Freudl R, Eschbach ML, Henning U (1988) Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide. J Biol Chem 263, 13291- 13296 17 Jackson HE, Pratt JE, Stocker NG, Holland IB (1985) An inner membrane protein N-terminal signal sequence is able to promote efficient Iocalisation of an outer membrane protein in Escherichia coil E M B O J 4, 2377-2383 18 Coulton, JW, Reid GK, Campana A (1988) Export of hybrid proteins FhuA-LacZ and FhuA-PhoA to the cell envelope of Escherichia coli K-12. J Bacteriol 170, 2267-2275 19 Bosch D, Scholten H, Verhagen C, Tommassen J (1989) The role of the earboxy-terminal membrane-spanningfragment in the biogenesis of Escherichia coli K-12 outer membrane protein PhoE. Mol & Gen Genet 216, 144-148 20 Magota K, Otsuji N, Miki T, Horiuchi T, Tsunasawa S, Kondo J, Sakiyama F, Amemura M, Morita T, Shinagawa H, Nakata A (1984) Nueleotide sequence of the phoSgene, the structural gene for the Phosphate-binding protein of Escherichia coli. J Bacteriol 157, 909-917 21 Anba J, Baty D, Lloubrs R, Pagrs JM, Joseph-Liauzun E, Shire D, Roskam W, Lazdunski C (1987) Expression vector promotinQ the gvnl'h~glgand ~vn,'sr~c~f*h. h. . . . . . . . . . . *k k. . . . . . releasing factor in Escherichia coli. Gene 53, 219-229 22 Tommassen J, van Tol H, Lugtenberg B (1983) The ultimate localization of an outer membrane protein of Escherichia coli K12 is not determined by the signal sequence. E M B O J 2, 1275-1279 23 Jackson ME, Pratt JM, Holland IB (1986) Intermediates in the assembly of the TonA polypeptide into the outer membrane of Escherichia coli K-12. J Mol Biol 189, 477-486 24 Pages JM, Pagrs C, Bernadac A, Princ6 P (1988) Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12. Mol lmmuno125, 555-563 25 Lupi N, Bourgois A, Bernadac A, Laboucari6 S, Pagrs JM (1989) Immunological analysis of porin polymorphism in Escherichia coli B and K-12. Mol lmmuno126, 1027-1036 26 Morita T, Amemura M, Makino K, Shinagawa H, Magota K, Otsuji N, Nakata A (1983) Hyperproduction of phosphatebinding protein, PhoS, and pre-PhoS protein in E coli carrying a cloned phoSgene. Eur J Biochem 130,427-435 27 Pagrs JM, Anba J, Bernadac A, Shinagawa H, Nakata A, Lazdunski C (1984) Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coll. Eur J Biochem 143,499-505 28 Cavard D, Lazdunski C (1981) Involvement of BtuB and OmpF proteins in binding and uptake of colicin A. FEMS Microbiol Left 12, 311-316 29 Anba J, Bernadac A, Pagrs JM, Lazdunski C (1984) The periseptal annulus in Escherichia coil Biol Cell 50, 2"13-278 --
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.
.
.
.
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Assembly of PhoS-OmpF hybrid proteins in E coli 30 Bosch D, Voorhout W, Tommassen J (1988) Export and localization of N-terminally truncated derivatives of Escherichia coil K-12 outer membrane protein PhoE. J Biol Chern 263, 9952-9957 31 Pagrs JM, Bolla JM, Bernadac A, Fourel D (1990) Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli. Biochimie 72, 169-176 32 Freudl R, Mac lntyre S, Degen M, Henning U (1986b) Cell surface exposure of the outer membrane protein OmpA of Escherichin coli K-12. J Moi Biol 188,491-494 33 Morona R, Tommassen J, Henning U (1985) Demonstration of a bacteriophage receptor site on the Escherichia coli K-12 outer membrane protein OmpC by the use of protease. E u r J Biochem 150, 161-169 34 Tommassen J, Lugtenberg B (1984) Amino terminus of outer membrane PhoE protein: localization by use of a bla-phoE hybrid gene. J Bacteriol 157, 327-329 35 Anba J, Pagrs JM, Lazdunski C (1986) Mode of transfer of the phosphate-binding protein through the cytoplasmic membrane in Escherichia coli. F E M S Microbiol Lett 34, 215-219 36 Tommassen J, Pugsley AP, Korteland J, Lugtenberg B (1984) Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K-12. Mol & Gen Genet 197, 503-508 37 Pugsley AP (1984) The ins and outs of colicins. Microbiol Sci 1, 168-176 & 203-205 38 Chai TJ, Wu V, Foulds J (1982) Colicin A receptor: role of two Escherichia coli outer membrane proteins (OmpF protein and btuB gene product) and lipopolysaccharide. J Bacteriol 151, 983-988 39 Tommassen J (1986) Fallacies of E coli cell fractionations and
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consequences thereof for protein export models. Microbiol Pathogenesis I, 225-228 40 Tommassen J, Lennissen J, van Damme-Jongsten M. Overdium P (1985) Failure of E coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm. E M B O J 4, 1041- 1047 41 Anba J, B~,rnadacA, Lazdunski C, Pages J (1988) Improving the stability of a foreign protein in the periplasmic space of Escherichin coli. Biochimie 70, 727-733 42 Rosenbusch JP (1974) Characterization of the major envelope protein from Escherichia coil J Biol Chem 249. 8019-8029 43 Minsky A, Summers RG, Knowles JR (1986) Secretion ofl3-1actamase into the periplasm of E coli: evidence for a distinct release step associated with a conformational change. Proc NatlAcad Sci USA 83, 4180-4184 44 Thom JR, Randall LL (1988) Role of the leader peptide of Maltose-Binding-Protein in two steps of the export process. J Bacteriol 170, 5654-5661 45 Benson SA, Occi JLL, Sampson BA (1988) Mutations that alter the pore function of OmpF porin of Escherichia coli K-12. J Mol Bio1203,961-970
46 Carlton P, Rosenbusch JP (1985) Folding patterns of porin and bacteriorhodopsin. E M B O J 4, 1593-1597 47 Hikita L, Satake Y, Yamada H, Mizuno T, Mizushima S (1989) Structural and functional characterization of the OtnpF and OmpC porins of the Escherichia coli outer membrane: studies involving chimeric proteins. Res Microbiol 140, 177-190 48 Shinkai A, Yamada H, Mizuno T, Miznshima S (1989) Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E coli. J Biochem 106, 323-330