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

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COMPARISON OF PROTEIN OprF FROM Pseudomonas WITH PROTEIN OprF FROM Pseudomonas  syringae  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 department or by  his or her  representatives.  be granted by the head of  It is understood  that copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  my  written  ii  ABSTRACT  The aeruginosa  major  OprF  from  Pseudomonas  was compared with OprF from the fluorescent phytopathogen  Pseudomonas  sequenced  outer membrane protein  syringae.  The P. syringae  oprF  gene was subcloned and  and found to code for a sequence  containing a 24 amino acid leader sequence.  of 344 amino acids  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  amino acid sequence with the P. aeruginosa  OprF  OprF and the E. coli OmpA  sequences showed that the sequences were most similar at the carboxyterrninal 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 pathovars and P. aeruginosa  OprF from the different  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 I.  II.  Title Bacterial strains and plasmids  Page 6  Amino acids in the P. syringae OprF sequence which are conservative substitutions for amino acids in the P. aeruginosa OprF sequence  III.  25  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  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  4.  18  Comparison of the P. syringae and the P. aeruginosa  oprF gene nucleotide sequences  5.  9  21  Comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA amino acid sequences  6.  23  Partial restriction maps of the oprF gene region from different P. syringae pathovars and from P. aeruginosa  7.  SDS-PAGE of the P. syringae outer membrane proteins and the corresponding Western immumoblot  8.  28  31  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). negative rod, P. aeruginosa  As a gram-  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 (10 to 5x10 4  s  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).  (Hancock and  This protein is heat and 2-mercaptoethanol modifiable 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 crossreact 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  structure  questions, especially  and mechanisms  in the areas  of porins.  To further  of molecular the knowledge  regarding outer membrane proteins and protein OprF, P. aeruginosa was  compared  Pseudomonas  with  OprF  from  another  fluorescent Pseudomonad,  syringae.  Bacteria belonging to the phytopathogenic are  OprF  subdivided  according  to host  range  into  over  40  (Palleroni, 1984). Outer membrane proteins of P. syringae relatively little study  (Hurlbert and Gross,  syringae  species P.  pathovars  have received  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.  opportunistic human pathogen and P.syringae aeruginosa  P. aeruginosa  is a plant pathogen. P.  has one flagella per cell whereas P. syringae  one flagella per cell. P. syringae  P. aeruginosa  cells, with P. aeruginosa  has more than  cells are longer and narrower than 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. is  oxidase  syringae aeruginosa,  positive  is an  and arginine dihydrolase positive  aeruginosa  whereas P.  is oxidase negative and arginine dihydrolase negative.  P.  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. syringae  aeruginosa  The nutritional spectrum of P.  strains is less extensive and more variable than that of P. strains. The G+C content of P. aeruginosa  DNA is 67.2% and  the G+C content of P. syringae DNA is 59 to 6 1 % (Palleroni, 1984). In order to compare P. aeruginosa the P. syringae  protein OprF with P. syringae OprF,  OprF gene was first cloned and sequenced.  Then, the  4 restriction  site  heterogeneity  of  the  syringae pathovars was determined.  oprF gene  among  different P.  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  plasmids used in this study are listed in Table I.  and their  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  PAOl Cm' prototroph; wild type reference strain  Hancock and Carey, 1979  H365  pv. syringae  ATCC #19310  H678  pv. papulans  (apple)  H679  pv. glycinea  (soybean)  H680  pv. syringae (lilac)  R. Moore (#5D19)  H681  pv. phaseolicola  R.-Moore (#HB6)  H682  pv. tomato  R. Moore (#3000)  H683  pv. tomato  R. Moore (#1108)  H684  pv. antirrhini  H685  pv. tabaci (tobacco)  H103 P.  syringae  (weakly pathogenic to lilac)  R. Moore (#3679) R. Moore (#B3)  (bean)  (snapdragon)  R. Moore (#2738) R. Moore (#GB1)  E. coli  DH5cxF'  F' endAl hsdR17(r \m ) supE44 thi-1 recAl gyrA96 relAl X' o80dIacZAMl5 A(lacZYAargF)U169  C483  DH5a/pGC31  This study  pTZ19U  GeneScribe cloning vector  BRL  pTZ19R  GeneScribe cloning vector  BRL  pGC31  pRK404 + 2.5 kb Psfl-BamHI fragment encoding P. syringae oprF gene  This study  +  k  k  Bethesda Research Laboratories (BRL)  Plasmids  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  (hexadecyltrimethyl ammonium bromide) (Ausubel et al,  using  1987).  CTAB 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 (1982).  E. coli DH5cxF cells were made competent using 0.1 M  (Maniatis, 1982).  al  CaCl  2  Transformants were screened for inserts by the slot-  lysis technique (Sekar, 1987). Agarose gel electrophoresis used the BioRad DNA  Sub  Cell and the Mini Sub  running buffer. Recovery of DNA  DNA  Cell with IX TBE  as the  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. and is shown as a circle.  A positive clone, pGC, was isolated  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  p38(pTZ19R)  p37(pTZ19U)  S  B  S  P  11 prepared according to the method in the manual GeneScribe-Z and  Experimental  Protocols  (United  States  Biochemical  Description 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 described In the  Sequencing  manual Sequenase:  with Sequenase  Step-by-Step  Protocols  used as for  DNA  (2nd edition). Sequencing of one short section  at the carboxy end of the gene used the kit TaqTrack (Promega) and the TaqTrack  Extension/Labeling  TaqTrack  Sequencing  Systems  Protocol as Technical  described  Manual  in the  manual  (Promega). Both  the  Sequenase and the TaqTrack kits used S-dATP (NEN/Dupont, Markham, 35  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. TGGCAAGGTCTGGTAGAC ' S  3  2. ^TGTACGACCAGCGCCCGT 3. CGACCAGAGCATTGGCCA 5  3  3  4. ^CGTCGTGCAAACGCCGTT ' 3  (upstream from start site) (bases 119-135) (bases 345-362) (bases 865-882)  5. ^TGATGTTCTTGCCATCAT '  (bases 242-259. opposite strand)  6. CGGCGTAGAAGTTGTCAG  (bases 431-449, opposite strand)  3  5  3  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. GACAGTTTTTCGTTGTAA 5  (bases 843-860, opposite strand)  3  8. CCACGCACAGCTGAATGCCGG 5  (bases  3  1126-1146, opposite  strand) Sequencing gels used were non-gradient, acrylamide:bisacrylamide (38:2), wedge gels. The top of the wedge was and the bottom of the wedge was  1.2 mm  Apparatus Model S2 was used with  0.4 mm  thick. The BRL  thick  Sequencing  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% and  run at constant voltage of 130 volts in IX TBE  containing 4 ug/ml ethidium bromide. DNA  agarose gels  running buffer  The alkaline blotting method of  capillary transfer, based on the procedure of Reed and Mann (1985),  was used to transfer the DNA The  to Zeta-Probe blotting membranes (Bio-Rad).  probe used in the hybridizations was  fragment containing the P. syringae  oprF gene.  a 2.5 kb Psfl-BamHI Radiolabelling of the  probe used a- P-dATP (NEN/Dupont, Markham, Ontario) and the random 32  oligonucleotide primer method as described by Feinberg and (1983).  Vogelstein  14 An Elutip-d column (Schleicher and Schuell, Inc., Keene, N.H.)  was  used to separate unincorporated reactants from the labeled probe.  The  from the technical literature Tips for  protocol followed was  Recovery  (Schleicher and Schuell, No. 208). Hybridization was based on the standard hybridization protocol in the Zeta-Probe Blotting Membranes Instruction Manual (Bio-rad). prehybidization solution consisted of 1.5X  SSPE, 1.0%  milk powder, 1.5 mg/ml salmon sperm DNA,  and  SDS,  10%  0.5%  The skim  dextran sulfate.  Membranes were sealed (often two together, back to back) in heat sealable pouches (Kapak Corporation, Minneapolis) and without shaking, for 0.5 to 4.0 hours. prehybridization solution, the bag without shaking, for 22 approximatedly 10  8  hybridization  to 36  was  probe was  resealed, and hours.  cpm/ug probe and  mixture  The  incubated at 45°  added to the  incubated at 45°  Probe specific activity  the probe concentration  approximately  10  6  C,  counts/ml.  C, was  in the At  the  completion of hybridization, the membranes were removed from the bags and  rinsed briefly in 2X  SSC.  temperature, shaking, for 15 SSC/0.1%SDS; 0.5X  They were then washed at room  minutes in each ofthe following:  SSC/0.1% SDS;  and  0.25X SSC/0.1% SDS.  2X 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 aeruginosa  membranes  from  nine P. syringae  pathovars and  P.  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 2mercaptoethanol 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% 2mercaptoethanol 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' dilution of the anti P.aeruginosa 3  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" in 3 % skim milk in PBS and added to the blot, which 3  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, alanine-proline rich area found in the P. aeruginosa conserved in the P. syringae  1988), and the  oprF gene were also  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 61.4%, 40.8%, and 78.2%, for the P. aeruginosa  oprF gene.  oprF gene and  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.  syringae  19  oprF  M  K  L  K  N  T  6  T C C C C A TGT GTG GGA C T G C T T A A T A A T CAT C A G A T G GGG A T T TAA CGG lATGl A A A CTG A A A A A C A C C  L  G  L  A  I  G  T  I  V  A  A  T  S  F  G  A  L  A  ^  Q  G  Q  G  2  18  8  T T G GGC T T G GCC A T T GGT ACT A T T G T T GCC GCA ACT T C G T T C GGC GCG CTG GCT CAA GGC C A A GGC  A  V  E  I  E  G  F  A  K  K  E  M  Y  D  S  A  R  D  F  K  N  N  84  5  0  GCA GTC GAA A T C GAA GGC T T C GCC A A G A A A GAA ATG T A C GAC AGC GCC CGT GAT TTC A A A A A C A A C  G  N  L  F  G  G  S  I  G  Y  F  L  T  D  D  V  E  L  R  L  G  Y  7  150  2  GGC AAC C T G T T C GGC GGC TCG A T T GGC T A C T T C CTG A C C GAC GAC GTT GAA T T G CGT C T G GGC T A C  D GAC  A  E  V  H  N  V  R  S  D  D  G  K  N  I  K  G  A  D  T  A  L  D  9  216  4  GAA GTC C A C AAC GTT CGT AGC G A T GAT GGC A A G A A C A T C AAG GGC GCA GAC ACT GCC C T G GAC  L  Y  H  F  N  N  P  G  D  M  L  R  P  Y  V  S  A  G  F  S  282  D  116  GCT C T C T A C C A C T T C A A C AAC C C A GGC GAC ATG C T G CGT C C A TAC GTT T C T GCC GGT T T C T C C G A C  Q  S  I  G  Q  N  G  R  N  G  R  N  G  S  T  F  A  N  I  G  G  348  G  138  C A G AGC A T T GGC C A G A A C GGT CGT A A C GGT CGT AAC GGT T C T ACC T T C GCC A A C A T C GGC GGC GGC  P CCC  Q CAA  V GTT  N AAC  A GCC  S AGC  E GAA  K  G  E  V  D  T  E  D  N  F  Y  A  A  R  G  ACT GAC A A C T T C T A C GCC CGT GCT  W  A  A  P  D  K  A  P  A  Q  E  V  V  S  P  P  R  GAA GTG GTT  V  G  I  V  A  E  G  V  ©  D  T  P  A  N  V  S  Y  G  D  V  E  CGT GTT  I  K  L  D  V  K  F  D  GAG CTG GAC GTG AAG TTC  N  L  A  D  F  M  Q  E  A  Q  Y  N  I  D  160  N  G  G  G  S  K  K  182  S  D  N  D  G  V  @  D  2  0  G  H  T  D  S  V  G  P  D  A  Y  N  V  226  D  A  D  G  (  C  )  P  A  V  GTT GAC GCT GAT GGC TGC CCA GCA GTT  D  Y  K  S  V  V  K  P  N  GAC A A A TCC GTA GTC AAG CCT A A C  P  Q  T  T  T  T  V  K  L  S  E  R  R  A  E  K  Q  V  L  S  K  P  V  N  CTG GTT  V  A  Q  Y  G  V  G  A A C CAG TAC GGT GTT  N  D  N  A  T  E  A  A  S  R  V  N  G  R  A  V  N  GAA AGC AAG C C A GTT GCT GAT AAC GCA ACT GAA GCT GGC CGC GCA GTT  E  V  E  A  Q  A  K  S  V  G  GGC GCT AGC CGC GTA A A C TCG GTT  R  R  V  TGC C T G  248 744  810  292  Y  876  314  GGT TAC  942  E  336  AAC CGT CGC GTA GAA  *  G A A G T A G A A G C T C A A G C T A A G |TAA| 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  TTTCTT  678  270  GGT CAC ACT GAC TCN GTC GGT CCT GAC GCT TAC A A C CAA AAA CTG TCC GAG CGT CGT GCA A A C  A A A CAG GTT  4 612  F  Q  546  GAC AGC GAC A A C GAC GGC GTG TGC GAC  GAT TTC  Q  F  480  GTA AAC TTC GGT GGC GGC AGC AAG A A A  D  T  V  V  GGC GTT G A A GCT C A A TAC A A C ATC GAC  TAC GGC GAC ATC AAG AAC CTC GCT GAC TTC ATG CAG CAG TAC CCA CAG ACC ACC ACC ACT GTT  V  A  T  GTC GAC A A G TGC CCG GAC A C C C C A GCC A A C GTT ACC  GTT  GCA  F  GAA GCA GCA C C A GCT CCA GTA GCT GAA GTG TGC TCC  A  G  Y  GGC GAC A C C GAG TGG GCT C C A A G C G T C GGT A T C GGC  GCC  GGC  L  AAG CTC TAC TTC  414  1008  344 1074  1086  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 aminoterrninal halves, and 49.2 for the carboxy-terminal halves.  All these  values were "significant (above 3SD) results". Conservative substitutions between the P. syringae aeruginsa  and the P.  OprF amino acid sequences are indicated in Figure 5 by dots  and are specified in Table II. From amino acid residues one to 184, 2 5 % 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 7 8 % for the amino-terrninal end, 9 5 % for the carboxyterminal end, and 8 6 % for the entire protein.  21  Figure 4.  Comparison of the P. syringae  nucleotide sequences.  and the P. aeruginosa  oprF gene  Vertical lines indicate nucleotide matches.  initiation codons (ATG) and the termination codons (TAA) are boxed. putative  Shine-Dalgarno  sequences  (GGGA) and  the  putative  The The 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.  22  opr F P.AER.F  GGCTGATTGTTGGACAACTAACTGACCATCAAGATGGGGATTTAACGG^TC^RAACTGAAGAACACCTTAGC^GTTGTCATCGCXn'CG  i P.SYR.F  P . AER. F  III  ii  39  1111111111111111111111111111 i l i u m i n i i i n I I i  i  TCCCCATGTGTGGGACTGCTTAATAATCATCAGATGGGGATTTAACGG(ATgAAACTGAAAAACACCTTGGGCTTGGCCATTGGTACT 39  CTGGTTGCCGCTTCGGCAATGAACGCCTTCGCCCAGGGCCAGAACTCGGTAGAGATCGAAGCCTTCGGCAAGCGCTACTTCACCGAC I  I IIIIIII  I  I  I  III  I  II  II  IIIII  I  I  I I  II  IIIIIII  I II II  III I  I  I  P.SYR.F  ATTGTTGCCGCAACTTCGTTCGGCGCGCTGGCTCAAGGCCAAGGCGCAGTCGAAATCGAAGGCTTCGCCAAGAAAGAAATGTACGAC  P.AER.F  AGCGTTCGCAACATGAAGAAC I III  II  I  I  II  126  III I 126  GCTGACCTGTACGGCGGCTCGATCGGCTACTTCCTGACCGACGACGTCGAGCTGGCTCTGTCC 210  III  I  I IIII I  IIIIIII II III  IIIIIIIIIII II II II  II I  III  II  II  IIII  P.SYR.F  AGCGCCCGTGATTTCAAAAACAACGGCAACCTGTTCGGCGGCTCGATTGGCTACTTCCTGACCGACGACGTTGAATTGCGTCTGGGC  213  P.AER.F  TACGGTGAGTACCACGATGTTCGTGGCACCTACGAAACCGGCAACAAGAAGGTCCATGGCAACCTGACCTCCCTGGACGCCATCTAC  297  I III P.SYR.F  P. AER. F  II  IIII  P.AER.F  P . AER. F  II  I  II  III  I IIIII II I I  IIII I  GGCAAGAACATCAAGGGCGCAGACACTGCCCTGGACGCTCTCTAC  291  I  III  IIII  IIII IIII  IIIII  II  II  I II  I  I  IIIIII  III  I  I  I  I  I  III  I  III  I  IIII IIIIIIIII  I I I  III  IIIII IIII  II  IIIII II  IIII  384  II 375  II  471  I I I  GGTCGTAACGGTTCTACCTTCGCCAACATCGGCGGCGGCCCCAAGCTCTACTTCACTGACAACTTCTACGCCCGTGCTGGCGTT 459  GACGGCCAGTACGGCCTGGAGAAGCGTGACAACGGTCACCAGGGTGAGTGG I  II  I II  I  I  II-  GAAGCTCAATACAACATC  I I  ATGGCTGGCCTGGGCGTCGGCTTCAACTTC 552  II IIII  I  I  II  I  IIIII  I III  I  GACCAAGGCGACACCGAGTGGGCTCCAAGCGTCGGTATCGGCGTAAACTTCGGTGGC  GGTGGTTCGAAAGCCGCTCCGGCTCCGGAACCGGTTGCCGACGTTTGCTCCGACTCCGACAACGACGGCGTCTGCGACAACGTCGAC  II P .SYR.F  IIII II  CAAGGCCGTCAGCAGATGACCATGGCCAACATCGGCGCTGGTCTGAAGTACTACTTCACCGAGAACTTCTTCGCCAAGGCCAGCCTC  I I  P . AER.F  I  CACTTCAACAACCCAGGCGACATGCTGCGTCCATACGTTTCTGCCGGTTTCTCCGACCAGAGCATTGGCCAGAACGGTCGTAAC  P.SYR.F  P.SYR.F  II  CACTTCGGTACCCCGGGCGTAGGTCTGCGTCCGTACGTGTCGGCTGGTCTGGCTCACCAGAACATCACCAACATCAACAGCGACAGC  I I  P.AER.F  IIIII I  TACGACGAAGTCCACAACGTTCGTAGCGATGAT  I IIIII P . SYR. F  I  I  I  lllll  I  I II II I  534  639  I I I I I I II I I II I I I I I I I I I I I I II llltl I I I I I  GGCAGCAAGAAAGTTGAAGCAGCACCAGCTCCAGTAGCTGAAGTGTGCTCCGACAGCGACAACGACGGCGTGTGCGACAACGTCGAC  AAGTGCCCGGACACCCCGGCCAACGTCACCGTTGACGCCAACGGCTGCCCGGCTGTCGCCGAAGTCSTACGCGTACAGCTGGACGTG  621  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 P .SYR.F  P . AER.F  AAGTGCCCGGACACCCCAGCCAACGTTACCGTTGACGCTGATGGCTGCCCAGCAGTTGCCGAAGTGGTTCGTGTTGAGCTGGACGTG  708  AAGTTCGACTTCGACAAGTCCAAGGTCAAAGAGAACAGCTACGCTGACATCAAGAACCTGGCCGACTTCATGAAGCAGTACCCGTCC  813  I I I I I I I I I I I I III I I I I  lllll  I I I III I I I I  I I I I I I III I I I I I II I I I I I I I I  P .SYR.F  AAGTTCGATTTCGACAAATCCGTAGTCAAGCCTAACAGCTACGGCGACATCAAGAACCTCGCTGACTTCATGCAGCAGTACCCACAG 795  P.AER.F  ACTTCCACCACCGTTGAAGGTCATACCGACTCCGTCGGTACCGACGCTTACAACCAGAAGCTGTCCGAGCGTCGTGCCAACGCCGTT  II P . SYR. F  P.AER.F  ACCACCACCACTGTTGAAGGTCACACTGACTCNGTCGGTCCTGACGCTTACAACCAAAAACTGTCCGAGCGTCGTGCAAACGCCGTT  CGTGACGTACTGGTCAACGAGTACGGTGTGGAAGGTGGTCGCGTGAACGCTGTCGGTTACGGCGAGTCCCGCCCGGTTGCCGACAAC I  P.SYR.F  P . AER. F  II  II III  III  II III IIIII I  I  I  I  III II  III  I  II  I I I I I III I I  I  I  II  I  I  II I  I  AAACAGGTTCTGGTTAACCAGTACGGTGTTGGCGCTAGCCGCGTAAACTCGGTTGGTTACGGCGAAAGCAAGCCAGTTGCTGATAAC  GCCACCGCTGAAGGCCGCGCTATCAACCGTCGCGTTGAAGCCGAAGTAGAAGCCGAAGCCAAGJIAarCGGCTGAGCCTTCAAAGAAA  lllll  I  GCAACTGAAGCTGGCCGCGCAGTTAACCGTCGCGTAGAAGCAGAAGTAGAAGCTCAAGCTAAGg^TTAGCCGCTTGTACTGAAAAG  P.AER.F  AACCGGCCCAGGCCGGGTTTTTCTTTGCCTGGAAAAAGACCGCTCGTCAGGCGCTCAGGGAAACCGGTT I I  I  I  II  882  987 I  'I I I  969  1074  I I III I I I I I I I I I I I I III I I I I I I I I I I I I I I I I III I I I I 1 1 1 I I I I  P.SYR.F  P . SYR. F  900  I I I III 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 II  I III  II  I  II  I  I  I I I  1056  114 3  i  CCCGGCTTAGGCCGGGCTTTTCTTTGCCTGCGATTTGGCATTGCGTCTGTTCAGGCGGGCTTGATGTCA  1125  23  Figure 5.  Comparison of the P. syringae  sequence with the P. aeruginosa OmpA protein sequence.  protein OprF amino acid  protein OprF sequence and the E. coli  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  (Schwartz and Dayhoff, 1978). al,  1988.  P. aeruginosa  E. coli data was from Chen et al,  as a  cutoff  data was from Duchene et 1980.  24 P.AER.F  MKLKNTLGWIGSLVAASAMNAFAQGQNSVEIEA-FGKRYFTDSV 44  P.SYR.F  MKLKNTLGLAIGTIVAATSFGALAQGQGAVEIEG FAKKEMYDSA 44  I I I I I I I I• .  OmpA  I I..I I I..  ..II.  .I.  I.I I I I  .  I I  .1111.  ..  |.|.  II  I  I. •  MKKTAIAIAVALAGFATVAQAAPKDNTWYTGAKLGWSQYHDT  P.AER.F  RNMKN ADL-  P.SYR.F  RDFKNNGNL  I •  II  42  -YGGSIGYFLTDDVELALSYGEYHDVRGTYE 82 . I I I I I I I I I I I I I I  •.I  I.I.I  I •I I •  FGGSIGYFLTDDVELRLGYDEVHNVRSD  ..III  I .  II.  I  • .I I I  .  81  .  OmpA  GFINNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYK - 85  P.AER.F  TGNKKVHGNLT S LDAIYHF -  P.SYR.F  DGKNIKGADTALDALYHF  •  I..  I  -GTPGVGLRPYVSAGLAHQN 120  I •I I I •I I I  II  I I I I I I I I...I •  NNPGDMLRPYVSAGFSDQS 118  OmpA  --GSVENGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTK  P.AER.F  ITNINSDSQGRQQMTMANIGAG—  P.SYR.F  IGQ  OmpA  SNV—YGKNHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDA 171  P.AER.F  YGLEKRDNGHQGEWMA  P.SYR.F  YNIDQGDT  OmpA  HTIGTRPD  P.AER.F  DSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEWRVQLDVKFDF  I  •  . . . I I .  -LKYYFTENFFAKASLDGQ 160 I  I I I I.I  ...  I  I  •  I I I •I I •I . I  II  I •  GLGVGFNFGGSK—AAPAPEPVADVCS 201  I I -  I.I I  I  I.I  .11111.111  EWAPSVGIGVNFGGGSKK  I .  .  I  PKLYFTDNFYARAGVEAQ 156  NGRNGRNGSTFANIGGG I.I  I  I  128  I. I..  VEAAPAPVAEVCS 195  I l l  I  •I I I I  .II  NGMLSLGVS YRFGQGEAAPWAPAPAPAPEV  210 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  P.SYR.F  DSDNDGVCDNVDKCPDTPANVTVDADGCPAVAEWRVELDVKFDF •  OmpA  II  240  I• I  QTKHFTLKSDVLFNF 225  P.AER.F  DKSKVKENSYADIKNLADFMKQ—YPSTSTTVEGHTDSVGTDAYN 289  P.SYR.F  DKSWKPNSYGDIKNLADFMQQ  OmpA  NKATLKPEGQAALDQLYSQLSNLDPKDGSVWLGYTDRIGSDAYN 270  P.AER.F  QKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEG  P.SYR.F  QKLSERRANAVKQVLVNQYGVGASRVNSVGYGESKPVADNATEAG  OmpA  QGLSERRAQSWDYLISK GIPADKISARGMGESNPVTGNTCDNV 314  P.AER.F P.SYR.F OmpA  III  II  . 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 I II • 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  I  RAVNRRVEAEVEAQAK* II  I I  . I. I I II  •  334 I  32 8  |I I •I I .• I • .  I I . I I I I I I I I I I • I II .1111  I  . • I I I . I I I I II • I I I I II I  RAINRRVEAEVEAEAK* I  I I II  YPQTTTTVEGHTDSVGPDAYN 283  KQRAALIDCLAPDRRVEIEVKGIKDWTQP OA  350 344 346  25  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). Table  P. aeruginosa OprF  alanine arginine asparagine aspartic acid  glutamic acid glutamine glycine histidine isoleucine leucine lysine phenylalanine proline serine  tyrosine valine  Substitution in P. syringae OprF  glycine proline serine lysine asparatic acid glutamine serine asparagine glutamic acid glutamine glycine aspartic acid glutamine asparagine glutamic acid alanine serine aspartic acid aspartic acid leucine valine isoleucine phenylalanine valine arginine asparagine glutamine leucine tyrosine alanine alanine asparagine glycine threonine phenylalanine isoleucine leucine  Number of substitutions  4 1 3 3 2 1 1 2 2 1 1 2 2 2 1 3 3 2 1 1 1 3 1 1 1 1 2 1 1 1 2 1 2 3 1 1 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 aminoterminal to residue 184 of P. syringae, the amino acid identity between the two proteins was 19.6%, and from residue 184 to the carboxyterminal 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  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.  T a b l e III.  Total  Amino acids 1 to Amino acids 185 184 (P. syringae) - end (P. syringae) Number of amino acids  Gaps introduced  160 P. syringae 344 P. syringae 184 P. syringae P. aeruginosa 190 P. aeruginosa 160 P. aeruginosa 350 144 E. colt 346 E. coli 202 E. coli 5  0 4  6 Amino acid identity  53%  Conservative substitutions  25%  Amino acid identity plus conservative substitutions  78%  Normalized alignment score (Doolittle, 1986) Alignment score (Needleman and Wunsch, 1970)  20%  ^ ^ 2 0 %  5  85%  68%  ^-^33%  ^ ^ 2 4 %  10%  18%  86%  ^^59%  114  2.2  172  264 49.2  31.2  ^^46%  653  875  459  .^22%  ^^26% 95%  ^^-"40%  10  61.9 7.9  7.5  28  Figure 6. syringae  Partial restriction maps of the oprF gene region from different P.  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  H678 (apple)  S  B  P i  P  B  P i  S i  B  P  S  _JL_  H680 (lilac)  S  t.  t,  H681 (bean) H685 (tobacco)  H103  P  B i  H682 (tomato) H684 (snapdragon)  P i  K  S  S  P i  B  S  —u.  S  B  P  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. H103; lane 4, P. syringae syringae  H365; lane 5, P. syringae  H680; lane 7, P. syringae  H678; lane 6, P.  H681; lane 8, P. syringae  molecular weight markers; lane 10, purified P. aeruginosa  aeruginosa  H685; lane 9,  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  ends, which had an amino acid identity of 85%.  carboxy-terminal  The overall amino acid  identity of the two proteins was 6 8 % 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 8 8 % to 9 2 % (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 further  end of the protein being most important.  suggested  by the similarity  observed  between  This is  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 identity  of 18.5%,  the ammo-terminal  ends  ends.  showed  With an  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 highly  cross-reactions,  conserved  Hancock, 1985).  suggesting  surface  that  there  epitopes on protein  are at least two  OprF  (Mutharia and  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 2mercaptoethanol modifiable.  OprF from P. syringae appeared to be more  easily heat modifiable than OprF from P. aeruginosa. could  indicate that  This difference  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. al  A preliminary study by Lawson et  (1986) used a 22 to 32 kb siderophore-related  restriction  fragment  length  polymorphism  gene probe and  (RFLP)  analysis  to  demonstrate that among some P. syringae pathovars and rhizosphereassociated  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 4 7 % 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. difference analysis  between  pairs  demonstrated  of strains  relationships  was  among  quantified  The RFLP and  P. syringae  cluster  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  Southern blots and dot blots.  The probe hybridized  probes in  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  infrequently  study and  used  field  inversion  distinctive patterns of large from P. syringae P. syringae  DNA  endonucleases which  gel electrophoresis  to  cut  generate  fragments (genomic fingerprinting)  pathovars (Cooksey and Graham, 1989) . Ten strains of  pv. tomato  distinguished.  restriction  were examined  and  two  groups could  be  Five strains in the first group, all carrying a copper  plasmid, showed similar fingerprints, and the other five in the second group,  all copper  had  some  differences  Seven strains from five other P. syringae  fingerprints. genomic  sensitive,  fingerprints  very  in  their  pathovars had  of P. syringae  different from those  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. 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 than for P. syringae 1988).  syringae  pv. syringae,  which has a wide host range,  pv. tomato, which has a single host (Denny et  al,  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  consistent with the above observations.  outside  the  oprF  gene are  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.  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