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Analysis of structual features of OprF from Pseudomonas aeruginosa using fusion proteins created by TnphoA… Finnen, Renée Louise 1991

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ANALYSIS OF STRUCTURAL FEATURES OF OprF FROM Pseudomonas aeruginosa USING FUSION PROTEINS CREATED BY TnphoA MUTAGENESIS by Renee Louise Finnen B.Sc.(Agr.), University of Guelph, 1988. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE in T H E FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA September 1991 © Renee Louise Finnen, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^/eolSrOLQ^/ The University of British Columbia Vancouver, Canada Date tf**. J9*l DE-6 (2/88) i i A B S T R A C T The major outer membrane protein OprF from Pseudomonas aeruginosa was mutagenized with the specialized transposon TnphoA in order to investigate structural features of the protein. TnphoA insertions in the oprF gene were mapped by Sail digestion and determined to be located at 16 different sites across the gene. The precise location of at least one example of each insertion was determined by sequencing. In most cases the molecular weight of the OprF::TnphoA fusion proteins predicted by sequencing corresponded well to the apparent molecular weight determined by Western immunoblots probed with anti-bacterial alkaline phosphatase polyclonal sera (for in-frame fusions) or anti-OprF monoclonal antibodies (for larger fusions). The 16 fusion proteins characterized differed in their lengths of OprF amino-terminal amino acid residues. Thus their reactivities with a panel of 10 anti-OprF MAbs permitted crude localization of the epitopes for these MAbs. The proposed epitope locations were complemented and further resolved by the MAb reactivity patterns of OprF derivatives expressed by various oprF subclones. A previously published structural model of OprF featured disulphide bridges between neighbouring cysteine residues and a surface-exposed carboxy-tenninus. Both of these features were supported by the results of this study. The results of this study also indicated that the sorting signal for transport of OprF to the outer membrane was contained within the first 180 amino acids. i i i TABLE OF CONTENTS Page ABSTRACT . . ii. LIST OF TABLES v. LIST OF FIGURES vi. ABBREVIATIONS vii. ACKNOWLEDGEMENTS viii. INTRODUCTION 1 MATERIALS AND METHODS A. Bacterial strains, bacteriophage and plasmids 10 B. Monoclonal antibodies and polyclonal sera 10 C. Media and growth conditions 10 D. XTnphoA mutagenesis 14 E. DNA techniques 1. Isolation of plasmid DNA 17 2. Quantitation of plasmid DNA 17 3. Sequencing 17 4. General DNA techniques 18 iv TABLE OF CONTENTS (continued) Page F. Protein techniques 1. Isolation of total cellular protein 19 2. Preparation of outer membranes 20 3. Quantitation of protein 20 G. Immunodetection techniques 1. Colony immunoblotting 21 2. Western blotting 22 RESULTS A. Primary screening 23 B. Secondary screening 24 C. Further characterization of fusion proteins 34 D. Location and characterization of OprF epitopes 47 E. Reactivity of other OprF derivatives with anti-OprF monoclonal antibodies 51 DISCUSSION 56 REFERENCES 61 V LIST OF TABLES Table Title Page I. Bacterial strains, plasmids and bacteriophage 11 II. Monoclonal antibodies and polyclonal sera 13 III. Insertion sites located by Sail restriction mapping 27 IV. Characteristics of OprF fusion proteins obtained 29 V. Molecular mass of immunoreactive OprF fusion proteins 37 VI. Effect of (3-mercaptoethanol treatment on OprF epitopes 48 VII. Immunoreactivity of OprF derivatives created by subcloning . . 52 vi LIST OF FIGURES Figure Title Page 1. Conceptual model of the structure of OprF 6 2. Plasmids used in mutagenesis 16 3. Sail digestion patterns of fusion plasmids 26 4. TnphoA mutagenesis of oprF 31 5. Immunoreactivity of OprF::PhoA fusion proteins with anti-bacterial alkaline phosphatase polyclonal sera 33 6. Location of fusions and monoclonal antibody reactivity patterns of P. aeruginosa OprF fusion proteins 36 7. Immunoreactivity of OprF fusions at amino acids 289 and 299 with MA7-2 39 8. Immunoreactive degradation products of OprF and OprF and OprF fusions at amino acids 289 and 299 42 9. Immunoreactivity of OprF fusions at amino acids 144 and 180 with MA7-1 44 10. Immunoreactivity of OprF fusion at amino acid 180 with MA7-1 in the presence of (3-mercaptoethanol 46 11. Immunoreactivity of OprF fusions at amino acids 180 and 204 with MA4-4 and MA7-8 in the presence and absence of p-mercaptoethanol 50 12. Proposed locations of anti-OprF epitopes 55 vii ABBREVIATIONS BAP bacterial alkaline phosphatase B M E [3-mercaptoethanol BSA bovine serum albumin DMSO dimethyl sulphoxide FepA Escherichia coli ferric enterobactin receptor IS50 insertion sequence 50 kan kanamycin MAb monoclonal antibody PA polyacrylamide PBS phosphate buffered saline PhoA Escherichia coli alkaline phosphatase 'phoA gene for alkaline phosphatase production minus promoter and signal sequence coding region OmpA Escherichia coli outer membrane protein A OprF Pseudomonas spp. outer membrane protein F SDS sodium dodecyl sulphate tet tetracycline X-P 5-bromo-4-chloro-3-indolyl-phosphate viii ACKNOWLEDGEMENTS The financial assistance of the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged. I wish to acknowledge my supervisor, R.E.W. Hancock, and members of my supervisory committee for their guidance. I would also like to acknowledge N. Martin and R. Siehnel for their technical advice and helpful discussions. A special acknowledgement goes out to A. Chu and S. Longman for diversions and excursions which helped me keep it all in perspective. 1 INTRODUCTION Pseudomonas aeruginosais a gram-negative rod which can be readily found in water, in soil and on vegetation. Despite its ubiquitous nature, P. aeruginosa is also an opportunistic human pathogen, causing disease in patients compromised by extensive burns or by conditions such as cystic fibrosis or leukemia (Ryan, 1984). Infections caused by this organism are particularly difficult to treat as it is resistant to many antibiotics. This intrinsic antibiotic resistance can be attributable in part to the low outer membrane permeability observed for P. aeruginosa (Yoshihara and Nikaido, 1982; Nicas and Hancock, 1983). The outer membrane of gram-negative bacteria acts as a selectively permeable barrier (Nikaido and Vaara, 1985). It consists of an asymmetric lipid bilayer, made up of lipopolysaccharide on the outside face and phospholipid on the periplasmic face, into which proteins are embedded. Some of these proteins, known as porins, form either specific or non-specific channels the size of which determine the permeability of the outer membrane. In the outer membrane of P. aeruginosa, 8 to 10 major species of protein are present in copy numbers of 5 x 104 to 2 x 105 copies per cell (Hancock and Carey, 1979). The most abundant of these major outer membrane proteins is OprF, encoded by the oprF gene. The oprF gene appears to be conserved within the family Pseudomonadacae. 2 Restriction mapping and southern blot analysis using P. aeruginosa oprF as a probe revealed that the restriction map is highly conserved in serotype strains and clinical isolates of P. aeruginosa (Ullstrom etal, 1991). Pseudomonas species belonging to the same rRNA homology group as P. aeruginosa were also able to to hybridize with the oprF probe (Ullstrom et al, 1991). More extensive characterization of oprFfrom P. syringae showed 72% DNA sequence identity with P. aeruginosa oprF and 68% overall amino acid identity between the two oprF gene products (Ullstrom et al, 1991). Also, monoclonal antibodies (MAbs) raised against P. aeruginosa OprF were able to cross react with OprFs from different Pseudomonas species (Mutharia and Hancock, 1985). P. aeruginosa OprF is a constitutively produced protein which is associated with the peptidoglycan (Mizuno and Kageyama, 1979; Hancock et al, 1981). This protein is both heat and p-mercaptoethanol (BME) modifiable (Hancock and Carey, 1979). The sequence of the P. aeruginosa oprF gene predicts a protein with a molecular weight of 32,250 (Duchene et al, 1988). On sodium dodecyl sulphate polyacrylamide gels, unheated, non-BME-treated OprF bands at a position equivalent to a molecular weight of 33,000; heated, non-BME-treated OprF bands at 39,000; unheated, BME-treated OprF bands at 36,000; and heated, B M E -treated OprF bands at 41,000 (Hancock and Carey, 1979). These multiple banding positions reflect the presence of SDS-stable (J-sheet structure (heat modifiability) and disulphide bonds (BME-modifiabiliry) (Hancock, 1986). OprF is believed to play a dual role, acting both as a porin fomiing water-filled channels 3 through the outer membrane (Hancock, 1986; Nikaido and Hancock, 1986) and as a structural protein important for maintenance of cell shape and outer membrane integrity (Gotoh et al, 1989; Woodruff and Hancock, 1988; 1989). Conflicting results of the in vitro systems used to determine the size exclusion limit of the pores formed by OprF has made its role as a porin a source of controversy. Black lipid bilayer studies by Woodruff et al (1986) led to a hypothesis that OprF may function as a heterogenous porin forming a small portion (< 1%) of large channels permeable to antibiotics, with the remaining channels being small and antibiotic impermeable. This hypothesis could explain the apparent contradiction between the large size exclusion limit reported for the P. aeruginosa outer membrane and the low outer membrane permeability observed. Liposome swelling assays performed by some researchers support this hypothesis (Yoshimura et al, 1983), however the same sort of assays performed by other investigators suggested that the exclusion limit of P. aeruginosa is much lower than originally reported (Yoneyama et al, 1986; Gotoh et al, 1989; Yoshihara and Nakae, 1989). Antibiotic uptake studies on an engineered oprF mutant (Woodruff and Hancock, 1988) failed to resolve this controversy as structural alterations resulting from the loss of OprF complicated the analyses. The nature of the channels formed by OprF, therefore, remains to be resolved. The structural role of OprF is analagous to that of OmpA, a major outer membrane protein of Escherichia coli. P. aeruginosa mutants lacking OprF show many of the same defects as E. coli lacking OmpA (in a Ipp mutant background) 4 including osmotic instability, rounded morphology and leakage of periplasmic contents (Sonntag et al, 1978; Gotoh et al, 1989; Woodruff and Hancock, 1989). Furthermore, OprF and OmpA are immunologically cross-reactive, the two proteins demonstrate 33% identity of amino acid sequences in their carboxy-terminal halves and cloned OprF appears to be able to substitute for the structural role of OmpA in an E. coli Ipp mutant background (Woodruff and Hancock, 1989). Despite the distinct similarities between the carboxy-terminal halves of OprF and E. coli OmpA, there is little direct amino acid homology in their amino-terminal halves. However, when the antigenic indices of the two proteins were compared, the results suggested that the tertiary structures of the ammo-terminal halves of the two proteins were similar (Siehnel et al, 1990). Using the model for the arrangement of the OmpA amino-terminus (Morona et al, 1984) as a basis, an analogous arrangement was predicted for OprF (Figure 1). The carboxy-terminal half of the OprF model is not analogous to that predicted for OmpA. The OmpA model placed most of the carboxy-terminus within the periplasm whereas the OprF model depicted a membrane-spanning carboxy-terminus. A membrane-spanning carboxy-terminus seemed to be more consistent with unpublished data that localized the epitope for the surface-reactive anti-OprF MAb MA5-8 to the carboxy terminus. The region between the disulphide bridges, outlined by the broken line on Figure 1, is not homologous to OmpA. This region was placed on the surface based on the observation that another surface-reactive anti-OprF 5 Figure 1. Conceptual model of the structure of P. aeruginosa OprF (Siehnel et al, 1990). Predicted membrane-spanning (3-sheet structures, determined by the method of Paul and Rosenbusch (1985), are boxed. The positions of the (3-strands connecting the (3-sheets in the amino terminus were determined by analogy to E. coli OmpA (Morona et al, 1984). The broken line defines the loop structures predicted to arise as a result of disulphide bonds between neighbouring cysteine residues, shown by the shaded areas. 6 R T D S E T G Y T G R V D H Y E G „ Y f S L A L E V '4 tr D T S D Y F 95 .S3 103 16 G G 28! I A V E R V R G A E N E V A A E V T A ^ G A E Y N A G c^- 299 D A V K R P 7 MAb, MA4-4 failed to react with the protien when it was treated with (3-mercaptoethanol. This treatment would destroy the predicted disulphide bridges. After proposing a model such as that depicted in Figure 1, the next logical step is to test it to ensure it is in agreement with experimental data. Of course the ultimate affirmation of the model would be a comparison to its crystalline structure. Porin proteins like OprF have proven to be difficult to crystallize. Until such time that crystalline structures become a reality, other means of analyzing protein structure must suffice. One alternative genetic method used to study protein topology is to fuse the protein of interest to E. coli alkaline phosphatase (PhoA). PhoA (EC3.1.3.1) is an easily assayable enzyme which is active when it is localized to the periplasm but not when it is localized to the cytoplasm. Hoffman and Wright (1985) took advantage of these two properties of PhoA to study protein secretion in bacteria, reasoning that PhoA activity in a protein fused to PhoA indicated that the protein in question was being transported out of the cytoplasm. PhoA activity can be easily monitored by including a chromogenic PhoA substrate such as 5-bromo-4-chloro-3-indolyl phosphate (X-P) in agar medium and screening for coloured colonies (blue in the case of X-P). Manoil and Beckwith (1985) took this concept one step further by developing the specialized transposon TnphoA, which consists of transposon Tn5 with the phoA gene minus its promoter and signal sequence CphoA) inserted into the IS50L element of that transposon. Transposition of TnphoA into genes can be used to generate fusions of PhoA to amino-terminal 8 fragments of the protein product of that gene. Since its incept ion, TnphoA mutagenesis has been employed by researchers for m a n y applicat ions i n c l u d i n g the s tudy of membrane protein topology. Mos t of the l i terature on u s i n g T n p h o A to s tudy membrane prote in topology deals w i t h cytoplasmic membrane proteins, w i t h considerably fewer reports deal ing w i t h outer membrane proteins. Outer membrane proteins successful ly mutagenized by T n p h o A inc lude FepA, the E. coli ferric enterobactin receptor (Murphy et al, 1990; M u r p h y and Klebba , 1989), F h u A , the E. coli ferrichrome-iron(III) receptor (Coulton et al, 1988; G u n t z a n d B r a u n , 1988), E. coli O m p A (Weiser a n d Gotsch l i ch , 1991) and OmpT, a n E. coli outer membrane protease (Song and Lundr igan , 1991). Of the aforementioned reports, only the investigations of M u r p h y et al (1990) employed T n p h o A fusions specifically to s tudy the topology of the protein i n question, FepA. The i r results indica ted that i n the case of some FepA: :PhoA fusions, the P h o A por t ion remained i n the pe r ip lasm whi le the F e p A por t ion became distorted w i t h respect to wild-type conformation. The ul t imate fate of P h o A i n a bacter ia l cel l wh e n i t is fused to a n outer membrane protein is an uncer ta in ty and is a n impor tant considera t ion w h e n ana lys ing an outer membrane protein by T n p h o A mutagenesis . In th is investigation, T n p h o A mutagenesis was carr ied out o n P. aeruginosa oprF w i t h the goal of creating a series of O p r F amino- te rmina l fragments of differing lengths w h i c h could then be used for deletion analysis . B y deterrnining the exact fus ion jo in t between oprF and T n p h o A a n d cross-referencing this w i t h 9 any loss in reactivity with anti-OprF monoclonal antibodies, the positions of some OprF epitopes were located. The predicted epitope locations were complemented by the immunoreactive patterns of OprF derivatives created by subcloning. Implications of the results on the proposed structural model of OprF and other characteristics of OprF are discussed. 10 MATERIALS AND METHODS A. Bacterial Strains, Bacteriophage and Plasmids The strains of Escherichia coli, bacteriophage and plasmids used in this study are listed in Table I. Strain LE392 and ATnphoA were generous gifts from Keith Poole, Dept. of Microbiology and Immunology, Queens University, Kingston, Ontario; strain CC118 was a generous gift from Brett Finlay, Dept. of Microbiology, University of British Columbia, Vancouver, British Columbia. B. Monoclonal Antibodies and Polyclonal Sera Monoclonal antibodies and polyclonal sera used in this study are listed in Table II. C. Media and Growth Conditions The culture media used for all E. coli strains used in this study was Luria Broth (LB) (1% tryptone, 0.5% yeast extract, 1% NaCl). Solidified LB media contained 2% agar. Media components were from Difco laboratories, Detroit, MI. All strains were grown at 37°C with agitation overnight. Cells destined to \ 10 MATERIALS AND METHODS A. Bacterial Strains, Bacteriophage and Plasmids The strains of Escherichia coli, bacteriophage and plasmids used in this study are listed in Table I. Strain LE392 and XTnphoA were generous gifts from Keith Poole, Dept. of Microbiology and Immunology, Queens University, Kingston, Ontario; strain CC118 was a generous gift from Brett Finlay, Dept. of Microbiology, University of British Columbia, Vancouver, British Columbia. B. Monoclonal Antibodies and Polyclonal Sera Monoclonal antibodies and polyclonal sera used in this study are listed in Table II. C. Media and Growth Conditions The culture media used for all E. coli strains used in this study was Luria Broth (LB) (1% tryptone, 0.5% yeast extract, 1% NaCl). Solidified LB media contained 2% agar. Media components were from Difco laboratories, Detroit, MI. All strains were grown at 37°C with agitation overnight. Cells destined to Table I. Bacterial strains, bacteriophage and plasmids Characteristics Reference or source E. coli strain TBI C328 C524 CC118 LE392 UT5600 C477 C508 -> C523 C525 HB101 MM294 C290 rec araA(lac pro AB) thi rpsL/Q>80dlac AMI 5hsdR TBl/pWW2200 T B l / p G C 3 1 F" araD139 A(ara,leu)7697 AlacX74phoAA20 galE galK thi rpsE rpoB argEam recAl F- hsdR514{v\ m'J supE44 supF58 lacYl or A(lacIZY]6 galK2 galT22 metBl trpR55 A," F" ara-14 leuB6 azi-6 lacYl proC14 tsx-67 A(ompT-fepC)266 entA403 X' trpE38 rfbDl rpsL109 xyl-5 mtl-1 thi-1 F" lacam trpam phoam maiam rpsL supC3 lori h£pR165-TnlO CC118 /pRFl -> pRF16 CC118 /pGWl F" hsdS20{v3 m g recA13 ara-14 proA2 lacYl galK2 rpsL20[SmI) xyl-5 mtl-1 supE44 X' supE44 hsdR endAl pro thi HB101/pWW5 Messing and Vieira, 1982 Woodruff and Hancock, 1989 This study Manoil and Beckwith, 1985 Maniatis et al, 1982 Elish et al, 1988 Goff et al, 1984 This study This study Maniatis et al, 1982 Sambrook et al, 1988 Woodruff et al, 1986 12 C299 C324 C321 Phage TnphoA Plasmid pWW2200 pGC31 p R F l -> pRF16 p G W l pWW5 pWW12 MM294/pWW12 TBl/pWW1602 TBl/pWW1901 b221 cl857 Pam3 with TnphoA in or near rex pRK404 + 2.4 kb PstI fragment containing P. aeruginosa oprF gene pRK404 + 2.5 kb Pstl-BamHI fragment containing P. syringae oprF gene pWW2200 + TnphoA insert in P. aeruginosa oprF gene pGC31 + TnphoA insert in P. syringae oprF gene pUC8 + 2.0 kb Sall-PstI fragment encoding a P. aeruginosa OprF with an internal deletion pLAFR + 11.2 kb EcoRI fragment encoding a truncated P. aeruginosa OprF pWW1602 pUC8 + 1.2 kb Sail fragment encoding the carboxy-terminus of P. aeruginosa OprF pWW1901 pUC8 + 3.0 kb Sail fragment encoding the amino-terminus of P. aeruginosa OprF Woodruff, Ph.D. thesis Woodruff, unpublished Woodruff, unpublished Gutierrez et al, 1987 Woodruff and Hancock, 1989 Ullstrom et al, 1991 This study This study Woodruff et al, 1986 Woodruff, Ph.D. thesis Woodruff, unpublished Woodruff, unpublished 13 Table II. Monoclonal antibodies and polyclonal sera Immunogen Reference or source Monoclonal  antibodies MA4-4, MA5-8 P. aeruginosa OprF Mutharia and Hancock, 1985 MA7-1 -> MA7-8 P. aeruginosa OprF or heat-killed cells M . Rosok, Oncogen, Seattle, WA Polyclonal  sera a-BAP E. coli PhoA 5 Prime -> 3 Prime, Boulder, CO be made compotent were grown at 37°C with agitation to an O . D . 6 0 0 of 0.2 to 0.4. Antibiotic concentrations used were 12.5 ug/ml of tetracycline (Tc) and 50 ug/ml of kanamycin (Km). Five-bromo-4-chloro-3-indolyl phosphate (X-P) (p-toluidine salt, BaChem, Phildelphia, PA) was dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 20 ug/ml. Strains were stored for short term on plates at 4°C and for long term in 8% DMSO at -70°C. Bacteriophage A-TnphoA was propogated by mixing 0.1 ml of a 108 PFU/ml stock with 0.1 ml of overnight E. coli LE392 host in four ml of prewarmed overlay agar (0.6% agar, 1% LB). Overlay was poured onto LB plates, allowed to set and incubated at 37°C overnight. Overlay was scraped off into five ml sterile LB and centrifuged at 1000 x g for 15 minutes. Supernatant was removed and 0.1 ml of 14 chloroform was added to the supernatant. Serial dilutions were performed to determine the titre of the phage stock. The original phage stock was stored at -70°C. Stocks propogated from the original stock were stored at 4°C. D. TnphoA mutagenesis TnphoA mutagenesis of plasmid pWW2200 (Figure 2) was carried out as follows. Cells grown in LB supplemented with 10 mM MgS04 to an O.D. 6 0 0 of 0.5 were infected with ArnphoA at a multiplicity of infection of 1. Successful transpositions were selected on LB plates containing Tc to select for maintenance of the plasmid and Km to select for the presence of the transposon. To separate successful transpositions onto plasmid DNA from chromosomal transposon mutants, plasmid DNA was isolated from the pool of doubly resistant colonies and the pooled plasmid preparation was used to transform strain CC118. Transformants were again selected for Tc and Km resistance on LB plates and screened for production of alkaline phosphatase (PhoA) by inclusion of X-Pin the medium. Both PhoA positive and negative clones were selected for further study. The mutagenesis of plasmid pGC31 (Figure 2) was carried out by Graham Wong who kindly provided the mutant C525 for inclusion in this study. Plasmid pGC31 had to be transformed into the appropriate TB1 background prior to mutagenesis by the same protocol. 15 Figure 2. Plasmids used in mutagenesis. Restriction sites: B = BamHl; E = EcoRI; H = Hindlll; M = Smal; P = PstI; S = Sail. Plasmid sizes are given in kilobases (kb). 16 1 7 E . DNA techniques 1. Isolation of plasmid DNA For large scale isolation of plasmid DNA, alkaline lysis followed by centrifugation in ethidium bromide cesium chloride density gradients was used (Sambrook et al, 1989). For rapid small scale isolation of plasmid DNA, an . alkaline lysis method was used (Sambrook et al, 1989). 2. Quantitation of plasmid DNA Plasmid DNA was quantitated by ultra violet spectrophotometry (Sambrook et al, 1989). In later experiments, a fluorescence spectrophotometric assay (Labarca and Paigen, 1980) using the DNA-binding fluorochrome H33258 (Hoefer, San Francisco, CA) was used. 3. Sequencing DNA sequencing was carried out in order to determine the precise locations of the fusions between oprF and TnphoA. Double stranded, cesium chloride gradient purified, whole plasmid DNA was used as the template for all sequencing reactions. The reaction primer used was a generous gift from Jeff Greenwood, 18 Dept. of Microbiology, University of British Columbia, Vancouver, British Columbia. This primer hybridized to a region 47 to 66 bases upstream of the left end of TnphoA, around the junction between IS50L and phoA (Figure 3). All sequencing was carried out with the Applied Biosystems (Foster City, CA) model 373A DNA Sequencing System. Preparation of sequencing gels, buffers and other reagents was done according to Applied Biosystem protocols. The Taq DyeDeoxy Terminator Cycle Sequencing Kit from Applied Biosystems was used for all sequencing reactions. Polymerase chain reactions were carried out in an Ericomp (San Diego, CA) model TCX15 thermal cycler. Excess DyeDeoxy Terminators from completed sequencing reactions were removed by passing the reaction mixture over a 1 ml Sephadex G-50 column (5 Prime -> 3 Prime, Boulder, CO). Column eluants were concentrated, loaded on to sequencing gels and run according to Applied Biosystem protocols. In order to locate fusion joints, the DNA sequences generated were comparatively aligned with the published DNA sequence of oprF from P. aeruginosa (Duchene et al, 1988) or oprFfrom P. syringae (Ullstrom et al, 1991) or the reverse complementary sequence to each gene. This analysis was assisted by the SeqEd 675 DNA Sequence Editor program from Applied Biosystems. 4. General DNA techniques Restriction enzyme digests, agarose gel electrophoresis and transformations 19 were carried out as described by Sambrook et al (1989). E. coli strains C268, UT5600, C477 and CC 118 were all made compotent using 0. IM CaCl 2 (Sambrook et al, 1989). Agarose gel electrophoresis was performed in BioRad (Richmond, CA) DNA Sub cells or Mini Sub DNA cells with IX T B E as the running buffer. F. Protein techniques 1. Isolation of total cellular protein Total cellular protein was isolated using a protocol derived from the method of Nicas and Hancock (1980). Cells grown on plates or in broth were suspended in sterile water to an O . D . 6 0 0 of 0.4. A sample of this suspension was pelleted and resuspended in a smaller volume of sterile water and mixed with an equal volume of lysis buffer consisting of 2% sodium dodecyl sulphate (SDS), 10% glycerol, bromophenol blue, 0.1 M Tris-HCl, pH 6.8. The ratio of cell suspension : sterile water : lysis buffer was kept constant at 1 m l : 50 u l : 50 ul. Lysates were heated at 100°C for 10 minutes and 10 ul was loaded per well. The proteins in the lysate were separated by SDS polyacrylamide gel electrophoresis (PAGE), using 11% PA gels run in BioRad Mini-PROTEAN II cells. The running buffer used was 25 m M Tris-Cl, 0.1% SDS, 192 mM glycine, pH 8.3. Protein gels were stained with Coomassie Blue. 20 2. Preparation of outer membranes Outer membranes were prepared using differential solubility in Triton X-100 as outlined by Schnaitman (1971). Overnight cultures (50 ml) were harvested by centrifugation, resuspended in 5 ml of 10 m M sodium phosphate buffer, pH 7.4, 5 mM MgS0 4 , treated with 50 ug/ml DNase and broken by two passages through a French pressure cell at 15,000 psi. Broken cells were centrifuged at 3,000 rpm for 10 minutes to remove cell debris and the resulting supernatant was centrifuged at 45,000 rpm for 1 hour. A sample of the supernatant resulting from this centrifugation was retained for each preparation. This supernatant represented the cytoplasmic and periplasmic fractions of the cell. The pellet of cell envelopes was resuspended in sterile water, checked for protein content (section F3) and adjusted to 10 mg/ml with sterile water. Cell envelope preparations were adjusted to 10 mM Tris-HCl, 5 m M MgS0 4 , pH 7.4 and Triton X-100 was added to a concentration of 2%. This preparation was centrifuged at 45,000 rpm for 1 hour and the resulting pellet of outer membrane was resuspended in sterile water. Outer membrane samples were run on SDS-PA gels as described in F l . 3. Quantitation of protein Protein was quantitated using the assay of Sandermann and Strominger 21 (1972). A 0.1% bovine serum albumin (BSA) solution was used to prepare standard curves. G. Immunodetection techniques 1. Colony immunoblotting Colony immunoblotting was performed by the method of Woodruff and Hancock (1986). Bacterial colonies were transferred from agar plates to nitrocellulose filters by contact. Filters were suspended in a chloroform vapour saturated chamber for 20 minutes to permeabilfze the cells, placed in empty petri dishes and covered with 10 ml of lysis buffer consisting of 10 mM Tris-HCl, 150 m M NaCl, 5 m M MgCl 2 , 3% BSA, 1 ug/ml DNase and 40 pg/ml lysozyme, pH 8.0. After incubating at least 45 minutes with shaking, filters were squirted with phosphate buffered saline (PBS) to remove bacterial debris and washed twice for 5 minutes in PBS. Filters were then covered with 10 ml of primary antibody solution (1% BSA in PBS plus 10 pi of primary antibody) and incubated 1 and 1/2 to 2 hours at 37°C or overnight at room temperature with shaking. Filters were washed 3 times with PBS for 5 minutes and covered with secondary antibody solution (PBS plus 5 ' ul of appropriate horseradish peroxidase-conjugated secondary antibody) and incubated 1 and 1/2 to 2 hours at 37°C with shaking. Filters were washed 3 times with PBS for 5 minutes and developed 22 using the system described by Harlow and Lane (1989). 2. Western blotting Proteins were separated by SDS-PAGE (section Fl) and electrophoretically transferred to nitrocellulose filters from the gel using the BioRad Mini Trans-Blot electrophoretic transfer cell. The blotting buffer used was 25 mM Tris-HCl, 192 m M glycine and 20% (v/v) methanol, pH 8.3. After transfer, the filters were treated as described in G l except that the chloroform treatment step was omitted and filters were initially blocked with 1% BSA in PBS for 30 minutes. 23 RESULTS A. Primary Screening A total of 96 blue colonies and 500 white colonies arising from 10 separate transfections of C328 with ATnphoA were collected. The observed frequency of blue colonies was approximately 1%. A similar frequency of blue colonies was observed when C524 carrying oprF from P. syringae on plasmid pGC31 was subjected to the same TnphoA mutagenesis protocol (Graham Wong, personal communication). All 596 colonies were screened by colony immunoblotting against all 10 anti-OprF MAbs to determine which colonies were producing immunodetectable OprF derivatives. Colonies which reacted with all 10 MAbs were not found, suggesting that all selected transposition events had probably occurred within the oprF gene. Plasmid DNA from all blue colonies, all immunoreactive white colonies and randomly selected non-immunoreactive white colonies was digested with Sail in order to approximate the location of transposition into oprF. Sail was a convenient enzyme to use for locating transposition sites. The oprF gene contains a Sail site in the centre which divides the gene into coding regions corresponding to the amino- and carboxy-terminal halves of OprF (Figure 2). This Sail site has been used as the dividing line between amino- and carboxy-24 terminus for the remainder of this thesis. Digestion of pWW2200 with Sail yields four bands with the two smallest bands 1.3 and 1.1 kb in size corresponding to the coding regions of the amino- and carboxy-terminus respectively. With Sail restriction mapping it was easy to determine whether transposition had occurred within the amino- or carboxy-terminal coding region by looking for the disappearance of the 1.3 or 1.1 kb Sail fragment respectively (Figure 3). The loss of one of the small Sail fragments was coupled with the appearance of two extra Sail fragments in the 3 to 6 kb range (Figure 3), as there is one Sail site in the TnphoA DNA. Based on the primary screening of colony immunoblotting followed by Sail restriction mapping, 16 different insertion sites were located across the oprF gene (Table III). The limited number of TnphoA insertion sites found in the 361 plasmids mapped with Sail suggests that either insertion of TnphoA into oprF is not a random event or that some insertion sites result in a lethal protein conformation and are therefore not selected. Representative mutants from each insertion category were collected for secondary screening. B. Secondary screening Plasmid DNA from each representative mutant was sequenced in order to determine the precise location of fusions between oprF and TnphoA and also to determine the orientation of transposition. Depending on the orientation and 25 Figure 3. Sail digestion patterns of fusion plasmids. Far right lane contains 1 kb ladder (BioRad) and second lane from the right contains the parental plasmid, pWW2200, digested with Sail. The remaining lanes contain pWW2200 carrying TnphoA insertions, digested with Sail. Lanes containing the parental plasmid and representative examples of insertions in the amino- and carboxy-terminal coding regions are indicated by P, N, and C respectively. All plasmids were prepared using the alkaline lysis mini-preparation procedure. 26 S a i l fragments from TnphoA insert 27 Table III. Insertion sites located by Sail restriction mapping Size of Sail fragments (kb)a Colony colour on X-P medium b Insertion location0 Observed frequency11 3.8, 5.4 B N 17 3.7, 5.5 B N 48 3.6, 5.6 W N 7 3.5, 5.7 B N 2 4.1, 5.1 W N 13 3.5, 5.7 W N 28 4.2, 5.0 W N 11 3.4, 5.8 ^ W N 4 4.3, 4.9 W N 43 3.3, 5.9 W N 1 4.4, 4.8 W N 11 3.2, 6.0 B N 22 4.6, 4.6 W N 2 3.3, 5.6 W C 39 3.5, 5.4 W C 75 4.0, 4.9 W C 38 a Sail fragments are those arising as a result of TnphoA insertion; kb = kilobases. b B = blue; W = white. c Insertion sites reported from most ammo-terminal to most carboxy-terminal; N = amino-terminal coding region; C = carboxy-terminal coding region. d Out of a total of 361 colonies screened by Sail restriction mapping. 28 reading frame of the insertion, the fusion proteins consisted of a truncated OprF region fused to either PhoA or to peptides of 1 to 20 amino acids in length derived from the IS50 elements of TnphoA (Table TV). As shown schematically in Figure 4, TnphoA insertions can occur in two different orientations. Insertions in the correct orientation will place 'phoA under the control of the oprF promoter. Consequently, insertions in the correct orientation can end up either with 'phoA in the same reading frame as oprF (in frame fusions) or in the 2 other reading frames (out of frame fusions). The product of in frame fusions to 'phoA would consist of a truncated OprF fused to PhoA while the product of out of frame fusions would consist of a truncated OprF fused to peptides of 1 or 6 amino acids in length. Insertions in the opposite orientation can occur in three different reading frames resulting in truncated OprF fused to peptide tags of either one, 18 or 20 amino acids in length. The size of the peptide tags were determined by the number of amino acids encoded before the first stop codon in the reading frame was reached. Only four transposition sites were recovered in the P. aeruginosa oprF gene which gave rise to functional expression of PhoA as defined by the production of blue colonies on X-P medium. Fusion joint sequencing further confirmed that the insertions were in frame with respect to the 'phoA coding region. Also, bands corresponding to the predicted molecular mass of the full fusion proteins were demonstrated on Western blots of whole cell lysates developed with anti-bacterial alkaline phosphatase (BAP) (Figure 5, lanes C - F). All functional fusions to 29 TABLE IV. Characteristics of OprF fusion proteins obtained Fusion Plasmid Fusion site Last oprF bp Last OprF aa Predicted size of peptide fused to OprF (aa)a p R F l 73 Ala-1 PhoA b pRF2 91 Val-6 PhoA pRF3 119 Tyr-15 1 pRF4 205 Ala-44 PhoA pRF5 206 Ala-44 20 pRF6 215 Tyr-47 1 pRF7 351 Ala-93 1 pRF8 360 Asn-96 6 pRF9 382 Asp-103 18 pRFlO 422 Gly-116 1 p R F l l 505 Asn-144 18 pRF12 532 Gly-153 , PhoA pRF13 614 Asp-180 1 pRF14 685 Gly-204 18 pRF15 941 Gly-289 20 pRF16 970 Ser-299 PhoA p G W l 916 Gly-268 PhoA bp = base pair; aa = amino acid; a Length of peptide determined by number of aa encoded before first stop codon. b Indicates predicted in frame fusion to PhoA. 30 Figure 4. TnphoA mutagenesis of oprF. Adapted from Taylor et al (1989). Dark boxes represent the IS50L and IS50R regions of Tn5; the lines connecting the dark boxes represents the Tn5 central region carrying genes for kanamycin resistance; open boxes represent the 'phoA gene inserted into the IS50L region of Tn5 to give TnphoA; hatched boxes represent the oprF gene. The two possible insertion orientations are shown alongside the corresponding phenotypes. 31 Tn5 Tn5 central IS50L region IS50R V/-TnphoA 'phoA transposition into oprF on plasmid pW¥2200 (Tcr) correct orientation: oprF' m m . 'oprF 3 Tc r K m r PhoA + / - OprF oprF' 'oprF incorrect orientation: V/- Tc r Km" PhoA OprF 32 Figure 5. Immunoreactivity of OprF::PhoA fusion proteins with anti-bacterial alkaline phosphatase polyclonal sera. Whole cell lysates were heated at 100°C for 10 minutes, run on 11% SDS PA gels and transferred to nitrocellulose. The position of purified PhoA (Sigma, St. Louis, MO) is indicated on the left and molecular size markers in kilodaltons (kD) are indicated on the right. Arrows indicate the bands used for apparent molecular mass determination (see Table V). Lane A, purified PhoA; lane B, CC118 control to show cross-reactive bands in the background strain; lane C, CC118 (pRFl); lane D, CC118 (pRF2); lane E , CC118 (pRF4); lane F, CC118 (pRF12); lane G, CC118 (pRF16); lane H, CC118 (pGWl). The predicted and apparent molecular weights are given in Table V. 33 - 2 4 34 PhoA occurred in the amino-terminus and none were observed in the carboxy-terminus of P. aeruginosa OprF. However one functional fusion at amino acid 268 in the carboxy-terminus of OprF from P. syringae was obtained by G. Wong and studied further here. This fusion was also in frame according to fusion joint sequencing although the full fusion protein could not be demonstrated with anti-BAP (Figure 5, lane H). The fusion at amino acid 299 was interesting in that it appeared from sequencing around the fusion joint to be in frame with respect to 'phoA yet did not give rise to blue colonies on X-P nor was a band corresponding to the full fusion protein of PhoA detectable with anti-BAP (Figure 5, lane G). MAb reactivity patterns of the OprF fusion proteins were determined by reacting whole cell lysates run on 11% SDS PA gels and blotted on to nitrocellulose with each of the 10 anti-OprF MAbs. Reactivity results are summarized in Figure 6 and Table V. C. Further characterization of OprF fusion proteins As indicated in Table V , the apparent molecular masses of the immunoreactive OprF fusion proteins corresponded well with the molecular masses predicted from sequencing data. The exceptions were the fusions at amino acids 289 and 299 where the main immunodetectable product did not correspond to the molecular masses predicted for the full fusion protein or the OprF portion of the fusion protein. The weakly immunodetectable products, 35 Figure 6. Location of fusions and MAb reactivity patterns of P. aeruginosa OprF fusion proteins. Dark arrows indicate functional expression of PhoA (blue colonies on X-P medium), open arrows indicate white colonies on X-P medium, and the direction of the arrow indicates the orientation of transposition. Restriction sites: K = Kpnl; M = Smal; S = Sail. 36 73 91 205 206 < > 351 532 360 422 k > 3 " K/M 382 > 505 6 U 679 941 970 1Q£ oprF LEADER OprF Fusions up to bp 532 all non-reactive 7-1 reactive 7-1 4 - 4 , 7 - 8 reactive 7 - 1 , 4 - 4 . 7 -8 reactive; 7 - 2 , 7 -6 weakly reactive No fusion proteins were reactive with 5 -8 , 7 - 3 , 7 -4 , 7 - 5 and 7 - 7 . 37 TABLE V. Molecular mass of immunoreactive OprF fusion proteins Fusion site Predicted molecular Apparent molecular in OprF (aa) mass (kD)a mass (kD)b Ala-T 47.0/ - 47.9 Val-6 47.6/0.6 48.4 Ala-44 51.5/4.5 49.3 Gly-153 62.5/15.5 53.2 Asp-180 18.1/18.2 18.5C Gly-204 22.8/20.7 24.5 Gly-289 31.6/29.3 23.5/28.5 Ser-299 77.3/30.3 23.5/29.3 Gly-268 74.1/27.1 49.3 aa = amino acid; kD = kilodalton; a Calculated from sequencing data. First number is size of full fusion protein and second number is the size of the OprF portion only. b Estimated by migration distance relative to prestained markers of known molecular size on Western blots developed with either anti-BAP or anti-OprF MAbs. First numbers are the main products detected, second numbers are other weakly detected products. c This is the product detected in BME-treated samples; in non-BME treated samples the main product detected is 45.2 kD in size while the 18.5 kD product is more weakly detectable (Figure 9). detectable only by MA7-2 and MA7-6 (Figure 7), corresponded better to the predicted molecular mass of the OprF portion of the fusion protein. The fusion proteins generated appeared to be unstable and subject to degradation. The 38 Figure 7. Imrnunoreactivity of OprF fusions at amino acids 289 and 299 with MA7-2. Whole cell lysates were heated at 100°C for 10 minutes, run on 11% SDS PA gels and transferred to nitrocellulose. The heated (F*) and unheated (F) forms of OprF are iondicated on the left and molecular size markers in kilodaltons (kD) are indicated on the right. Lane A, CC118 (pWW2200); lane B, CC118 (pRF16); lane C, CC118(pRF15). 39 40 degradation products observed in the case of both fusions ran at the same molecular weight as an OprF degradation product normally seen when this protein is expressed in E. coli (Figure 8). Attempts to overcome degradation by expressing the fusion proteins in E. coli backgrounds deficient in protease activity, namely an ompT mutant (Elish et al, 1988) and a Ion htpR double mutant (Goff et al, 1984), were unsuccessful. The main immunodetectable product in both backgrounds was still the degradation product. In the case of fusions at amino acid 180, the main immunodetectable product corresponded to twice the predicted molecular mass (Figure 9). This product was lost upon treatment with B M E (Figure 10). Fusions here would interrupt the predicted disulphide bond between Cys-176 and Cys-185 (see Figure 1). The monomelic form, therefore, would possess one unpaired cysteine residue which could bond to the cysteine of another monomer resulting in stable dimer formation. Outer membranes were prepared from strains producing fusion proteins detectable by anti-OprF MAb(s). These fusion proteins were detectable in the outer membrane fraction of the cell but not in the combined cytoplasmic and periplasmic fractions. This indicated that these products were transported to the E. coli outer membrane. The carboxy-terminal half of OprF, therefore, appeared to be non-essential for transport to the outer membrane. 4 1 Figure 8. Immunoreactive degradation products of OprF and OprF fusions at amino acids 289 and 299. Outer membrane preparations were heated at 100°C for 10 minutes, run on 11% SDS PA gels, transferred to nitrocellulose and reacted with MA4-4. The heated (F*) and unheated (F) forms of OprF are indicated on the left, molecular size markers in kilodaltons (kD) are indicated on the right, and the relevant OprF degradation product is indicated by the arrow. Lane A, CC118 (pRF16); lane B, CC118 (pRF15); lane C, CC118 (pWW2200). 42 - 16 43 Figure 9. Immunoreactivity of OprF fusions at amino acids 144 and 180 with MA7-1. Outer membrane preparations were heated at 100°C for 10 minutes, run on 11% SDS PA gels and transferred to nitrocellulose. The unheated form (F) of OprF, and the monomelic (M) and dimeric (D) form of the fusion at amino acid 180 are indicated on the left. Molecular size markers in kilodaltons (kD) are indicated on the right. Lane A, CC118 (pRF13); lane B, CC118 (pRFll); lane C, CC118 (pWW2200). kD 110 84 47 33 24 16 45 Figure 10. Immunoreactivity of the OprF fusion at amino acid 180 with MA7-1 in the presence of p-mercaptoethanol (BME). Outer membrane preparations were heated at 100°C for 10 minutes, run on 11% SDS PA gels and transferred to nitrocellulose. The unheated, BME-modified (F) and heated, BME-modified (F'*) forms of OprF and the monomeric (M) form of the fusion at amino acid 180 are indicated on the left. Molecular size markers in kilodaltons (kD) are indicated on the right. Lane A, CC118 (pWW2200); lane B, CC118 (pRF13). 46 - 110 - 8 4 - 47 I * 4 7 D. Location and characterization of OprF epitopes The MA7-1 epitope appeared to be contained within the first 180 amino acids of OprF as reactivity was only seen with fusions at Asp-180 onwards (Figure 9). The tendency of fusions at amino acid 180 to form dimers did not interfere with the formation of the MA7-1 epitope (Figure 9). The localization predicted for this epitope is consistent with fact that B M E treatment of OprF, which destroys predicted disulphide bonds between cysteine residues does not result in loss of the MA7-1 epitope (Table VI). As indicated in Table VI, the MA4-4 epitope and the MA7-8 epitope are both lost upon treatment of OprF with B M E . This implicated one or both of the predicted disulphide bonds as being essential for the formation of these epitopes. Fusions at Asp-180, which retain only the first cysteine residue, lost their reactivity with MA4-4 and MA7-8 while fusions at Gly-204, which retain the first 3 cysteine residues, were still reactive (Figure 11). Consequently, the MA4-4 and MA7-8 epitopes can be localized in part to between Cys-176 and Gly-204. MA4-4 and MA7-8 were raised against OprF in separate procedures in separate laboratories. No evidence thus far has indicated that these two monoclonal antibodies are recognizing different epitopes, so there is a distinct possibility that they recognize the same epitope. The epitopes for MA7-2 and MA7-6 can be localized in part to the region between Gly-204 and Gly-289 based on the observation that OprF fusions at Gly 48 T A B L E VI. Effect of (3-mercaptoethanol treatment on OprF epitopes Monoclonal Antibody Reactivity with non-BME-treated OprF Reactivity with B M E -treated OprF MA4-4 + -MA5-8 + + MA7-1 + + MA7-2 + + MA7-3 + W MA7-4 + w MA7-5 + + MA7-6 + w MA7-7 + w MA7-8 + -+ = positive reaction; - = no reaction; W = weak reaction 204 were non-reactive while OprF fusions at Gly-289 and Ser-299 both retained activity with these MAbs. The immunoreactive product in the case of both of these fusions was not the degradation product but the more weakly detectable band corresponding to the predicted molecular weights of the OprF portions (Figure 7). The degradation products were hot detectable by either MA7-2 or MA7-6. The limits of the epitopes could certainly be better defined if the exact 49 Figure 1 1 . Immunoreactivity of OprF fusions at amino acids 180 and 204 with MA4-4 and MA7-8 in the presence and absence of f3-mercaptoethanol (BME). Outer membrane preparations were heated at 100°C for 10 minutes, run on 11% SDS PA gels and transferred to nitrocellulose. The heated (F*) and unheated (F) forms of OprF are indicated on the left, molecular size markers in kilodaltons are indicated at the top. Samples in lanes D, E , F, J , K, and L were treated with B M E . Samples in lanes A through F were reacted with MA4-4; samples in lanes G through L were reacted with MA7-8. Lanes A, D, G, and J , CC118 (pRF13); lanes B, E , H , and K, CC118 (pRF14); lanes C, F, I, and L, CC118 (pWW2200). 50 A B C D E F G H I J K L lc D - 1 6 51 nature of these degradation products were known. The degradation products extend past Gly-204 as they run slightly larger than OprF fusions at Gly-204 but exactly how far they extend is at the moment unresolved. The MA7-2 and MA7-6 epitopes appear to be distinct as reactivity of intact OprF with MA7-2 is unaffected by BME-treatment whereas reactivity with MA7-6 is weakened by B M E treatment (Table VI). Also, MA7-2 and MA7-6 have different cross reactivity patterns with OprFs from other Pseudomonas species (Martin, N., and R.E.W. Hancock, unpublished data). All OprF fusion proteins generated in this study were non-reactive with MA5-8, MA7-3, MA7-4, MA7-5 and MA7-7 (Figure 6). The epitopes for these MAbs are either located from Ser-299 onwards or require the presence of more of the OprF carboxy-terminus for their formation. E . Reactivity of other OprF derivatives with anti-OprF monoclonal antibodies The locations of the epitopes recognized by the anti-OprF MAbs as proposed by analysis of the OprF derivatives created by TnphoA mutagenesis were complemented by the MAb reactivity patterns of OprF derivatives created by subcloning. These subclones were all constructed or isolated by Wendy Woodruff during her Ph.D. research. The amino-terminal (start to Sail) and carboxy-terminal (Sail to end) coding regions of oprFwere subcloned to give pWW1901 and pWW1602 respectively. The other two OprF derivatives, pWW5 and pWW12, 52 Table VII. Immunoreactivity of OprF derivatives expressed by oprF subclones oprF subclone oc-OprF MAb pWW5 pWW12 a pWW1901b pWW1602 c MA4-4 + + -MA5-8 + - + MA7-1 + + + -MA7-2 W - + MA7-3 - - + MA7-4 - - + MA7-5 - - + MA7-6 w - + MA7-7 - - + MA7-8 + + -+ = positive reaction; - = negative reaction; W = weak reaction a T h e product of this subclone is subject to degradation; degradation products are not immunoreactive with MA7-2 or MA7-6. b Immunoreactivity checked by colony immunoblot only. c Data kindly provided by Nancy Martin. arose during the original cloning of the oprF gene. Sequencing of pWW5 and pWW12, carried out by Richard Siehnel, revealed that pWW5 encoded an OprF with an internal deletion from amino acid 171 through 300 whereas pWW12 encoded and OprF which was truncated at amino acid 273. 53 The proposed locations of some epitopes can be further defined by taking these OprF derivatives into consideration as well. MA7-1 still reacted with the protein product expressed by pWW5 so the MA7-1 epitope can be narrowed down to the first 171 amino acids of OprF. MA4-4 and MA7-8 both retained reactivity with the amino-terminal half of OprF. Consequently the limits of the epitope(s) can be narrowed down to between Cys-176 and Asn-187, containing only the first pair of cysteine residues, since Asn-187 is the last amino acid encoded before the Sail site. The reactivity pattern of the truncated OprF expressed by pWW12 with MA7-2 and MA7-6 narrows the epitopes for these MAbs to between Gly-204 and Ala-273. The epitope for the MA5-8 can be localized to the last 26 amino acids of OprF based on the observation that the protein products expressed by pWW5 and pWW1602 retained reactivity with this MAb. Finally, the epitopes for MA7-3, MA7-4, MA7-5, and MA7-7 are contained in the carboxy-terminus of the protein as indicated by their reactivity with the product of pWW1602. The proposed locations of the epitopes recognized by these anti-OprF MAbs are shown schematically in Figure 12. 54 Figure 12. Proposed locations of anti-OprF epitopes. Arrows on the linear map indicate the positions of TnphoA fusions across oprF [see Figure 6). The proposed epitope locations are shown above the linear map and the predicted products expressed by the various oprF subclones are shown underneath. The hatched region indicates the region of OprF which is homologous to OmpA. C = cysteine residues; restriction sites: K = Kpnl; M = Smal; S = Sail. 55 M A 7 - 1 M A 7 - 3 , 7 - 4 , 7 - 5 , 7 - 7 M A 4 - 4 M A 7 - 8 M A 7 - 2 . 6 M A 5 - 8 1 2 4 8 10 12 • 11 13 14 < < < GENE PROTEIN K/M < pWW1901 pWW1602 pWW12 pWW5 C C C C 187 188 170 15 16 > 1050 ' 326 326 273 301 326 H 56 DISCUSSION TnphoA mutagenesis of P. aeruginosa oprF yielded a limited number of distinct insertion sites. One explanation for this might be that transposition was not occurring randomly. This seems to be possible in light of the fact that the parent transposon of TnphoA, Tn5, recognizes preferred insertion sites within genes, known as hotspots (Berg et al, 1983). However, the possibility that some insertion sites are lethal should not be overlooked. Consistant with this concept, very few in frame insertions of the 'phoA gene occurred. Further, it is apparent from Figure 5 that very few insertions occurred within the region encoding the carboxy-terminus (from the Sail site through to the end of the gene) in comparison with the larger number of insertions in the region encoding the amino-terminus. Insertions in the carboxy-terminus may give rise to lethal effects if protein folding and/or export is disrupted. Only four different OprF::PhoA fusion proteins resulted from TnphoA mutagenesis. This number is considerably less than the results of Murphy and Klebba (1989) who reported 21 different in-frame insertion sites across the fepA gene after TnphoA mutagenesis. Consequently, they were able to rely mainly on FepA::PhoA fusion proteins in order to study FepA surface topology. FepA is 2.5 times larger than OprF and may simply contain more suitable insertion sites. The limited number of OprF::PhoA fusion proteins made it necessary, in this study, 57 to utilize mainly the protein products resulting from insertions of TnphoA in the opposite orientation to the oprF gene and insertions which were out of frame with respect to 'phoA. The size range of the peptide tags which could be fused to OprF was predicted from the reading frame and known sequence of the ends of TnphoA to be one to 20 amino acids. Using these OprF derivatives could possibly avoid the potential complications arising from the intrinsic properties of PhoA. For example, Murphy et al. (1990) found in their study of FepA::PhoA fusion proteins that certain fusion sites gave rise to fusion proteins in which the PhoA portion remained periplasmic while the FepA portion became distorted with respect to wild type conformation. Two of the main features of the working model of OprF presented in Figure 1 are in good general agreement with the results of this study. The first feature is the membrane spanning carboxy-terminus. Many of the anti-OprF MAbs used in this study were surface reactive (Martin, N., E . Rawling, and R.E.W. Hancock, manuscript in preparation). Consequently, the epitopes recognized by these MAbs should be presented at the cell surface. If the carboxy-terminus of OprF resided entirely within the periplasm, like the OmpA model (Morona, 1984), it should follow that epitopes for surface reactive MAbs would not be located in this region. The results of this study indicate that at least two of the surface reactive anti-OprF MAbs (MA7-6 and MA5-8) and as many as five (MA5-8, MA7-3, MA7-5, MA7-6 and MA7-7) are recognizing epitopes in the carboxy-terminus of OprF. A membrane-spanning carboxy-terminus like that depicted in Figure 1 is more 58 consistent with these observations. Recently, investigators studying the PHI protein of Neisseria gonorrhoeae, a protein which shows carboxy-terminal homology with OprF, also presented evidence of a surface exposed carboxy-terminus (Gulati et al, 1991). Based on mobility shifts in the presence of B M E , Hancock and Carey (1979) proposed that OprF contained two disulphide bonds. In the model (Figure 1), these bonds were placed for convenience between neighbouring disulphide residues. The resultant loop regions between the bonds were arranged at the cell surface based on the observation that reactivity with MA4-4, a MAb that recognizes surface epitopes, was lost upon treatment of OprF with B M E (Mutharia and Hancock, 1985). This surface arrangement is supported by the data which localized the MA4-4 and MA7-8 epitope to the region between amino acids 176 and 187 (Figure 11). The secondary structure created by a disulphide bond between Cys-176 and another cysteine residue forms the epitope recognized by these two MAbs. The other cysteine residue involved is probably not Cys-205 or Cys-191 as evidenced by the fact that the ammo-teirninal half of OprF, which would lack both these cysteines, is still reactive with MA4-4 and MA7-8. Also, fusions at amino acid 204 (which would lack Cys-205) retained reactivity with both MA4-4 and MA7-8. Although it should be pointed out that there are cysteine residues in the peptide tag of this particular fusion protein which may compensate for the lack of Cys-205. In general, the proposed arrangement of the disulphide bonds is supported by this study. 59 During the course of this research other observations regarding the transport and assembly of OprF were made which, although they do not directly address the main objective of mapping OprF epitopes, still merit some discussion. The carboxy-terminus of OprF, at least from amino acid 180 onwards, appeared to be unnecessary for transport to the outer membrane. This observation has also been made with some outer membrane proteins including LamB (Hall et al, 1982; Benson and Silhavy, 1983; Benson et al, 1984), and OmpA (Bremer et al, 1982; Henning et al, 1983). Sorting signals for transport to the outer membrane in the case of these proteins are believed to be contained within the amino-terminus, although it is unclear at present whether it is the conformation of the amino-terminus or a distinct amino acid sequence within the ammo-terminus that is responsible (Freudl et al, 1985; Struyve et al, 1991). The results of this study suggest that sorting signals for OprF transport to the outer membrane are also contained in the amino-terminus. The fusion at amino acid 180 had a tendency to form dimers which were still immunoreactive. Perhaps this region plays a key role in the assembly of OprF into multimeric forms. The latter portion of the carboxy-terminus, from Ala-273 onwards, may play a role in stabilizing the portion of the carboxy-terminus between amino acids 204 and 273 from degradation by proteases. OprF derivatives which were lacking amino acids 273 and onwards, namely the fusions at amino acids 289 and 299 and the truncated OprF expressed by pWW12, were observed to be more readily 60 processed into degradation products as compared to whole OprF. Finally, as discussed above, insertions into some sites in the carboxy-terminus may result in lethality. These important issues can only be resolved by further studies on OprF. The OprF derivatives created or further characterized in this study would be useful for continued study of OprF. The derivatives could be used to study transport and localization of OprF in greater detail and to identify regions of the protein important for the structural and functional roles of OprF. For these sorts of studies, the derivatives should be expressed in P. aeruginosa as well as E. coll This may require making more stable subclones of the TnphoA mutants by removing the transposase gene in the IS50R region. Since the locations of the MA4-4, MA7-8 and MA5-8 epitopes have been narrowed down considerably, it may now be feasible to use nested synthetic peptides to further zero in on the exact residues responsible for formation of the epitopes. Site-directed mutagenesis of OprF could also be used for this purpose. Furthermore, site-directed mutagenesis of the cysteine residues in particular could be used to answer questions about the role of the disulphide bridges in epitope formation and function of OprF as a porin. 61 REFERENCES Benson, S.A., E . Bremer, and T . J . Silhavy. 1984. Intragenic regions required for LamB export. Proc. Natl. Acad. Sci. USA. 81:3830-3834. Benson, D . E . and T . J . Silhavy. 1983. Information within the mature LamB protein necessary for localization to the outer membrane of E. coli K12. Cell 32:1325-1335. Berg, D .E . , M.A. Schmandt, and J .B . Lowe. 1983. Specificity of Tn5 insertion. Genetics 105:813-828. Bremer, E . , S T . Cole, I. Hindennach, U. 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Probing Fhu'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K-12. Mol. Gen. Genet. 215:69-75. Gutierrez, C , J . Barondess, C. Manoil, and J . Beckwith. 1987. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. J . Mol. Biol. 195:289-297. Hall, M.N., M . Schwartz, and T . J . Silhavy. 1982. Sequence information within the lamB gene is required for proper routing of the bacteriophage X receptor protein to the outer membrane of Escherichia coliK-12. J . Mol.Biol. 156:93-112. Hancock, R.E.W. 1986. Model membrane studies of porin function, p. 187-224. In M . Inouye (ed.), Bacterial outer membranes as model systems. John Wiley & Sons, N.Y. 6 4 Hancock, R.E.W., and A . M . Carey. 1979. Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifiable proteins. J . Bacteriol. 140:902-910. Hancock, R.E.W., R.T. Irvin, J.W. Costerton, and A . M . Carey. 1981. Pseudomonas aeruginosa outer membrane: peptidoglycan associated proteins. J . Bacteriol. 145:628-631. Harlow, E . and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbour Laboratory Cold Spring Harbour, NY. Henning, U. , S.T. Cole, E . Bremer, I. Hindennach and H. Schaller. 1983. Gene fusions using the ompA gene coding for a major outer membrane protein of Escherichia coli. Eur. J . Biochem! 136:233-240. Hoffman, C.S. and A. Wright. 1985. Fusions of secreted proteins to alkaline phosphatase: an approach for studying proteins secretion. Proc. Natl. Acad. Sci. USA 82:5107-5111. Labarca, C , and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352. 65 Maniatis, T., E . F . Fritsch, andJ. Sambrook. 1982. Molecular cloning: a laboratory-manual. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY. Manoil, C , and J . Beckwith. 1985. TnphoA: A transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82: 8129-8133. Messing, J . , and J . Vieira. 1982. A new pair of M13 vectors for selecting either DNA strand of double-digest fragments. Gene 19:269-276. Morona, R., M . Klose, and U. Henning. 1984. Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins. J . Bacteriol. 159:570-578. Murphy, C.K., and P.E. Klebba. 1989. Export of FepA:PhoA fusion proteins to the outer membrane of Escherichia coli K-12. J . Bacteriol. 171:5894-5900. Murphy, C.K., V.I. Kalve, and P.E. Klebba. 1990. Surface topology of the Escherichia coli K-12 ferric enterobactin receptor. J . Bacteriol. 172:2736-2746. 66 Mutharia, L .M. , and R.E.W. Hancock. 1985. Characterization of two surface localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can. J . Microbiol. 31:381-386. Nicas, T.I. and R.E.W. Hancock. 1980. Outer membrane protein HI of Pseudomonas aeruginosa: involvement in adaptative and mutational resistance to ethylenediaminetetraacetate, polymixin B and gentamycin. J . Bacteriol. 143:872-878. Nicas, T.I., and R.E.W. Hancock. 1983. Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant. J . Bacteriol. 153:281-285. Nikaido, H . , and R.E.W. Hancock. 1986. Outer membrane permeability of Pseudomonas aeruginosa, p. 145-193. In J.R. Sokatch and L.N. Ornston (eds.). The Bacteria, vol. 10. The Biology of Pseudomonas. Academic Press, Inc., Orlando FL. Nikaido, H . and M . Vaara. 1985. Molecular basis of outer membrane permeability. Microbiol.-Rev. 49:1-32. 67 Paul, C. and J.P. Rosenbusch. 1985. Folding patterns of porin and bacteriorhodopsin. E M B J O J . 4:1593-1597. Ryan, K . J . 1984. Pseudomonas and other opportunistic gram-negative bacilli, p. 264-270. In J . C . Sherris (ed.). Medical microbiology an introduction to infectious disease. Elsevier Press, NY. Sambrook, J . , E . F . Fritsch, andT. Maniatis. 1989. Molecular cloning: a laboratory manual. Second ed. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY. Sandermann, and Strominger. 1972. Purification and properties of C5 5-Isoprenoid alcohol phosphokinase from Staphylococcus aureus. J . Biol. Chem. 247:5123-5131. Schnaitman, C A . 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J . Bacteriol. 108:545-552 Siehnel, R.J . , N.L. Martin, and R.E.W. Hancock. 1990. Function and structure of the porin proteins OprF and OprP of Pseudomonas aeruginosa, p. 328-342. In S. Silver (ed.), Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, DC. 68 Song, R., and M, D. Lundrigan. 1991. TnphoA mutagenesis of the Escherichia coli OmpT endopeptidase. Abstracts of the 91st General Meeting of the American Society of Microbiology. American Society for Microbiology. Washington, DC. Sonntag, I., H . Schwartz, Y. Hirota, and U. Henning. 1978. Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J . Bacteriol. 136:280-285. Struvye, M . , M . Moons, and J . Tommassen. 1991. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J . Mol. Biol. 218:141-148. Taylor, R.K., C. Manoil, and J . J . Mekalanos. 1989. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence deteraiinants of Vibro cholerae. J . Bacteriol. 171:1870-1878. Ullstrom, C.A., R. Siehnel, W. Woodruff, S. Steinbach, a n d R E . W . Hancock. 1991. Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene. J . Bacteriol. 173:768-775. 69 Weiser, J .N. , and E . C . Gotschlich. 1991. Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli. Infect, and Immun. 59:2252-2258. Woodruff, W.A. 1988. Cloning of the oprF gene of Pseudomonas aeruginosa. Ph.D. thesis. University of British Columbia. Woodruff, W.A., and R.E.W. Hancock. 1988. Construction and characterization of Pseudomonas aeruginosa porin protein F-deficient mutants after in vivo and in vitro insertion mutagenesis of the cloned gene. J . Bacteriol. 170:2592-2598. Woodruff, W.A., and R.E.W. Hancock. 1989. Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein. J . Bacteriol. 171:3304-3309. Woodruff, W.A., T.R. Parr, R.E.W. Hancock, L. Hanne, T.I. Nicas, and B. Iglewski. 1986. Expression in Escherichia coli and function of porin protein F of Pseudomonas aeruginosa. J . Bacteriol. 167:473-479. 70 Yoneyama, H . , A. Akatsuka, and T. Nakae. 1986. The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of dissacharides. Biochem. Biophys. Res. Commun. 134:106-112. Yoshihara, E . , and T. Nakae. 1989. Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores. J . Biol. Chem. 264:6297-6301. Yoshimura.F. and H. Nikaido. 1982. Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. J . Bacteriol. 152:636-642. Yoshimura, F., L.S. Zalman, and H. Nikaido. 1983. Purification and properties of Pseudomonas aeruginosa porin. J . Biol. Chem. 1258:2308-2314. SCHOLARSHIPS Awarded by the University of Guelph: 1987 F . E . Chase Memorial Scholarship 1986 & Ontario Agricultural College Scholarship 1987 1986 Ontario Food Protection Association Scholarship Awarded by NSERC: 1985 & Industrial Scholarships 1986 1989 & Graduate Scholarships 1990 PUBLICATIONS Refereed publications: Bellido, F., R.L. Finnen, N.L. Martin, R.J . Siehnel, and R.E.W. Hancock. 1992. The function and structure of Pseudomonas aeruginosa outer membrane protein OprF. In: S. Silver, A. Chakrabarty, B. Iglewski, and S. Kaplan, eds., Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, DC. Finnen, R.L., B.N. Dhanvantari, and J .T. Trevors. 1990. Plasmid profiles and restriction endonuclease analysis of genomic DNA of Clavibacter michiganensis ssp. michiganensis. J . Microbiol. Meth. 12: 57-64. Abstracts and submitted publications: Finnen, R.L., N.L. Martin, R.J . Siehnel, W.A. Woodruff, M . Rosok, and R.E.W. Hancock. 1992. Analysis of structural features of the major outer membrane protein OprF from Pseudomonas aeruginosa using derivatives created by TnphoA mutagenesis and subcloning. J . Bacteriol. (manuscript submitted). Bellido, F„ R.L. Finnen, N.L. Martin, R.J . Siehnel, and R.E.W. Hancock. 1991. Study of the function and structure of OprF from Pseudomonas aeruginosa using the raffinose operon, TnphoA fusions and peptide mapping. Abstracts of the Pseudomonas 1991 Meeting, Trieste, Italy. Finnen, R.L., N.L. Martin, M.J . Rosok, and R.E.W. Hancock. 1991. Use of TnphoA fusions, monoclonal antibodies, and peptides to map the structural features of OprF from Pseudomonas aeruginosa. Abstracts of the 91st General Meeting of the American Society for Microbiology, Dallas, TX. 

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