UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Comparison of protein OprF from Pseudomonas syringae with protein OprF from Pseudomonas aeruginosa Ullstrom, Catherine Ann MacDonald 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 U44.pdf [ 2.84MB ]
Metadata
JSON: 831-1.0098191.json
JSON-LD: 831-1.0098191-ld.json
RDF/XML (Pretty): 831-1.0098191-rdf.xml
RDF/JSON: 831-1.0098191-rdf.json
Turtle: 831-1.0098191-turtle.txt
N-Triples: 831-1.0098191-rdf-ntriples.txt
Original Record: 831-1.0098191-source.json
Full Text
831-1.0098191-fulltext.txt
Citation
831-1.0098191.ris

Full Text

COMPARISON OF PROTEIN OprF FROM Pseudomonas syringae WITH PROTEIN OprF FROM Pseudomonas aeruginosa by Catherine Ann MacDonald Ullstrom B.Sc.(Microbiology), University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies Department of Microbiology We accept this thesis as conforming to the required standard The University of British Columbia April 1990 ©Catherine Ann MacDonald Ullstrom, 1990 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT The major outer membrane protein OprF from Pseudomonas aeruginosa was compared with OprF from the fluorescent phytopathogen Pseudomonas syringae. The P. syringae oprF gene was subcloned and sequenced and found to code for a sequence of 344 amino acids containing a 24 amino acid leader sequence. The mature protein, with a deduced molecular weight of 34,225, contained four cysteine residues and an alanine-proline rich area. Comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA sequences showed that the sequences were most similar at the carboxy-terrninal ends. Restriction enzyme site heterogeneity near the oprF gene from nine different P. syringae pathovars was determined. All pathovars had a conserved San site within the gene and conserved Psti. and BamHI sites near the ends of the gene. The location of the PstL and the Sail sites outside the gene was variable, although similar. Immunological relatedness between P. syringae OprF from the different pathovars and P. aeruginosa OprF was confirmed. Protein OprF from all the pathovars was shown to be 2-mercaptoethanol modifiable and more easily heat modifiable than was OprF from P. aeruginosa. iii TABLE OF CONTENTS Page ABSTRACT . ii. TABLE OF CONTENTS iii. List of Tables v. List of Figures vi. ACKNOWLEDGEMENTS vii. INTRODUCTION 1 MATERIALS AND METHODS A. Bacterial strains and plasmids 5 B. Media and growth conditions 5 C. DNA techniques 1. Bacterial chromosomal isolation 7 2. Plasmid DNA isolation 7 3. Quantitation of DNA 7 4. General DNA techniques 8 5. Sequencing 8 6. Nucleic acid blotting 13 D. Outer membrane techniques 1. Outer membrane preparation 15 2. Heat and 2-mercaptoethanol modifiability 15 3. Immunology 16 iv TABLE OF CONTENTS (continued) Page RESULTS A. Sequencing 17 B. Mapping 26 C. Immunological Relatedness 30 D. Heat and 2-Mercaptoethanol Modifiability 30 DISCUSSION 36 REFERENCES 41 V LIST OF TABLES Table Title Page I. Bacterial strains and plasmids 6 II. Amino acids in the P. syringae OprF sequence which are conservative substitutions for amino acids in the P. aeruginosa OprF sequence 25 III. Summary of the comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA sequences 27 vi LIST OF FIGURES Figure Title Page 1. Subcloning strategy for P. syringae oprF gene 9 2. Sequencing strategy for the P. syringae oprF gene .... 12 3. Nucleotide sequence and the deduced amino acid sequence of the P. syringae oprF gene 18 4. Comparison of the P. syringae and the P. aeruginosa oprF gene nucleotide sequences 21 5. Comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA amino acid sequences 23 6. Partial restriction maps of the oprF gene region from different P. syringae pathovars and from P. aeruginosa 28 7. SDS-PAGE of the P. syringae outer membrane proteins and the corresponding Western immumoblot 31 8. SDS-PAGE profiles of outer membrane preparations solubilized without 2-mercaptoethanol at 100°C for five minutes 34 vii ACKNOWLEDGEMENTS The guidance and the financial assistance of my supervisor, Dr. R.E.W. Hancock, is gratefully acknowledged. I would also like to acknowledge G. Crockford for providing the P. syringae oprF clone and the Hancock lab members, especially R.J. Siehnel, for their technical advice. 1 INTRODUCTION Pseudomonas aeruginosa is an opportunistic human pathogen causing disease in immunocompromised patients (Hancock, 1985). As a gram-negative rod, P. aeruginosa has a cell envelope consisting of three layers: the inner cytoplasmic membrane, the peptidoglycan layer, and the outer membrane (Lugtenberg and Van Alphen, 1983). The outer membrane, consisting of a lipid bilayer embedded with proteins, acts as a selective permeability barrier (Hancock, 1987). Five to nine of the outer membrane proteins (depending on growth conditions) are present in high copy number (104 to 5x10s copies per cell) and are called major outer membrane proteins (Hancock, 1987). Some of the proteins in the outer membrane form trans-membrane, water-filled channels called porins (Hancock, 1986). These porins allow the passage of hydrophilic molecules that are smaller in size than the exclusion limit of the porin (Hancock, 1987). The major outer membrane protein of P. aeruginosa, protein OprF, is constitutively expressed and peptidoglycan associated (Hancock et al, 1981; Mizuno and Kageyama, 1979). Protein OprF has been cloned and sequenced, and has a deduced molecular weight of 35,250 (Duchene et al, 1988). This protein is heat and 2-mercaptoethanol modifiable (Hancock and Carey, 1979). In the absence of 2-mercaptoethanol, unheated OprF bands on an SDS-PAGE gel at a molecular weight of 33,000 and heated (100°C for 60 minutes) OprF bands at 39,000. In the presence of 2-mercaptoethanol, unheated OprF bands at a molecular weight of 36,000 and heated (100°C for 60 minutes) OprF bands at 2 41,000 (Hancock and Carey, 1979). Modification of mobility by 2-mercaptoethanol is due to the disruption of two intrachain disulfide bonds (Hancock and Carey, 1979). Protein OprF has an important function in outer membrane stability and in cell shape determination (Gotoh et al, 1989; Woodruff and Hancock, 1989). In addition, studies have shown OprF to function as a porin (Hancock et al, 1979; Benz and Hancock, 1981; Yoshimura et al, 1983; Godfrey and Bryan, 1987). Data have suggested the OprF molecules form both large channels (single channel conductance of 5.6 nS in 1 M KC1 or a diameter of 2.0 nm) and small channels (0.36 nS in 1 M KC1) (Nikaido and Hancock, 1986), with the larger channels being far less prevalent (< 1%) than the smaller channels (Woodruff et al, 1986). The small number of large channels and thus the small area for diffusion of antibiotics, together with other resistance determinants such as periplasmic (3-lactamases, has been proposed as the basis of the high intrinsic resistance of P. aeruginosa to certain antibiotics (Woodruff and Hancock, 1988). In contrast to the above findings, other studies have shown that protein OprF has no function as a porin (Yoneyama et al, 1986; Yoshihara et al, 1988; Gotoh et al, 1989; Yoshihara and Nakae, 1989). At present, the controversy regarding the role of protein OprF as a porin and its role in antibiotic resistance remains unresolved. Comparison of the amino acid sequence of protein OprF with the amino acid sequence of the E. coli outer membrane protein OmpA showed significant similarity between the carboxy terminal halves of the two proteins (Woodruff and Hancock, 1989). Proteins OprF and OmpA cross-react immunologically and both have a role in the structural integrity of the bacterial cells (Gotoh et al, 1989; Woodruff and Hancock, 1989). 3 Although much is known about outer membrane proteins, there are still unanswered questions, especially in the areas of molecular structure and mechanisms of porins. To further the knowledge regarding outer membrane proteins and protein OprF, P. aeruginosa OprF was compared with OprF from another fluorescent Pseudomonad, Pseudomonas syringae. Bacteria belonging to the phytopathogenic species P. syringae are subdivided according to host range into over 40 pathovars (Palleroni, 1984). Outer membrane proteins of P. syringae have received relatively little study (Hurlbert and Gross, 1983), with most work having been done on iron regulated proteins (Cody and Gross, 1987). Although belonging to the same rRNA homology group, P. aeruginosa and P. syringae have notable differences. P. aeruginosa is an opportunistic human pathogen and P.syringae is a plant pathogen. P. aeruginosa has one flagella per cell whereas P. syringae has more than one flagella per cell. P. aeruginosa cells are longer and narrower than P. syringae cells, with P. aeruginosa cells being 0.5 to 0.7 um by 1.5 to 3.0 um and P. syringae cells being 0.7 to 1.2 um by 1.5 um. P. aeruginosa is oxidase positive and arginine dihydrolase positive whereas P. syringae is oxidase negative and arginine dihydrolase negative. P. aeruginosa, with an optimal growth temperature of 37°C, can grow at 41°C whereas P. syringae, with an optimal growth temperature of 25° to 30°C, cannot grow at 37°C. The maximum growth rate of P. syringae strains is slow relative to that of P. aeruginosa. The nutritional spectrum of P. syringae strains is less extensive and more variable than that of P. aeruginosa strains. The G+C content of P. aeruginosa DNA is 67.2% and the G+C content of P. syringae DNA is 59 to 61% (Palleroni, 1984). In order to compare P. aeruginosa protein OprF with P. syringae OprF, the P. syringae OprF gene was first cloned and sequenced. Then, the 4 restriction site heterogeneity of the oprF gene among different P. syringae pathovars was determined. Immunological relatedness between P. syringae OprF from the pathovars and P. aeruginosa OprF was confirmed. Finally, the heat and 2-mercaptoethanol modifiability of protein OprF in P. syringae outer membrane preparations was determined. 5 MATERIALS AND METHODS A. BACTERIAL STRAINS AND PLASMIDS The strains of P. aeruginosa, P. syringae, and E. coli and their plasmids used in this study are listed in Table I. The P. syringae pathovars were a generous gift from Richard Moore, Agriculture Canada, Guelph, Ontario. B. MEDIA AND GROWTH CONDITIONS The culture media used was Luria broth (1% tryptone, 0.5% yeast extract, 1% NaCl) for E. coli strains, proteose peptone No.2 (1%) for P. aeruginosa, and NBYG (0.8% nutrient broth, 0.5% yeast extract, and 0.5% glucose) for P. syringae strains. For single stranded DNA preparation, the media used was 2XYT (1.6% tryptic peptone, 1% yeast extract, and 0.5% NaCl). Solid media contained 2% agar. All media components were from Difco Laboratories, Detroit, Michigan. E. coli and P. aeruginosa strains were grown at 37°C overnight. P. syringae strains were grown at 25° to 27°C overnight or longer if growth was • slow. Most strains of P. syringae would not grow at 30°C. All cultures were agitated during growth. Antibiotic concentrations for cultures of E. coli were 20 pg/ml of tetracycline and 50 pg/ml of ampicillin. Short term storage of strains was on plates at 4°C and long term storage was in 7% DMSO at -70°C. 6 Table I. Bacterial strains and plasmlds Strain or plasmid Characteristics Reference or source P. aeruginosa H103 PAOl Cm' prototroph; wild type reference strain Hancock and Carey, 1979 P. syringae H365 pv. syringae (weakly pathogenic to lilac) ATCC #19310 H678 pv. papulans (apple) R. Moore (#3679) H679 pv. glycinea (soybean) R. Moore (#B3) H680 pv. syringae (lilac) R. Moore (#5D19) H681 pv. phaseolicola (bean) R.-Moore (#HB6) H682 pv. tomato R. Moore (#3000) H683 pv. tomato R. Moore (#1108) H684 pv. antirrhini (snapdragon) R. Moore (#2738) H685 pv. tabaci (tobacco) R. Moore (#GB1) E. coli DH5cxF' F' endAl hsdR17(rk\mk+) supE44 thi-1 Bethesda Research recAl gyrA96 relAl X' Laboratories o80dIacZAMl5 A(lacZYAargF)U169 (BRL) C483 DH5a/pGC31 This study Plasmids pTZ19U GeneScribe cloning vector BRL pTZ19R GeneScribe cloning vector BRL pGC31 pRK404 + 2.5 kb Psfl-BamHI fragment This study encoding P. syringae oprF gene 7 C. DNA TECHNIQUES 1. Bacterial Chromosomal Isolation Early in the study, bacterial genomic DNA was isolated using a modified technique based on the procedure of Meade et al (1982). This procedure involved numerous phenol:chloroform extractions. Later in the study, the genomic DNA was isolated by a method using CTAB (hexadecyltrimethyl ammonium bromide) (Ausubel et al, 1987). In this method, the protease incubation is followed by a CTAB extraction. CTAB complexes with polysaccharides and proteins, which are removed by emulsification and extraction with chloroform/isoamyl alcohol. The DNA is then removed from the supernatent by isoprpanol precipitation. 2. Plasmid DNA Isolation Large scale plasmid isolations used the alkaline lysis method followed by centrifugation in ethidium bromide cesium chloride gradients (Maniatis et al, 1982). Small scale plasmid isolation (from a few ml of culture) used a modification of the boiling method of Holmes and Quigley (1981). Plasmid isolation from cultures of 30 ml used a CTAB method (Helen Jost, personal communication) based on the methods of Holmes and Quigley(1981) and Del Sal et al (1987). 3. Quantitation of DNA Quantitation of bacterial chromosomal DNA was by spectrophotometric determination as described by Maniatis et aL(1982). 8 Quantitation of plasmid DNA was by the Saran Wrap method of the ethidium bromide fluorescent spot test as described by Maniatis et al (1982). 4. General DNA Techniques Restriction enzyme digests, agarose gel electrophoresis, ligations, and transformations were carried out as described by Maniatis et al (1982). E. coli DH5cxF cells were made competent using 0.1 M CaCl 2 (Maniatis, 1982). Transformants were screened for inserts by the slot-lysis technique (Sekar, 1987). Agarose gel electrophoresis used the Bio-Rad DNA Sub Cell and the Mini Sub DNA Cell with IX TBE as the running buffer. Recovery of DNA fragments from agarose gels used NA-45 DEAE membrane (Schleicher and Schuell, Inc., Keene, N.H.) and the band interception method of Winberg and Hammerskjold (1980). 5. Sequencing A 2.5 kb Psfi-BamHI fragment containing the P. syringae oprF gene had previously been ligated into the vector pRK404 to give the plasmid pGC31. From the plasmid pGC31, 1.6 kb SaR-PsO. and 0.9 kb BamHI-SaU. fragments were isolated and ligated into the GeneScribe-Z vectors pTZ19R and pTZ19U (United States Biochemical Corporation, Cleveland, Ohio), as shown in Figure 1. These pTZ19 clones were then treated with exonuclease III to give progressive unidirectional deletions of the insert DNA. The Erase-a-Base System (Promega), which is based on the method developed by Henikoff (1984), was used for this procedure. Single-stranded plasmid DNA was used as the template for the sequencing reactions. Single-stranded DNA from the pTZ19 clones was 9 Figure 1. Subcloning strategy for P. syringae oprF gene. First, 9 to 10 kb BamHI-Hindni fragments of P. syringae chromosomal DNA were ligated into the plasmid pRK404. The recombinant plasmids were introduced into E. coli DH5aF' cells by transformation. A positive clone, pGC, was isolated and is shown as a circle. The subclone pGC31 was then isolated. The BamHI-Psfl fragment was then further subcloned into pTZ19 cloning vectors as shown. The diagrams of the pTZ19 clones show the strand produced when single stranded DNA is produced by helper phage infection and the arrows beneath the restriction maps indicate the direction of primer extension during the sequencing reactions. The abbreviations for the restriction sites are as follows: B, BamHI; C, HincIP. H, HincUll', P, Psfl; S, Sail; V, PuuII. PBS indicates the primer binding sites. 10 p 3 7 ( p T Z 1 9 U ) S B p38(pTZ19R) S P 11 prepared according to the method in the manual GeneScribe-Z Description and Experimental Protocols (United States Biochemical Corporation, Cleveland, Ohio). This procedure is a modification of the method of Dente et al (1983), using the ammonium acetate/PEG precipitation step of Carlson and Messing (1984). The helper phage used was M13K07. The Sanger dideoxy-mediated chain termination method of DNA sequencing (Sanger, 1977) was used to sequence both strands of the P. syringae oprF gene. For most of the sequencing, the kit Sequenase (United States Biochemical Corporation, Cleveland, Ohio) was used as described In the manual Sequenase: Step-by-Step Protocols for DNA Sequencing with Sequenase (2nd edition). Sequencing of one short section at the carboxy end of the gene used the kit TaqTrack (Promega) and the TaqTrack Extension/Labeling Protocol as described in the manual TaqTrack Sequencing Systems Technical Manual (Promega). Both the Sequenase and the TaqTrack kits used 35S-dATP (NEN/Dupont, Markham, Ontario). The gene sequencing strategy is shown in Figure 2. The universal primers used were those provided with the Sequenase kit (United States Biochemical, Cleveland, Ohio). Synthetic oligonucleotide primers were synthesized by T. Atkinson, Department of Biochemistry, University of British Columbia. Purification of the oligonucleotides used SEP-PAK cartidges (Waters Associates) and a protocol adapted from Atkinson and Smith (1984). The oligonucleotide primers used were as follows: 1. STGGCAAGGTCTGGTAGAC3' (upstream from start site) 2. ^ TGTACGACCAGCGCCCGT3 (bases 119-135) 3. 5CGACCAGAGCATTGGCCA3 (bases 345-362) 4. ^ CGTCGTGCAAACGCCGTT3' (bases 865-882) 5. ^ TGATGTTCTTGCCATCAT3' (bases 242-259. opposite strand) 6. 5CGGCGTAGAAGTTGTCAG3 (bases 431-449, opposite strand) 12 BamHI Sail Pstl • — — • Figure 2. The sequencing strategy of the P. syringae oprF gene. The 1.6 kb Psfl-Sott and the 0.9 kb Sail-BamHI fragments containing the oprF gene were subcloned into pTZ19 cloning vectors. The arrows indicate the sequences obtained using universal primers (-<—•), synthetic oligonucleotide primers ( < •). or Exonuclease HI deletion clones and universal primers (-<— ). 13 7. 5GACAGTTTTTCGTTGTAA3 (bases 843-860, opposite strand) 8. 5CCACGCACAGCTGAATGCCGG3 (bases 1126-1146, opposite strand) Sequencing gels used were non-gradient, acrylamide:bis-acrylamide (38:2), wedge gels. The top of the wedge was 0.4 mm thick and the bottom of the wedge was 1.2 mm thick. The BRL Sequencing Apparatus Model S2 was used with the running buffer IX TBE and the power at 70 watts. After the run, gels were soaked in a solution of 5% methanol and 5% glacial acetic acid for twenty minutes, dried at 80°C under a vacuum onto Whatman 3MM paper, and then left overnight in contact with Kodak diagnostic film X-Omat RP. DNA and protein sequence data analysis was assisted by the programmes ESEE, The Eyeball Sequence Editor Version 1.03 (Eric Cabot, Simon Fraser University, Burnaby, 1988), and PC/Gene, Release 6.01 (Amos Bairoch, University of Geneva, Switzerland. 1989). 6. Nucleic Acid Blotting Chromosomal digests were loaded into 30 well, 0.65% agarose gels and run at constant voltage of 130 volts in IX TBE running buffer containing 4 ug/ml ethidium bromide. The alkaline blotting method of DNA capillary transfer, based on the procedure of Reed and Mann (1985), was used to transfer the DNA to Zeta-Probe blotting membranes (Bio-Rad). The probe used in the hybridizations was a 2.5 kb Psfl-BamHI fragment containing the P. syringae oprF gene. Radiolabelling of the probe used a-32P-dATP (NEN/Dupont, Markham, Ontario) and the random oligonucleotide primer method as described by Feinberg and Vogelstein (1983). 14 An Elutip-d column (Schleicher and Schuell, Inc., Keene, N.H.) was used to separate unincorporated reactants from the labeled probe. The protocol followed was from the technical literature Tips for Recovery (Schleicher and Schuell, No. 208). Hybridization was based on the standard hybridization protocol in the Zeta-Probe Blotting Membranes Instruction Manual (Bio-rad). The prehybidization solution consisted of 1.5X SSPE, 1.0% SDS, 0.5% skim milk powder, 1.5 mg/ml salmon sperm DNA, and 10% dextran sulfate. Membranes were sealed (often two together, back to back) in heat sealable pouches (Kapak Corporation, Minneapolis) and incubated at 45° C, without shaking, for 0.5 to 4.0 hours. The probe was added to the prehybridization solution, the bag resealed, and incubated at 45° C, without shaking, for 22 to 36 hours. Probe specific activity was approximatedly 108 cpm/ug probe and the probe concentration in the hybridization mixture was approximately 106 counts/ml. At the completion of hybridization, the membranes were removed from the bags and rinsed briefly in 2X SSC. They were then washed at room temperature, shaking, for 15 minutes in each ofthe following: 2X SSC/0.1%SDS; 0.5X SSC/0.1% SDS; and 0.25X SSC/0.1% SDS. The membranes were wrapped in Saran Wrap, placed between two intensifying screens, and exposed to Kodak diagnostic X-Omat RP film at -70° C for an average of 24 hours. Used blots were stripped of the probe by washing twice for 20 minutes in 0.1X SSC/0.5% SDS at 95°C (Gatti et al, 1983). 15 D. OUTER MEMBRANE PROTEIN TECHNIQUES 1. Outer Membrane Preparations Outer membranes from nine P. syringae pathovars and P. aeruginosa were prepared according to the method of Hancock and Carey (1979). Briefly, a 400 ml overnight culture was resuspended in a 4 ml solution of 20% sucrose in 10 mM Tris-HCL (pH 8.0) and 50 micrograms/ml DNase I. Cells were broken by passage twice through a small French Pressure cell at 15,000 p.s.i. Cell debris was removed by centrifugation and the outer and inner membranes were separated by overnight centrifugation in a two step gradient of 70% and 60% sucrose. The concentration of protein in the outer membrane preparations was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories), which is based on the method of Bradford (1976). 2. Heat and 2-Mercaptoethanol Modifiability Approximately 12 micrograms of each outer membrane preparation was solubilized under the following ten conditions: 2% SDS plus (A) no 2-mercaptoethanol and (i) no heat, (ii) 65° C, five minutes, (iii) 65° C, 30 minutes, (iv) 100° C, five minutes, (v) 100° C, 30 minutes; or (B) 5% 2-mercaptoethanol and conditions (i) to (v). After solubilization, the outer membrane proteins were separated by electrophoresis through 12% SDS-polyacrylamlide gels as described by Hancock and Carey (1979). Proteins were visualized by staining with Commasie Brilliant Blue R250 (Bio-Rad) or by Western blotting. A Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) was used for transfer of SDS-PAGE protein onto Zeta-probe membrane (Bio-Rad) by a modification of the method of Towbin et al 16 (1979). The transfer buffer used was 25 mM Tris, 192 mM glycine, and 20% methanol. 3. Immunology Immunoblotting of the nylon membranes used a procedure modified from O'Connor and Ashman (1982). Briefly, the blot was incubated in 3% (w/v) skim milk powder in phosphate buffered saline (PBS) for 30 minutes at room temperature, then incubated with rocking for two hours at 37° C, in a 10'3 dilution of the anti P.aeruginosa OprF monoclonal antibody MA4-10 (Mutharia and Hancock, 1985) in 3% skim milk powder in PBS. The blot was then washed three times (5 minutes each time) at room temperature with PBS. The second antibody, alkaline phosphatase conjugated goat anti mouse IgG (Kirkegaard and Perry Laboratories, Inc.), was diluted 2X10"3 in 3% skim milk in PBS and added to the blot, which was then incubated, rocking, at 37° C for one hour. The blot was washed three times with PBS as above, and once with 50mM Tris-HCl, pH 8.0. Finally, the blot was incubated at room temperature with the substrate (1 mg/ml Naphthol AS MX phosphate, disodium salt and 2 mg/ml Fast Red TR salt in 50 mM Tris-HCl, pH 8.0) until bands appeared and was then washed with distilled water and air dried. 17 RESULTS A. SEQUENCING The sequence of the oprF gene encoding the major outer membrane protein F from P. syringae is shown in Figure 3. The gene had one continuous reading frame of 1032 base pairs coding for a sequence of 344 amino acids starting with an initiator methionine. The methionine initiates a 24 amino acid leader sequence, which ends with the common feature of Ala-X-Ala, a potential cleavage site for a procaryotic signal peptidase (Watson, 1984; Perlman and Halvorson, 1983). The deduced molecular weight of the mature protein is 34,225. The most likely transcription termination site is the region between nucleotides 1050 and 1079 (between 15 and 29 bases after the TAA stop codon), which could potentially form a hairpin loop and thus act as a rho-independent terninator. Compared with the P. syringae oprF gene, the P. aeruginosa oprF gene was slightly longer, having 1050 base pairs and 350 amino acids (Duchene et al, 1988). The P. aeruginosa oprF gene encoded a 24 amino acid leader sequence, as did the P. syringae oprF gene. A SaU site was identically placed in the oprF gene from both species. The four cysteine residues, which form two disulfide bonds (Woodruff et al, 1988), and the alanine-proline rich area found in the P. aeruginosa oprF gene were also conserved in the P. syringae oprF gene. The overall G+C content of the P. syringae oprF gene was 55.3%, which is lower than the 60.2% value for the P. aeruginosa oprF gene. The G+C content of nucleotides one, two. and three of the coding triplets is 62.5%, 41.6%, and 61.7%, resepectively, for the P. syringae oprF gene and 61.4%, 40.8%, and 78.2%, for the P. aeruginosa oprF gene. 18 Figure 3. The nucleotide sequence and the deduced amino acid sequence of the P. syringae oprF gene. Single-letter amino acid codes are used. The initiation codon (ATG) and the termination codon (TAA) are boxed. The arrow indicates the end of the signal peptide. The putative Shine-Dalgarno sequence (GGGA) and the putative rho-independent terminator region are underlined. The four cysteine residues are circled and the SaU site is indicated by a wavy line. P . s y r i n g a e o p r F 19 M K L K N T 6 TCC CCA TGT GTG GGA CTG CTT AAT AAT CAT CAG ATG GGG ATT TAA CGG lATGl AAA CTG AAA AAC ACC 18 L G L A I G T I V A A T S F G A L A ^ Q G Q G 2 8 TTG GGC TTG GCC ATT GGT ACT ATT GTT GCC GCA ACT TCG TTC GGC GCG CTG GCT CAA GGC CAA GGC 84 A V E I E G F A K K E M Y D S A R D F K N N 5 0 GCA GTC GAA ATC GAA GGC TTC GCC AAG AAA GAA ATG TAC GAC AGC GCC CGT GAT TTC AAA AAC AAC 150 G N L F G G S I G Y F L T D D V E L R L G Y 7 2 GGC AAC CTG TTC GGC GGC TCG ATT GGC TAC TTC CTG ACC GAC GAC GTT GAA TTG CGT CTG GGC TAC 216 D E V H N V R S D D G K N I K G A D T A L D 9 4 GAC GAA GTC CAC AAC GTT CGT AGC GAT GAT GGC AAG AAC ATC AAG GGC GCA GAC ACT GCC CTG GAC 282 A L Y H F N N P G D M L R P Y V S A G F S D 116 GCT CTC TAC CAC TTC AAC AAC CCA GGC GAC ATG CTG CGT CCA TAC GTT TCT GCC GGT TTC TCC GAC 348 Q S I G Q N G R N G R N G S T F A N I G G G 138 CAG AGC ATT GGC CAG AAC GGT CGT AAC GGT CGT AAC GGT TCT ACC TTC GCC AAC ATC GGC GGC GGC 414 P K L Y F T D N F Y A R C C C A A G C T C T A C T T C A C T G A C A A C T T C T A C G C C C G T Q G D T E W A P S V G I C A A G G C G A C A C C G A G T G G G C T C C A A G C G T C G G T A T C V E A A P A P V A E V © G T T G A A G C A G C A C C A G C T C C A G T A G C T G A A G T G T G C N V D K Q P D T P A N V A A C G T C G A C A A G T G C C C G G A C A C C C C A G C C A A C G T T A G V E A Q Y N I D 1 6 0 G C T G G C G T T G A A G C T C A A T A C A A C A T C G A C 4 8 0 G V N F G G G S K K 1 8 2 G G C G T A A A C T T C G G T G G C G G C A G C A A G A A A 5 4 6 S D S D N D G V @ D 2 0 4 T C C G A C A G C G A C A A C G A C G G C G T G T G C G A C 6 1 2 T V D A D G ( C ) P A V 2 2 6 A C C G T T G A C G C T G A T G G C T G C C C A G C A G T T 6 7 8 A E V V R V E L D V K F D F D K S V V K P N 2 4 8 G C C G A A G T G G T T C G T G T T G A G C T G G A C G T G A A G T T C G A T T T C G A C A A A T C C G T A G T C A A G C C T A A C 7 4 4 S Y G D I K N L A D F M Q Q Y P Q T T T T V 2 7 0 A G C T A C G G C G A C A T C A A G A A C C T C G C T G A C T T C A T G C A G C A G T A C C C A C A G A C C A C C A C C A C T G T T 8 1 0 E G H T D S V G P D A Y N Q K L S E R R A N 2 9 2 G A A G G T C A C A C T G A C T C N G T C G G T C C T G A C G C T T A C A A C C A A A A A C T G T C C G A G C G T C G T G C A A A C 8 7 6 A V K Q V L V N Q Y G V G A S R V N S V G Y 3 1 4 G C C G T T A A A C A G G T T C T G G T T A A C C A G T A C G G T G T T G G C G C T A G C C G C G T A A A C T C G G T T G G T T A C 9 4 2 G E S K P V A D N A T E A G R A V N R R V E 3 3 6 G G C G A A A G C A A G C C A G T T G C T G A T A A C G C A A C T G A A G C T G G C C G C G C A G T T A A C C G T C G C G T A G A A 1 0 0 8 A E V E A Q A K * 3 4 4 G C A G A A G T A G A A G C T C A A G C T A A G | T A A | T T A G C C G C T T G T A C T G A A A A G C C C G G C T T A G G C C G G G C T 1 0 7 4 T T T C T T TGC CTG 1 0 8 6 20 Figure 4 compares the nucleotide sequences of the two oprF genes and Figure 5 compares the deduced amino acid sequences. From the beginning of the protein to amino acid residue 184 (just before the ala-pro rich region), the nucleotide sequence for the P. syringae oprF gene and the nucleotide sequence for the P. aeruginosa oprF gene were found to be 65.9% identical, and the deduced amino acid sequences, 53.3% identical. From amino acid residue 185 to the carboxy-terminal end of the protein (160 amino acids total), the nucleotide sequences for the two genes were found to be 79.3% identical, and the amino acid sequences, 83.8% identical. The overall identity between the two proteins was 72.3% for the nucleotide sequences and 67.4% for the amino acid sequences. The high normalized alignment scores of 647 for the entire sequence, 459 for the amino-terrninal halves, and 875 for the carboxy-terminal halves, indicated "certain" significant matches between the two amino acid sequences (Doolittle, 1986). Comparison of the sequences by the Needleman and Wunsch method (Needleman and Wunsch, 1970), which used the genetic code matrix with a bias of 0, a gap penalty of 4, and 100 random runs, gave alignment scores of 61.9 for the entire sequence, 31.2 for the amino-terrninal halves, and 49.2 for the carboxy-terminal halves. All these values were "significant (above 3SD) results". Conservative substitutions between the P. syringae and the P. aeruginsa OprF amino acid sequences are indicated in Figure 5 by dots and are specified in Table II. From amino acid residues one to 184, 25% of the amino acids were conservative substitutions and from residues 185 to 344, 10% were conservative substitutions. In the entire sequence, 18% of the amino acids were conservative substitutions. When conservative substitutions were included in the amino acid identity values, these values increased to 78% for the amino-terrninal end, 95% for the carboxy-terminal end, and 86% for the entire protein. 21 Figure 4. Comparison of the P. syringae and the P. aeruginosa oprF gene nucleotide sequences. Vertical lines indicate nucleotide matches. The initiation codons (ATG) and the termination codons (TAA) are boxed. The putative Shine-Dalgarno sequences (GGGA) and the putative rho-independent terminator regions are underlined. The sequences coding for the cysteine residues are doubly underlined. The wavy line indicates the Sail site. P. aeruginosa data were from Duchene et al.,1988. opr F 22 P.AER.F GGCTGATTGTTGGACAACTAACTGACCATCAAGATGGGGATTTAACGG^TC^RAACTGAAGAACACCTTAGC^GTTGTCATCGCXn'CG 39 i i i I I I i 1111111111111111111111111111 i l i u m i n i i i n I I i P.SYR.F TCCCCATGTGTGGGACTGCTTAATAATCATCAGATGGGGATTTAACGG(ATgAAACTGAAAAACACCTTGGGCTTGGCCATTGGTACT 39 P . AER. F CTGGTTGCCGCTTCGGCAATGAACGCCTTCGCCCAGGGCCAGAACTCGGTAGAGATCGAAGCCTTCGGCAAGCGCTACTTCACCGAC 126 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I P.SYR.F ATTGTTGCCGCAACTTCGTTCGGCGCGCTGGCTCAAGGCCAAGGCGCAGTCGAAATCGAAGGCTTCGCCAAGAAAGAAATGTACGAC 126 P.AER.F AGCGTTCGCAACATGAAGAAC GCTGACCTGTACGGCGGCTCGATCGGCTACTTCCTGACCGACGACGTCGAGCTGGCTCTGTCC 210 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I P.SYR.F AGCGCCCGTGATTTCAAAAACAACGGCAACCTGTTCGGCGGCTCGATTGGCTACTTCCTGACCGACGACGTTGAATTGCGTCTGGGC 213 P.AER.F TACGGTGAGTACCACGATGTTCGTGGCACCTACGAAACCGGCAACAAGAAGGTCCATGGCAACCTGACCTCCCTGGACGCCATCTAC 297 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I P.SYR.F TACGACGAAGTCCACAACGTTCGTAGCGATGAT GGCAAGAACATCAAGGGCGCAGACACTGCCCTGGACGCTCTCTAC 291 P. AER. F CACTTCGGTACCCCGGGCGTAGGTCTGCGTCCGTACGTGTCGGCTGGTCTGGCTCACCAGAACATCACCAACATCAACAGCGACAGC 384 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I P . SYR. F CACTTCAACAACCCAGGCGACATGCTGCGTCCATACGTTTCTGCCGGTTTCTCCGACCAGAGCATTGGCCAGAACGGTCGTAAC 375 P.AER.F CAAGGCCGTCAGCAGATGACCATGGCCAACATCGGCGCTGGTCTGAAGTACTACTTCACCGAGAACTTCTTCGCCAAGGCCAGCCTC 471 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I P.SYR.F GGTCGTAACGGTTCTACCTTCGCCAACATCGGCGGCGGCCCCAAGCTCTACTTCACTGACAACTTCTACGCCCGTGCTGGCGTT 459 P.AER.F GACGGCCAGTACGGCCTGGAGAAGCGTGACAACGGTCACCAGGGTGAGTGG ATGGCTGGCCTGGGCGTCGGCTTCAACTTC 552 I I I I I I I I I I II- I I I I I I I I I I I I I I I I I I I I I I I P.SYR.F GAAGCTCAATACAACATC GACCAAGGCGACACCGAGTGGGCTCCAAGCGTCGGTATCGGCGTAAACTTCGGTGGC 534 P . AER.F GGTGGTTCGAAAGCCGCTCCGGCTCCGGAACCGGTTGCCGACGTTTGCTCCGACTCCGACAACGACGGCGTCTGCGACAACGTCGAC 639 I I I lllll I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I llltl I I I I I I I I I I I I I I I I I I P .SYR.F GGCAGCAAGAAAGTTGAAGCAGCACCAGCTCCAGTAGCTGAAGTGTGCTCCGACAGCGACAACGACGGCGTGTGCGACAACGTCGAC 621 P . AER. F AAGTGCCCGGACACCCCGGCCAACGTCACCGTTGACGCCAACGGCTGCCCGGCTGTCGCCGAAGTCSTACGCGTACAGCTGGACGTG 72 6 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 111111111 I I I I I I I I I I I I It I I I I I I I I I I I I I I I P .SYR.F AAGTGCCCGGACACCCCAGCCAACGTTACCGTTGACGCTGATGGCTGCCCAGCAGTTGCCGAAGTGGTTCGTGTTGAGCTGGACGTG 708 P . AER.F AAGTTCGACTTCGACAAGTCCAAGGTCAAAGAGAACAGCTACGCTGACATCAAGAACCTGGCCGACTTCATGAAGCAGTACCCGTCC 813 I I I I I I I I I I I I I I I I I I I lllll I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I P .SYR.F AAGTTCGATTTCGACAAATCCGTAGTCAAGCCTAACAGCTACGGCGACATCAAGAACCTCGCTGACTTCATGCAGCAGTACCCACAG 795 P.AER.F ACTTCCACCACCGTTGAAGGTCATACCGACTCCGTCGGTACCGACGCTTACAACCAGAAGCTGTCCGAGCGTCGTGCCAACGCCGTT 900 I I I I I I I I I I I I I I I I I I I I II lllll I I I I I I I I I I I I I I I I I I I I I II I I I I I 1 I I I I I I I I I I I I I I I I I I I | P . SYR. F ACCACCACCACTGTTGAAGGTCACACTGACTCNGTCGGTCCTGACGCTTACAACCAAAAACTGTCCGAGCGTCGTGCAAACGCCGTT 882 P.AER.F CGTGACGTACTGGTCAACGAGTACGGTGTGGAAGGTGGTCGCGTGAACGCTGTCGGTTACGGCGAGTCCCGCCCGGTTGCCGACAAC 987 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I  I I I I I 'I I I P.SYR.F AAACAGGTTCTGGTTAACCAGTACGGTGTTGGCGCTAGCCGCGTAAACTCGGTTGGTTACGGCGAAAGCAAGCCAGTTGCTGATAAC 969 P . AER. F GCCACCGCTGAAGGCCGCGCTATCAACCGTCGCGTTGAAGCCGAAGTAGAAGCCGAAGCCAAGJIAarCGGCTGAGCCTTCAAAGAAA 1074 l l l l l I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 1 1 I I I I I I I I I P.SYR.F GCAACTGAAGCTGGCCGCGCAGTTAACCGTCGCGTAGAAGCAGAAGTAGAAGCTCAAGCTAAGg^TTAGCCGCTTGTACTGAAAAG 1056 P.AER.F AACCGGCCCAGGCCGGGTTTTTCTTTGCCTGGAAAAAGACCGCTCGTCAGGCGCTCAGGGAAACCGGTT 114 3 I I I I I I I I I I I I I I I I I I I I i P . SYR. F CCCGGCTTAGGCCGGGCTTTTCTTTGCCTGCGATTTGGCATTGCGTCTGTTCAGGCGGGCTTGATGTCA 1125 2 3 Figure 5. Comparison of the P. syringae protein OprF amino acid sequence with the P. aeruginosa protein OprF sequence and the E. coli OmpA protein sequence. Vertical lines indicate amino acid identities and dots indicate conservation substitutions. The spaces in the sequence represent gaps introduced and the dashes were added to keep the two sequences in correct alignment when a gap was introduced into the other sequence. Conservative substitutions were determined by the Dayhoff minimum-mutation matrix, using a matching score of 0.9 as a cutoff (Schwartz and Dayhoff, 1978). P. aeruginosa data was from Duchene et al, 1988. E. coli data was from Chen et al, 1980. 24 P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA P.AER.F P.SYR.F OmpA MKLKNTLGWIGSLVAASAMNAFAQGQNSVEIEA-FGKRYFTDSV 44 I I I I I I I I • I I . . I I I . . I . I I I I . 1 1 1 1 . | . | . II MKLKNTLGLAIGTIVAATSFGALAQGQGAVEIEG FAKKEMYDSA 44 . . . I I . . I . . I I . . I I . • MKKTAIAIAVALAGFATVAQAAPKDNTWYTGAKLGWSQYHDT 4 2 RNMKN ADL-I • II • . I RDFKNNGNL . . I I I -YGGSIGYFLTDDVELALSYGEYHDVRGTYE 82 . I I I I I I I I I I I I I I I . I . I I • I I • FGGSIGYFLTDDVELRLGYDEVHNVRSD 81 I . I I . I • . I I I . . GFINNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYK - 85 TGNKKVHGNLT S LDAIYHF -• I . . I I • I I I • I I I DGKNIKGADTALDALYHF -GTPGVGLRPYVSAGLAHQN 120 II I I I I I I I I . . . I • NNPGDMLRPYVSAGFSDQS 118 --GSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTK 128 ITNINSDSQGRQQMTMANIGAG— I • . . . I I . I I I I I . I IGQ NGRNGRNGSTFANIGGG I.I I • II I • SNV—YGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDA 171 -LKYYFTENFFAKASLDGQ 160 I I I I • I I • I . I I PKLYFTDNFYARAGVEAQ 156 YGLEKRDNGHQGEWMA GLGVGFNFGGSK—AAPAPEPVADVCS 201 I . . . I I I - I . I I I I . I . 1 1 1 1 1 . 1 1 1 YNIDQGDT EWAPSVGIGVNFGGGSKK VEAAPAPVAEVCS 195 I . . I . I . . I l l I • I I I I . I I HTIGTRPD NGMLSLGVS YRFGQGEAAPWAPAPAPAPEV 210 DSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEWRVQLDVKFDF 24 6 II I I I I I I I I I I I I I I I I I I I I I I I • I I I I I I I I I I I . I I I I I I I DSDNDGVCDNVDKCPDTPANVTVDADGCPAVAEWRVELDVKFDF 240 • II I • I QTKHFTLKSDVLFNF 225 DKSKVKENSYADIKNLADFMKQ—YPSTSTTVEGHTDSVGTDAYN 289 I I I II I I I . I I I I I I II I . I II I . I I I I I I I I I I I I I I I DKSWKPNSYGDIKNLADFMQQ YPQTTTTVEGHTDSVGPDAYN 283 . I . . I I I • • . . I I I I . I . I I I I NKATLKPEGQAALDQLYSQLSNLDPKDGSVWLGYTDRIGSDAYN 270 QKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEG 334 I I I I I I I I I I I • . I I II • I I I . • I I I . I I I I II • I I I I II I I QKLSERRANAVKQVLVNQYGVGASRVNSVGYGESKPVADNATEAG 32 8 I I I I I I I I . I • • • I . I I | I I • I I . • I • . QGLSERRAQSWDYLISK GIPADKISARGMGESNPVTGNTCDNV 314 350 344 346 RAINRRVEAEVEAEAK* I I . I I I I I I I I I I • I II RAVNRRVEAEVEAQAK* I . 1 1 1 1 II • KQRAALIDCLAPDRRVEIEVKGIKDWTQP OA 25 Table n. Amino acids in the P. syringae OprF sequence which are conservative substitutions for amino acids in the P. aeruginosa OprF sequence. Conservative substitutions were determined by the Dayhoff minimum-mutation matrix using a matching score of 0.9 as a cutoff (Schwartz and Dayhoff. 1978). P. aeruginosa OprF Substitution in Number of P. syringae OprF substitutions alanine glycine 4 proline 1 serine 3 arginine lysine 3 asparagine asparatic acid 2 glutamine 1 serine 1 aspartic acid asparagine 2 glutamic acid 2 glutamine 1 glycine 1 glutamic acid aspartic acid 2 glutamine 2 glutamine asparagine 2 glutamic acid 1 glycine alanine 3 serine 3 aspartic acid 2 histidine aspartic acid 1 isoleucine leucine 1 valine 1 leucine isoleucine 3 phenylalanine 1 valine 1 lysine arginine 1 asparagine 1 glutamine 2 phenylalanine leucine 1 tyrosine 1 proline alanine 1 serine alanine 2 asparagine 1 glycine 2 threonine 3 tyrosine phenylalanine 1 valine isoleucine 1 leucine 1 62 total 26 The carboxy-terminal portion of P. aeruginosa OprF was previously shown to be similar to the carboxy-terminal end of the E. coli protein OmpA (Woodruff and Hancock, 1989). This similarity was also found between P. syringae OprF and E. coli OmpA, as shown in Figure 5, which compares the amino acid sequences of the two proteins. From the amino-terminal to residue 184 of P. syringae, the amino acid identity between the two proteins was 19.6%, and from residue 184 to the carboxy-terminal end, the identity was 33.3%. When conservative substitutions were included in the identities, these values changed to 39.7% and 59.0%, respectively. The overall identity between the two proteins was 24.4% and, when conservative substitutions were included, was 45.9%. The normalized alignment score (Doolittle, 1986) of 172 for the entire protein indicated a "marginal to improbable" significant match, whereas a score of 264 for the carboxy-terminal halves indicated a "probable" significant match. The score of 114 for the ammo-terminal halves indicated an "improbable" significant match. Comparison of the two sequences by the Needleman and Wunsch method (Needleman and Wunsch, 1970), using the same parameters listed previously, gave alignment scores of 7.5 for the entire sequence, 2.2 for the amino terminal halves, and 7.9 for the carboxy-terminal halves. The scores of 7.5 and 7.9 were "significant (above 3SD) results", although they were much lower than the values for the P. syringae and P. aeruginosa OprF sequences. The sequence comparison results are summarized in Table III. B. MAPPING Restriction maps of the oprF gene from the nine different P. syringae pathovars and the P. aeruginosa type strain are shown in Figure 6. Unlike the oprF genes from 17 different P. aeruginosa serotypes (Woodruff, 1988), the oprF genes from the P. syringae pathovars did not 27 Table III. Summary of the comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF (top left) and the E. coli OmpA (bottom right) sequences. Amino acids 1 to 184 (P. syringae) Amino acids 185 - end (P. syringae) Total Number of amino acids P. syringae 184 P. aeruginosa 190 E. coli 202 P. syringae 160 P. aeruginosa 160 E. coli 144 P. syringae 344 P. aeruginosa 350 E. colt 346 Gaps introduced 5 6 0 4 5 10 Amino acid identity 53% 20% 85% ^ - ^ 3 3 % 68% ^ ^ 2 4 % Conservative substitutions 25% ^ ^ 2 0 % 10% ^ ^ 2 6 % 18% . ^ 2 2 % Amino acid identity plus conservative substitutions 78% ^^-"40% 95% ^ ^ 5 9 % 86% ^ ^ 4 6 % Normalized alignment score (Doolittle, 1986) 459 114 875 264 653 172 Alignment score (Needleman and Wunsch, 1970) 31.2 2.2 49.2 7.9 61.9 7.5 28 Figure 6. Partial restriction maps of the oprF gene region from different P. syringae pathovars and from P. aeruginosa (HI03). The plant for which a strain is pathogenic is in brackets. The one letter codes for the restriction enzymes are as follows: B, BamHI; K, Kpnl; M. Smal; P. PsfJ: S, Sail. The coding region of the oprF gene is represented by a thick line. 29 H365 (lilac, weak) {type strain) S P i B P i S H678 (apple) S P _JL_ B P S i i H680 (lilac) S t, B t. P S H681 (bean) H685 (tobacco) B i P —u. S H682 (tomato) H684 (snapdragon) P i B S B P H103 K S K 1 kb H679 (soybean): Psfl would not cut chromosomal DNA. BanitO. and Sail sites mapped the same as for H365. H683 (tomato): SaB. would not cut chromosomal DNA. BamHl and Psfl sites mapped the same as for H682 and H684. 30 contain Kpnl or Smal sites. All pathovars had a Sail site in the gene, as did the P. aeruginosa strains. The location of the BamHI and PsrJ sites outside the ammo-terminal end of the gene are conserved between the different pathovars. The location of the Psfl site outside the carboxy-terminal end of the gene varied slightly, with two different positions seen among the pathovars. The location of the SaU sites outside the gene varied among the pathovars, with three different positions for the site outside the amino-terminal end and four different positions for the site outside the carboxy-terminal end. This variation is greater than the highly conserved restriction site map for the 17 P. aeruginosa serotypes (Woodruff, 1988). C. IMMUNOLOGICAL RELATEDNESS As shown in Figure 7, interaction of a Western immunoblot of outer membrane proteins from nine P. syringae pathovars with P. aeruginosa protein OprF monoclonal antibody MA4-10 demonstrated that OprF from the P. syringae pathovars was immunologically related to P. aeruginosa OprF. D. HEAT AND 2-MERCAPTOETHANOL MODIFIABILITY Outer membranes from nine P. syringae pathovars and from P. aeruginosa were prepared and run on SDS-polyacrylamide gels after solubilization under the ten conditions described in the Methods section. Protein OprF from P. syringae ran at the same molecular weight as OprF from P. aeruginosa when solubilized under the same conditions. As previously described for P. aeruginosa OprF (Hancock and Carey, 1979). the omission of 2-mercaptoethanol during solubilization also caused an increase in mobility (lower apparent molecular weight) of P. syringae OprF. 31 Figure 7. (A) SDS-PAGE of the outer membrane proteins and (B) the corresponding Western immumoblot after transfer to a nyon membrane and interaction with P. aeruginosa protein OprF monoclonal antibody MA4-10. Lane 1, E. coli DH5aF(pGC31); lane 2, E. coli DH5ccF'; lane 3, P. aeruginosa H103; lane 4, P. syringae H365; lane 5, P. syringae H678; lane 6, P. syringae H680; lane 7, P. syringae H681; lane 8, P. syringae H685; lane 9, molecular weight markers; lane 10, purified P. aeruginosa protein OprF. 32 33 Previous studies have shown that boiling P. aeruginosa outer membrane preparations in SDS, with or without 2-mercaptoethanol, for very long periods of time (30 to 60 minutes) caused an alteration in the mobility of protein OprF from a faster to a slower running form, F* (Hancock and Carey, 1979; Mizuno and Kageyama, 1978). This heat modification to the F* form also occurred with the P. syringae outer membrane preparations, although the P. syringae protein OprF (from all nine strains) was more easily heat modified to the F* form than was the P. aeruginosa OprF. Figure 8 shows that after five minutes at 100°C, none of the P. aeruginosa OprF had been modified to the F* form whereas a substantial amount of the P. syringae OprF had been modified to the F* form. Heat modification was observed at 100°C, both in the presence and the absence of 2-mercaptoethanol, but not at 65°C. 34 Figure 8. SDS-PAGE profiles of outer membrane preparations solubilized without 2-mercaptoethanol at 100°C for five minutes. The heat modified (F*) and unmodified (F) forms of protein OprF are indicated. Lane 1, E. coli DH5aF'; lane 2, E. coli DH5aF'(pGC31); lane 3, P. aeruginosa H103; lane 4, purified P. aeruginosa protein OprF; lane 5, P. syringae H365; lane 6, P. syringae H678; lane 7, P. syingae H679; lane 8, P. syringae H680; lane 9, P. syringae H681; lane 10, molecular weight markers; lane 11, P. syringae H682; lane 12, P. syringae H683; lane 13, P. syringae H684; lane 14, P. syringae H685. 35 36 DISCUSSION The P. aeruginosa and the P. syringae OprF protein sequences showed a high degree of similarity, especially at their carboxy-terminal ends, which had an amino acid identity of 85%. The overall amino acid identity of the two proteins was 68% and if conservative substitutions were included, the identity was 86%. This similarity is remarkable since the DNA homology between the two Pseudomonas species is much lower and, as discussed in the Introduction, the two species are different in many ways. Although P. syringae and P. aeruginosa have an rRNA-DNA similarity of 88% to 92% (Palleroni et al, 1973), they have DNA-DNA similarity levels of 0% to 14% as determined by hybridization experiments (Johnson and Palleroni, 1989; Palleroni et al, 1972; Pecknold and Grogan, 1973). The conservation of the oprF gene between these two species, in contrast to the divergence of the chromosomal DNA, suggests a critical role for protein OprF in the cell, with the carboxy-terminal end of the protein being most important. This is further suggested by the similarity observed between P. syringae protein OprF and E. coli protein OmpA, which were 33.3% identical at their carboxy-terminal ends and when conservative substitutions were included, were 58.6% similar at their carboxy-terminal ends. With an identity of 18.5%, the ammo-terminal ends showed much less similarity. Immunological relatedness between OprF from P. aeruginosa and OprF from the different P. syringae pathovars was demonstrated. Previous studies have shown that OprF specific monoconal antibodies raised against P. aeruginosa OprF fall into two classes on the basis of 37 antigenic cross-reactions, suggesting that there are at least two highly conserved surface epitopes on protein OprF (Mutharia and Hancock, 1985). One class of antibodies contains a single monoclonal antibody which interacts only with P. aeruginosa OprF and the other class contains three monoclonal antibodies which cross-react with OprF from P. syringae and P. putida (Mutharia and Hancock, 1985). Comparison of the P. aeruginosa and the P. syringae OprF sequences will help determine the location of the two epitopes on the protein sequence. As previously demonstrated with P. aeruginosa OprF, protein OprF from the different P. syringae pathovars was shown to be heat and 2-mercaptoethanol modifiable. OprF from P. syringae appeared to be more easily heat modifiable than OprF from P. aeruginosa. This difference could indicate that the secondary or tertiary structure of the P. syringae OprF protein is less stable than that of P. aeruginosa OprF. Modification of mobility by 2-mercaptoethanol is due to the disruption of the two intrachain disulfide bonds formed between the cysteine residues. Restriction site mapping of the oprF gene from different P. syringae pathovars demonstrated a conserved Sail site within the gene and conserved Psfl and BamHI sites close to the amino terminal end of the gene for all the pathovars but variability in the location of the other restriction sites surrounding the OprF gene. From the nine different P. syringae pathovars, five different maps were seen. Restriction site maps of the two strains of P. syringae pv. tomato and the strain P. syringae pv. antirrhini (snapdragon) were notably different than the maps of the other six P. syringae pathovars. These three pathovars had an additional BamHI site just outside the carboxy-terminal end of the gene and the flanking Sail sites were twice the distance from the middle SaH site than were the flanking Sail sites of the other 38 pathovars. The genetic diversity of P. syringae pathovars has been explored to some degree in previous studies. A preliminary study by Lawson et al (1986) used a 22 to 32 kb siderophore-related gene probe and restriction fragment length polymorphism (RFLP) analysis to demonstrate that among some P. syringae pathovars and rhizosphere-associated fluorescent pseudomonads, genotypic organization of siderophore related DNA is highly diversified. DNA hybridization experiments by Denny et al (1988) showed that 26 strains of P. syringae pv. tomato and seven strains of P. syringae pv. syringae were, respectively, 86 to 100% and 37 to 47% homologous to DNA from a P. syringae pv. tomato reference strain when tested under stringent conditions. In the same study, RFLPs with three cloned, 20 kb hybridization probes demonstrated that each of the P. syringae pv. tomato and P. syringae pv. syringae strains was unique. The RFLP difference between pairs of strains was quantified and cluster analysis demonstrated relationships among P. syringae pv. tomato strains but not among P. syringae pv. syringae strains (Denny et al, 1988). In another study, Denny (1988) used cloned 3.5 and 3.6 kb EcoRI fragments from P. syringae pv. tomato as hybridization probes in Southern blots and dot blots. The probe hybridized strongly to P. syringae pv. tomato DNA from 31 strains but did not hybridize to P. syringae pv. syringae DNA from 68 strains, nor to selected strains of P. aeruginosa, P. cichoni, P. Jluorescens, P. viridiflava, or X. campestris pv. vesicatoria. The probe did hybridize to one or more strains of ten of the 20 addditional pathovars of P. syringae examined, including pathovars antirrhini glycinea, papulans, phaseolicola, and tabacl 39 A fourth study used restriction endonucleases which cut infrequently and field inversion gel electrophoresis to generate distinctive patterns of large DNA fragments (genomic fingerprinting) from P. syringae pathovars (Cooksey and Graham, 1989) . Ten strains of P. syringae pv. tomato were examined and two groups could be distinguished. Five strains in the first group, all carrying a copper plasmid, showed similar fingerprints, and the other five in the second group, all copper sensitive, had some differences in their fingerprints. Seven strains from five other P. syringae pathovars had genomic fingerprints very different from those of P. syringae pv. tomato. These studies have shown that the strains of P. syringae pv. tomato are more closely related to each other than to strains of P. syringae pv. syringae and that these two pathovars are genetically very distinct (Denny et al, 1988). The level of genetic diversity among strains is much greater for P. syringae pv. syringae, which has a wide host range, than for P. syringae pv. tomato, which has a single host (Denny et al, 1988). The studies also suggest genetic diversity between the other pathovars of P. syringae in addition to the diversity between the tomato and syringae pathovars. The location of restriction sites outside the oprF gene are consistent with the above observations. Specifically, the two tomato pathovars had maps which were different from all but one of the other pathovars, the two tomato pathovars appeared to have similar maps, the two syringae pathovars had maps different from each other, and some diversity was seen among the different pathovars. The similarity between the antirrhini pathovar map and the tomato pathovar maps is surprising. It would be interesting to determine whether or not the antirrhini pathovar was pathogenic on a tomato host. 40 A previous study showed that all 17 of the P. aeruginosa serotypes tested had the same Kpnl, San, and Smal sites in the oprF gene (one serotype had an extra Kpnl site) (Woodruff, 1988). Likewise, all nine P. syringae pathovars studied had the same Sail site in the oprF gene and similar BamHI and Psfl sites close to the gene. Sixteen of the 17 P. aeruginosa serotypes had identical restriction site maps in the area surrounding the oprF gene. In contrast, the P. syringae pathovars had variable, but similar, restriction site maps in the area surrounding the oprF gene. The stability of the P. syringae oprF gene in the background of genetic variability is interesting and again suggests a critical role for protein OprF in the cell. Because of the stability of the oprF gene in the P. syringae pathovars tested and the variability of restriction sites surrounding the gene, restriction fragment length polymorphism analysis using the P. syringae oprF gene as a probe maybe useful in determining the genetic relatedness of P. syringae pathovars and other Pseudomonas strains 41 REFERENCES Ausubel. F.M., R. Brent, R.E. Kingston, D.D. Moore. J.G. Seidman, J.A. Smith, and K. Struhl (ed.). 1987. Current protocols in molecular biology, vol. 1. John Wiley & Sons, New York. Atkinson. T., and M. Smith. 1984. Purification of oligonucleotides obtained by small scale (0.2 mol) automated synthesis by gel electrophoresis, p.35-81. In M.J. Gait (ed.), Oligonucleotide synthsis: a practical approach. IRL Press, Oxford. Benz, R., and R.E.W. Hancock. 1981. Properties of the large ion-permeable pores formed from protein F of P. aeruginosa in lipid bilayer membranes. Biochim. Biophys. Acta 646:298-308. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Caulcott, C.A., M.R.W. Brown, and I. Gonda. 1984. Evidence for small pores in the outer membrane of P. aeruginosa. FEMS Microbiol. Lett. 21:119-123. Chen, R., W. Schmidmayr. C. Kraemer, U. Chen-Schmeisser, and U. Henning. 1980. Primary structure of major outer membrane protein n (OmpA protein) of Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 77:4592-4596. 42 Cody, Y.S.. and D.C. Gross. 1987. Outer membrane protein medlationg iron uptake via ovoverdin^, the fluorescent siderophore produced by Pseudomonas syringae pv. syringae. J.Bacteriol. 169:2207-2214. Cooksey, D.A., and J.H. Graham. 1989. Genomic fingerprinting of two pathovars of phytopathogenic bacteria by rare-cutting restriction enzymes and field inversion gel electrophoresis. Phytopath. 79:745-750. Del Sal, G., G. Manfioletti, and C. Schneider. 1988. A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 16:9878. Denny, T.P. 1988. Differentiation of Pseudomonas syringae pv. tomato from P. s. syringae with a DNA hybridization probe. 1988. Phytopath. 78:1186-1193. Denny, T.P., M.N. Gilmour, and R.K. Selander. 1988. Genetic diversity and relationships of two pathovars of Pseudomonas syringae. J. Gen. Microbiol. 134:1949-1960. Dente, L., G. Cesareni, R. Cortese. 1983. pEMBL: a new family of single stranded plasimds. Nucleic Acids Res. 11:1645-1655. Doolittle, R.F. 1986. Of URFS and ORFS: a primer on how to analyze derived amino acid sequences. University Science Books, Mill Valley, Calif. 43 Duchene, M., A. Schweizer. F. Lottspeich, G. Krauss, M. Marget, K. Vogel, B. von Specht, and H. Domdey. 1988. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J. Bacteriol. 170:155-162. Feinberg, A.P., and B. Vogelstein. 1982. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. Gatti, R.A., P. Concannon, and W. Salser. 1984. Multiple use of Southern blots. Biotechniques 2:148-155. Godfrey. A.J., and L.E. Bryan. 1987. Penetration of P-lactams through Pseudomonas aeruginosa porin channels. Antimicrob. Agents Chemother. 31:1216-1221. Gotoh, N., W. Hirokazu. E. Yoshihara. T. Nakae. and T. Nishino. 1989. Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane. J. Bacteriol. 171:983-990. Hancock, R.E.W. 1985. Hie role of cell surface components of Pseudomonas aeruginosa in virulence, p. 247-256. In Bayer-Symposiium VHI: The pathogenesis of bacterial infections. Springer-Verlag Berlin Heidelberg. Hancock, R.E.W. 1986. Model membrane studies of porin fuction, p. 187-225. In M. Inouye (ed.), Bacterial outer membranes as model systems. John Wiley & Sons, N.Y. 44 Hancock, R.E.W. 1987. Role of porins in outer membrane permeability (minireview). J. Bacteriol. 169:929-933. 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., G.M. Decad, and H. Nikaido. 1979. Identification of the protein producing transmembrane clifiusion pores in the outer membrane of Pseudomonas aeruginosa PAOl. Biochim. Biophys. Acta 554:323-331. Hancock, R.E.W.. R.T. Irvin, J.W. Costerton, and A.M. Carey. 1981. Pseudomonas aeruginosa outer membrane: peptidoglyccan associated proteins. J. Bacteriol. 145:628-631. Henikoff, S. 1984. Unidirectional digestion wirth exonuclease in creates targeted breakpoints for DNA sequencing. Gene 28:351-359. Holmes, D.S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197. Hurlbert. R.E., and D.C. Gross. 1983. Isolation and partial characterization of the cell wall of Pseudomonas syringae pv. syringae HS191: comparison of outer membrane proteins of HS191 with those of two plasmidless derivatives. J. Gen. Microbiol. 129:2241-2250. 45 Johnson, J.L., and N.J. Palleroni. 1989. Deoxyribonuceic acid similarities among Pseudomonas species. Int. J. Syst. Bacteriol. 39:230-235. Lawson, E.C, C.B. Jonsson, and B.C. Hemming. 1986. Genotypic diversity of fluorescent Pseudomonads as revealed by Southern hybridization analysis with siderophore-related gene probes, p. 315-329. In T.R. Swinburne (ed.), Iron, siderophores, and plant diseases. Plenum Press, N.Y. Lugtenberg, B„ and L. Van Alphen. 1983. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biocim. Biophys. Acta. 737:51-115. Maniatis, R, E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Meade, H.M., S.R. Long, G.B. Brown, and R.M. Ausubel. 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium melilott induced by transposon Tn5 mutagenesis. J. Bacteriol. 149:114-122. Mizuno. T.. and M. Kageyama. 1978. Separation and characterization of the outer membrane of Pseudomonas aeruginosa. J. Biochem. 84:179-191. Mizuno, T., and M. Kageyama. 1979. Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein. J. 46 Biochem. 86:979-989. Mutharia, L.M., and R.E.W. Hancock. 1985. Characterization of two surface-localized antigenic sites of porin protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 31:381-386. Needleman, S.B., and CD. Wunsch. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Molec. Biol. 48:443-453. Nikaido, H., and R.E.W. Hancock. 1986. Outer membrane permeability of Pseudomonas aeruginosa, p. 145-193. In I.C. Gunsalus and R.Y. Stanier (ed.), The bacteria: a treatise on structure and function. Academic Press, N.Y.. O'Connor, G.G., and L.K. Ashman. 1982. Application of the nitrocellulase transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of athe specificity of monoclonal antibodies to protein mixtures. J. Immunol. Methods 54:267-271. Palleroni, N.J. 1984. Genus I: Pseudomonas, p. 141-199. In N.R. Krieg (ed.), Bergey's manual of systemic bacteriology, vol.1. Williams and Wilkins Company, Baltimore. Palleroni, N.J.. R.W. Ballard, E. Ralston, and M. Doudoroff. 1972. Deoxyribonucleic acid homologies among some Pseudomonas species. J. Bacteriol. 110:1-11. 47 Palleroni, N.J., R. Kunisawa, R. Contopoulou, and M. Doudoroff. 1973. Nucleic acid homologies in the genus Pseudomonas. Int. J. Syst. Bacteriol. 23:333-339. Pecknold, P.C., and R.G. Grogan. 1973. Deoxyribonucleic acid homology groups among phytopathogenic Pseudomonas species. Int. J. Syst. Bacteriol. 23:111-121. Perlman, D., and H.O. Halvorson. 1983. A putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides. J. Mol. Biol. 16Z:391-409. Reed. K.C., and D.A. Mann. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids Res. 13:7207-7221. Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with cham-terminating inhibitors. Proc. Nat. Acad. Sci. USA 74:5463-5467. Schwartz, R.M., and M.O. Dayhoff. 1978. Matrices for detecting distant relationships, p. 353-358. In M.O. Dayhoff (ed.). Atlas of protein sequences and structure, vol. 5. National Biomedical Research Foundation, Washington, D.C. Sekar, V. 1987. A rapid screening procedure for the identification of recombinant bacterial clones. BioTechniques 5:11-13. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacryaminde gels to nitrocellulose sheets: 48 procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. Watson, M.E.E. 1984. Compilation of published signal sequences. Nucl. Acids Res. 12:5145. Winberg, G., and M.L. Hammerskjold. 1980. Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA. Nucl. Acids Res. 8:253-264. Woodruff, W.A. 1988. Cloning and characterization of the oprF gene for protein F from Pseudomonas aeruginosa. Ph.D. Thesis, University of British Columbia. Woodruff, W.A., and R.E.W. Hancock. 1988. Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo 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 .Jr., R.E.W. Hancock, L.F. Hanne, T.I. Nicas, and B.H. Iglewski. 1986. Expression in E. coli and function of Pseudomonas aeruginsa outer membrane porin protein F. J. Bacteriol. 167:473-479. 49 Yoneyama, H., A. Akatsuka, and T. Nakae. 1986. The outer membrane of Pseudomonas aeruginosa is a barrier against the penetratioin of disaccharides. Biochem. Biophys. Res. Commun. 134:106-112. Yoshihara, E., N. Gotoh, and T. Nakae. 1988. In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 156:470-476. 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. 258:2308-2314. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098191/manifest

Comment

Related Items