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• W2099685623 abstract "Porin (341 amino acids; mass of 37,782 Da) in the outer membrane of Haemophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da. Molecular modeling of Hib porin identified its structural similarities to OmpF of Escherichia coli and disclosed for Hib porin a shorter length of loop 3 and a longer length of loop 4. By site-directed mutagenesis of the porin geneompP2, mutant porins were constructed to contain 6 or 12 amino acid deletions either in loop 3 or in surface-exposed loop 4. Wild type Hib porin and mutant porins were expressed in a nontypeableH. influenzae strain deleted for the ompP2gene. The mutant porins were purified and reconstituted into planar bilayers, tested for channel formation and compared with wild type Hib porin. Mutant Haemophilus porin possessing a 6-amino acid deletion in loop 3 displayed a broad distribution of single channel conductance values, while deletion of 12 amino acids from the same loop destabilized the porin channel. By comparison, deletion of 6 or of 12 amino acids from loop 4 of Hib porin resulted in an increased single channel conductance (1.15 and 1.05 nanosiemens, respectively) compared with wild type Hib porin (0.85 nanosiemens). The C3 epitope of the poliovirus VP1 capsid protein was inserted either into loop 3 or into loop 4 of Hib porin. By flow cytometry, the C3 epitope was detected as surface-exposed in strains expressing C3 insertion in loop 4; in strains expressing C3 insertion in loop 3, the epitope was inaccessible. We propose that loop 4 of Hib porin, although surface-accessible, is oriented toward the central axis of the pore and that deletions in this loop increase the single channel conductance by widening the pore entrance. Porin (341 amino acids; mass of 37,782 Da) in the outer membrane of Haemophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da. Molecular modeling of Hib porin identified its structural similarities to OmpF of Escherichia coli and disclosed for Hib porin a shorter length of loop 3 and a longer length of loop 4. By site-directed mutagenesis of the porin geneompP2, mutant porins were constructed to contain 6 or 12 amino acid deletions either in loop 3 or in surface-exposed loop 4. Wild type Hib porin and mutant porins were expressed in a nontypeableH. influenzae strain deleted for the ompP2gene. The mutant porins were purified and reconstituted into planar bilayers, tested for channel formation and compared with wild type Hib porin. Mutant Haemophilus porin possessing a 6-amino acid deletion in loop 3 displayed a broad distribution of single channel conductance values, while deletion of 12 amino acids from the same loop destabilized the porin channel. By comparison, deletion of 6 or of 12 amino acids from loop 4 of Hib porin resulted in an increased single channel conductance (1.15 and 1.05 nanosiemens, respectively) compared with wild type Hib porin (0.85 nanosiemens). The C3 epitope of the poliovirus VP1 capsid protein was inserted either into loop 3 or into loop 4 of Hib porin. By flow cytometry, the C3 epitope was detected as surface-exposed in strains expressing C3 insertion in loop 4; in strains expressing C3 insertion in loop 3, the epitope was inaccessible. We propose that loop 4 of Hib porin, although surface-accessible, is oriented toward the central axis of the pore and that deletions in this loop increase the single channel conductance by widening the pore entrance. The outer membrane of Gram-negative bacteria forms a selective permeability barrier to substances that are present in their environment. Solutes such as sugars, amino acids, nucleosides, and small antibiotics diffuse across the outer membrane, whereas substances such as proteins, detergents, and large antibiotics do not readily gain access to the periplasm. Porins are trimeric proteins located in the outer membrane and are largely responsible for the molecular sieve properties of this bilayer. They form water-filled channels, which allow the diffusion of hydrophilic molecules into the periplasm; large antibiotics are excluded from this compartment (1Benz R. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers, Amsterdam1994: 397-423Google Scholar, 2Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Google Scholar). The maximum size of a solute molecule that can permeate the pores defines a value termed the molecular mass exclusion limit. Solutes lower in molecular mass than this value are considered to diffuse through porins into the periplasm; solutes whose molecular mass exceed the value of the exclusion limit are apparently impeded in their passage. The variety of porins and their exclusion limits differ from one bacterial genus to another (3Jeanteur D. Lakey J.H. Pattus F. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers, Amsterdam1994: 363-380Google Scholar). Hib 1The abbreviations used are: Hib, H. influenzae type b; mAb, monoclonal antibody; PBS, phosphate-buffered saline; Hi, H. influenzae; PAGE, polyacrylamide gel electrophoresis; nS, nanosiemen(s). is an encapsulated Gram-negative bacterium that until recently was the leading cause of meningitis in infants under 18 months. The most abundant protein in the outer membrane of Hib is porin, encoded by the ompP2 gene. Sequencing of the ompP2 gene (4Hansen E.J. Hasemann C. Clausell A. Capra J.D. Orth K. Moomaw C.R. Slaughter C.A. Latimer J.L. Miller E.E. Infect. Immun. 1989; 57: 1100-1107Google Scholar, 5Munson Jr., R. Tolan Jr., R.W. Infect. Immun. 1989; 57: 88-94Google Scholar) revealed an open reading frame for a signal sequence of 20 amino acids followed by 341 amino acids of the mature protein. Whereas the outer membrane ofEscherichia coli contains at least three proteins (OmpF, OmpC, and PhoE) that are diffusion channels (6Nikaido H. Mol. Microbiol. 1992; 6: 42-435Google Scholar), only one of the six major outer membrane proteins from Hib apparently has channel activity. Hib porin has a molecular mass exclusion limit of 1400 Da (7Vachon V. Lyew D.J. Coulton J.W. J. Bacteriol. 1985; 162: 918-924Google Scholar), considerably larger than the value of 600 Da (6Nikaido H. Mol. Microbiol. 1992; 6: 42-435Google Scholar) for the pore formed by OmpF of E. coli. Significant advances in our understanding of porin functions derive from the crystal structures of Rhodobacter capsulatus porin (8Weiss M.S. Abele U. Weckesser J. Welte W. Schiltz E. Schulz G.E. Science. 1991; 254: 1627-1630Google Scholar), Rhodopseudomonas blastica porin (9Kreusch A. Neubuser A. Schiltz E. Weckesser J. Schulz G.E. Protein Sci. 1994; 3: 58-63Google Scholar), and E. coli OmpF, PhoE (10Cowan S.W. Schirmer T. Rummel G. Steiert M. Ghosh R. Pauptit R.A. Jansonius J.N. Rosenbusch J.P. Nature. 1992; 358: 727-733Google Scholar), and LamB (11Schirmer T. Keller T.A. Wang Y.-F. Rosenbusch J.P. Science. 1995; 267: 512-514Google Scholar) porins. From these analyses at atomic resolution, the folding pattern of bacterial porins was demonstrated to be 16 (or 18, in the case of specific porins) anti-parallel β strands that traverse the outer membrane and loops that connect the β strands on both sides of the membrane. The whole structure forms a β barrel. The connecting loops on the extracellular surface are longer than the turns on the periplasmic surface. In all five porin structures, loop 3 folds back into the channel and produces a constriction of the channel. This structural feature is considered to determine pore sizes and thus the molecular mass exclusion limits of porins. Based on parameters of hydrophilicity and amphiphilicity, we exploited computer-assisted algorithms to generate a model for the secondary structure of Hib porin (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar). Even though the amino acid sequences of porin from nontypeable Haemophilus did not show extensive homology to any known porins (3Jeanteur D. Lakey J.H. Pattus F. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Science Publishers, Amsterdam1994: 363-380Google Scholar), our model of Hib porin is in agreement with an emerging consensus for the channel-forming motif of porins. We predicted 16 anti-parallel β strands that traverse the outer membrane and eight long loops that connect the β strands on one side of the membrane. Experiments that used a panel of nine mAbs against Hib porin (13Hansen E.J. Pelzel S.E. Orth K. Moomaw C.R. Radoff J.D. Slaughter C.A. Infect. Immun. 1989; 57: 3270-3275Google Scholar, 14Srikumar R. Chin A.C. Vachon V. Richardson C.D. Ratcliffe M.J.H. Saarinen L. Käyhty H. Mäkelä P.H. Coulton J.W. Mol. Microbiol. 1992; 6: 665-676Google Scholar) supported our computer-assisted predictions of secondary structure and allowed for orientation of the Hib porin model; the eight long, connecting loops were assigned to the extracellular surface. These studies also identified two surface-exposed regions in Hib porin, amino acids 162–172 and amino acids 318–325, which were assigned to the fourth loop (loop 4) and to the eighth loop (loop 8) in our secondary structure model. Two regions between amino acids 112–126 and 148–153 were buried or inaccessible at the surface of the outer membrane. We proposed (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar) that residues 112–126 contribute to the third loop (loop 3) of Hib porin and that the inaccessibility of these residues at the cell surface was due to loop 3's forming a constriction inside the pore. This paper describes experimental strategies for isolation of an Haemophilus influenzae strain that is deleted for the ompP2 gene and the construction of mutant Hib porins by site-directed mutagenesis. Furthermore, we propose a homology-based model of Hib porin that provides the structural framework for our discussion of the channel-forming activity of these mutant Hib porins. The strains and plasmids used in this study are listed in TableI. Nontypeable H. influenzae (Hi) strain DB117 is a recombination-deficient derivative from parent Hi strain KW20.Haemophilus strains were grown routinely on chocolate agar plates (36 g·liter−1 GC base (Difco), 10 g·liter−1 hemoglobin, and 20 ml·liter−1Vitox supplements (Oxoid)) containing 150 μg·ml−1 of bacitracin. Liquid cultures of Haemophilus strains were propagated in brain heart infusion (Oxoid) broth supplemented with hemin (10 μg·ml−1) and NAD+ (10 μg·ml−1); this medium is designated supplemented brain heart infusion. Media for E. coli strains have been described (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Antibiotic concentrations used for selection of chromosomal or plasmid markers after transformation ofHaemophilus and E. coli were 20 μg·ml−1 of kanamycin and 10 μg·ml−1of tetracycline. For selection of plasmid-encoded resistance inE. coli, ampicillin concentration was 100 μg·ml−1.Table IBacterial strains, phages, and plasmids used in this studyStrain, phage, or plasmidRelevant characteristicsSource or referenceE. coli strainsDH5αsupE44ΔlacU169(φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1Life Technologies, Inc.CJ236dut1 ung1 thi-1 relA1/pCJ105(cam rF′)Bio-RadMV1190Δ(lac-proAB) thi supEΔ(srl-recA)306∷Tn10(tet r) F′[traD36 proAB lac1 q lacZΔM15]Bio-RadHaemophilus strainsATCC9795Wild type Hib subtype 1H ompP2 +(7)KW20Wild type Hi Rd ompP2 + rec-1 +(30Alexander H. Leidy G. J. Exp. Med. 1951; 93: 345-359Google Scholar)DB117KW20 rec-1(31Setlow J.K. Boling M.E. Beattie K.L. Kimball R.F. J. Mol. Biol. 1972; 68: 361-378Google Scholar)DL42Wild type Hib subtype 1H ompP2 + rec-1 +(4)DL42/2F4−DL42ompP2(24)RSFA21KW20 ΔompP2 kan rThis studyRS01RSFA21 containing pEJH39-1-35This studyRS03 to RS08RSFA21 containing pRS03 to pRS08This studyPhageM13K07M13 carrying a mutation in gene IIBio-RadPlasmidspBluescript SK(−)Phagemid bla +StratagenepUC-CIpACYC184 Ω(EcoRI∷EcoRI pUC4K 1.1-kb kan r(32Vieira J. Messing J. Gene ( Amst. ). 1982; 19: 259-268Google Scholar)pEJH39-1-35pGB103 Ω(PstI∷EcoRI-PstI DL42 2.5-kbompP2 +)ColE1 Hi Rep(24)pFFA02pBluescript SK(−) Ω(PvuII∷PvuII-SspI pEJH39-1-35 (1-kb sequences coding for mature Hib porin)This studypFFA03 to pFFA08pFFA02 carrying mutations in sequences coding for mature Hib porinThis studypRS02pEJH39-1-35 Ω(PvuII-MluI∷PvuII-SalI-MluI adaptor)This studypRS03 to pRS08pEJH39-1-35 carrying mutations in sequences coding for mature Hib porinThis studypRS21pRS02 Ω(SalI∷SalI pUC-CI 1.1-kb kan r)This study Open table in a new tab Restriction endonuclease digestions, ligations, and DNA manipulations were performed as described by Sambrook et al.(15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). E. coli strain DH5α or Hi strain DB117 were used as hosts for large scale isolation of plasmid DNA using the plasmid maxi kit from QIAGEN. DNA was extracted from agarose gels using the GeneClean kit (Bio 101). To transform E. coli, cells were made competent with calcium chloride (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Haemophilusstrains were made competent for DNA uptake using calcium chloride; alternatively, Haemophilus strains were induced to be naturally competent (16Barcak G.J. Chandler M.S. Redfield R.J. Tomb J.-F. Methods Enzymol. 1991; 204: 321-342Google Scholar). The entire Hib porin gene (ompP2) together with upstream sequences (1.1 kb at the 5′-end of the gene) and downstream sequences (0.37 kb at the 3′-end of the gene) are contained in the shuttle vector pEJH39-1-35 as an EcoRI-PstI fragment. This plasmid was linearized with PvuII and then subjected to partial digestion with MluI. The digestion products were electrophoresed on an agarose gel; the 11.6-kbPvuII-MluI DNA fragment was isolated. A double-stranded DNA adaptor with PvuII and MluI ends and containing an internal SalI site (underlined) was constructed by annealing two single-stranded oligonucleotides: 5′-CTGGTCGAC A-3′ and 5′-CG CGT GTCGAC CAG-3′. Ligation of the adaptor to the 11.6-kb DNA fragment therefore introduced a novel SalI site. The ligation mixture was used to transform Hi strain DB117; selection was for tetracycline resistance. Plasmid DNA was isolated from transformants and was designated pRS02. Plasmid pRS02 was deleted for 98% of the coding sequences for Hib porin, and it retained sequences flanking the excised porin gene. To overcome SalI site modification in Hi strain DB117, pRS02 was used to transform E. coli strain DH5α and plasmid DNA was re-isolated. E. coli-passaged pRS02 was digested with SalI and ligated to a 1.1-kb SalI-restricted DNA fragment containing a kanamycin resistance gene derived from transposon Tn903. The ligation mixture was used to transform E. coli strain DH5α with selection for both tetracycline and kanamycin resistances. Plasmid pRS21 harbors the kanamycin resistance cassette with its own promoter in the same orientation as the Hib ompP2 promoter. Plasmid pRS21, digested to completion with BamHI, was used to transform naturally competent Hi strain KW20; selection was for kanamycin resistance. Transformants were screened for absence of porin in outer membrane preparations. From Hi strains that were missing porin, chromosomal DNA was subjected to Southern blotting using as probes the ompP2 gene and the kanamycin resistance gene. Genomic DNA was isolated from Haemophilus strains by a microscale procedure (16Barcak G.J. Chandler M.S. Redfield R.J. Tomb J.-F. Methods Enzymol. 1991; 204: 321-342Google Scholar). DNA restriction fragments were separated electrophoretically in 0.7% agarose gels containing TAE (0.01m Tris acetate plus 0.01 m EDTA) buffer and transferred to Nytran hybridization membranes (Schleicher & Schuell). Enzymes and the digoxigenin DNA labeling kit (The Genius system) for Southern hybridization were obtained from Boehringer Mannheim. Southern hybridizations were carried out with digoxigenin-labeled probes at 25 ng·ml−1 of hybridization solution. Hybridizations were at 68 °C and a final wash with 0.1 × SSC containing 0.1% SDS at 68 °C for 15 min was included to reduce background. Plasmid pEJH39-1-35 was digested with PvuII andSspI. The 1.1-kb PvuII-SspI DNA fragment was isolated; it contained only the coding sequences for the mature form of Hib porin. This DNA fragment was ligated to the 2.5-kbPvuII fragment from pBluescript SK(−) and used to transformE. coli strain DH5α; selection was for ampicillin resistance. A recombinant plasmid in which thePvuII-SspI DNA fragment from pEJH39-1-35 was cloned in the same orientation as the lacZ gene in pBluescript SK(−) was designated pFFA02. It was used for mutagenesis experiments. Site-directed deletions in Hib porin were constructed with the Muta-Gene phagemid in vitromutagenesis kit, version 2 (Bio-Rad), using single-stranded mutagenic oligonucleotides. Two regions in Hib porin were selected for mutagenesis (Fig. 1), and they correspond to loop 3 and to loop 4 in the proposed topological model for Hib porin (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar). Plasmid pFFA02 was used to transform E. coli strain CJ236. After infection of a transformant with helper phage M13K07, uracil-containing single-stranded phagemid DNA was isolated and used for the mutagenesis. Two mutagenic oligonucleotides created deletions of 6 amino acids and 12 amino acids in loop 3: 5′-ACA AGT GCA GAA GAT AAA GAGCTC GAC TAT ATT CCT ACT AGT-3′ and 5′-GAT GGC ATA ACA AGT GCA GAGCTC CCT ACT AGT GGT AAT ACC-3′, respectively. Two mutagenic oligonucleotides created deletions of 6 amino acids and 12 amino acids in loop 4: 5′-AAG CGT GAG GGT GCA AAAGAGCTC AAG GCT GGT GAA GTA CGT-3′ and 5′-TTA GCA CAA AAG CGT GAG GAGCTC GAA GTA CGT ATA GGT GAA-3′, respectively. Each mutagenic oligonucleotide also incorporated a unique SacI site (underlined). After in vitromutagenesis, the reactions were used to transform E. colistrain MV1190. Plasmids were isolated from transformants and mapped using restriction enzyme digestions. All candidates for each mutation were confirmed by DNA sequencing of plasmid DNA. Plasmids (pFFA05 to pFFA08, Table I) were digested to completion withPvuII and MluI, and the 1.0-kbPvuII-MluI DNA fragments containing the mutations were isolated. These fragments were ligated to the 11.6-kbPvuII-MluI DNA fragment from pEJH39–1-35 and used to transform competent Hi strain RSFA21, with selection for kanamycin and tetracycline resistances. Plasmid pFFA02 contains unique sites for the restriction enzymes SpeI and SnaBI within the coding sequences for the mature form of Hib porin. Restriction sites for SpeI and SnaBI are found within sequences encoding amino acids of loop 3 and loop 4 of Hib porin, respectively. These sites were used to construct in-frame insertions of the C3 epitope of the VP1 protein of poliovirus (17Charbit A.J. Ronco J. Michel V. Werts C. Hofnung M. J. Bacteriol. 1991; 173: 262-275Google Scholar, 18Moeck G.S. Fazly Bazzaz B.S. Gras M.F. Ravi T.S. Ratcliffe M.J.H. Coulton J.W. J. Bacteriol. 1994; 176: 4250-4259Google Scholar) in Hib porin. Plasmid pFFA02, digested with either SpeI orSnaBI, was ligated to double-stranded oligonucleotides. Linker SpeI-C3 was constructed by annealing two single-stranded oligonucleotides: 5′-CT AGT GAT AAC CCG GCG TCG ACC ACT AAC AAG GAT AAG A-3′ and 5′-CT AGT CTT ATC CTT GTT AGT GGT CGA CGC CGG GTT ATC A-3′. Linker SnaBI-C3 was constructed by annealing two single-stranded oligonucleotides: 5′-GT GAT AAC CCG GCG TCG ACC ACT AAC AAG GAT AAG C-3′ and 5′-G CTT ATC CTT GTT AGT GGT CGA CGC CGG GTT ATC AC-3′. Linkers SpeI-C3 and SnaBI-C3 created the codons for the C3 epitope in-frame to the codons for Hib porin. The ligation mixtures were used to transform E. coli strain DH5α with selection for ampicillin resistance. Plasmids from transformants were isolated, mapped, and sequenced. Plasmids pFFA03 and pFFA04 were digested to completion with PvuII andMluI; the isolated mutagenized fragment was ligated to the 11.6-kb PvuII-MluI DNA from pEJH39-1-35. For dideoxy sequencing (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), the T7 sequencing kit (Pharmacia Biotech Inc.) was used. One oligonucleotide was adequate for all DNA sequencing across regions encoding amino acids corresponding to loop 3 and loop 4: 5′-T(584) GAA GTA AAA CTT GGT CGT (603)G-3′; the numbers correspond to the published sequence of the Hib porin gene (5Munson Jr., R. Tolan Jr., R.W. Infect. Immun. 1989; 57: 88-94Google Scholar). Outer membrane vesicles were obtained by treatment of cells with Tris-lysozyme-EDTA (19Hantke K. Mol. Gen. Genet. 1981; 182: 288-292Google Scholar). Samples of vesicles containing outer membrane proteins were suspended in electrophoresis sample buffer with 2% (w/v) SDS, heated for 5 min at 100 °C, and run on 10% (w/v) polyacrylamide gels. For immunoblotting, outer membrane proteins were subjected to SDS-PAGE, transferred to nitrocellulose paper (Schleicher & Schuell), and probed with monoclonal antibodies or polyclonal antibodies (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar) that were diluted 1/2000. Bacteria from mid-log phase cultures were washed in PBS and suspended in PBS to 2 × 109cells·ml−1. Affinity-purified anti-Hib porin mouse mAbs (14Srikumar R. Chin A.C. Vachon V. Richardson C.D. Ratcliffe M.J.H. Saarinen L. Käyhty H. Mäkelä P.H. Coulton J.W. Mol. Microbiol. 1992; 6: 665-676Google Scholar) or anti-peptide (C3 epitope of poliovirus) rabbit hyper-immune serum (18Moeck G.S. Fazly Bazzaz B.S. Gras M.F. Ravi T.S. Ratcliffe M.J.H. Coulton J.W. J. Bacteriol. 1994; 176: 4250-4259Google Scholar) at 1/100 dilution was mixed with 2 × 108cells and incubated at room temperature for 1 h. Bacteria were pelleted, washed, and incubated at room temperature for 1 h with anti-mouse or anti-rabbit immunoglobulins conjugated to fluorescein. Bacteria were diluted ten-fold in PBS and analyzed for green fluorescence intensity using a FACScan flow cytometer (Becton Dickinson) with LysisII software. For each sample, 104cells were analyzed. Planar bilayer studies were executed as described previously (20Dahan D. Vachon V. Laprade R. Coulton J.W. Biochim. Biophys. Acta. 1994; 1189: 204-211Google Scholar) but with the following change in instrumentation: an Axopatch-1D amplifier (Axon Instruments) was used to measure the ionic current across the membrane. Our proposed model for the secondary structure of Hib porin (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar) is consistent with the consensus fold that is derived from five high resolution x-ray structures of nonspecific bacterial porins. We therefore explored possibilities to map the sequence of Hib porin onto the homology-derived scaffold of these structures. Using Hib porin as a target for the FASTA sequence alignment program, only one significantly scoring sequence homolog was found in SWISSPROT, identifying OmpF from E. coli as the closest currently known sequence relative. Hib porin and OmpF are 341 and 340 amino acids, respectively; identities are found at 58 amino acids (17% of the sequence), and there is no extended clustering of amino acid identities. This initial alignment was improved as follows. (i) A family alignment of structurally known porins (R. capsulatus porin and E. coli OmpF and PhoE) was constructed based on their three-dimensionally equivalent residues and using SUPERIMPOSE (21Diederichs K. Proteins Struct. Funct. Genet. 1995; 23: 187-195Google Scholar). (ii) The sequences of three other Hi porins were included and the alignment modified so as to place insertions, deletions, and other variable regions of those sequences primarily within loops and turns, while maximizing the number of identities with OmpF and with other members of the structure-based family alignment. A similar strategy to that described here was successfully employed to identify a molecular replacement model, thereby solving the x-ray structure of porin from Paracoccus denitrificans (22Hirsch A. Breed J. Saxena K. Richter O.-M.H. Ludwig B. Diederichs K. Welte W. FEBS Lett. 1997; 404: 208-210Google Scholar). Atomic coordinates of OmpF (10Cowan S.W. Schirmer T. Rummel G. Steiert M. Ghosh R. Pauptit R.A. Jansonius J.N. Rosenbusch J.P. Nature. 1992; 358: 727-733Google Scholar) were obtained from the Protein Data Bank (Brookhaven National Laboratory, Upton, NY), entry code 1OMF. Using the Swiss-Model Server (23Peitsch M.C. Bio/Technology. 1995; 13: 658-660Google Scholar) 2The Swiss-Model server is accessible on the World-wide Web (URL: http://expasy.hcuge.ch). and the alignment shown in Fig. 1, a homology model for porin of Hib was generated (Fig. 2). The model maps the sequence of Hib porin onto the structural scaffold of the porin family. This model is not meant to represent the detailed conformation of the loops but rather to indicate the length and position of loops within the overall β-barrel framework. Highlighted in the stereo figure is the modeled Cα backbone of sequences that are proposed to form loop 3. The length of loop 3 for Hib porin is apparently shorter than the length of loop 3 for OmpF of E. coli. While there is currently no high resolution structure for Hib porin and therefore no direct evidence for the position of loop 3 within the pore, observations on the differences in the lengths of loops 3 are in agreement with the measured molecular mass exclusion limits for OmpF (600 Da) and Hib porin (1400 Da). The molecular model predicts that deletions of 6 amino acids or of 12 amino acids in loop 3 of Hib porin might alter properties of ion conductivity. The experiments that follow were designed in part to test this prediction. Plasmid pRS21 was constructed by replacing the sequences coding for the mature form of Hib porin in pEJH39-1-35 with a kanamycin resistance cassette. The kanamycin resistance cassette in this construct was flanked by sequences upstream and downstream of the Hib ompP2 gene. ABamHI fragment derived from pRS21 and containing the kanamycin resistance cassette was used to transform nontypeable Hi; selection was for kanamycin resistance. Hi strain RSFA21, a representative transformant, was subjected to SDS-PAGE analysis and Western blotting of outer membrane preparations. Gain of kanamycin resistance coincided with loss of porin (Fig. 4, lane 4). Analyses by Southern blotting (data not shown) confirmed the replacement of the chromosomal copy of the Hi porin gene with the kanamycin resistance cassette. The Hib ompP2 probe hybridized to a chromosomal DNA fragment from wild type Hi strain KW20 but not to any chromosomal DNA fragment from the porin deletion strain. Conversely, the kanamycin resistance gene probe hybridized to a chromosomal DNA fragment from the porin deletion Hi strain RSFA21 but not to any chromosomal DNA fragment from the wild type Hi strain. We chose to introduce deletions and insertions into the proposed loop 3 and the proposed loop 4 of Hib porin. The amino acids deleted were those that correspond to epitopes (12Srikumar R. Dahan D. Gras M.F. Ratcliffe M.J.H. van Alphen L. Coulton J.W. J. Bacteriol. 1992; 174: 4007-4016Google Scholar) recognized by the anti-Hib porin mAbs POR.1 (loop 3) and POR.4 (loop 4). The gene sequences of the mutations created by site-directed mutagenesis and the resulting mutant amino acid sequences of Hib porins are shown in Fig. 3. Since all four oligonucleotides used for construction of the deletions contained 6 nucleotides that introduced a SacI site, each deletion protein gained 2 additional amino acids, Glu and Leu. For example, to create a net deletion of 6 amino acids in loop 3 as encoded by pRS05, 8 amino acids were removed; they were replaced by 2 amino acids, Glu and Leu. The ends of the linkers SpeI-C3 and SnaBI-C3 were compatible for insertion at SpeI and SnaBI restriction sites, respectively. There were net insertions of 13 amino acids and 12 amino acids in the loop 3-C3 and loop 4-C3 mutant proteins. Plasmids containing the cloned Hib porin gene (pEJH39-1-35) or containing deletions and insertions in Hib ompP2 (pRS03 to pRS08) were used to transform the porin deletion Hi strain RSFA21. Proteins expressed in the transformants (RS01 and RS03 to RS08) were initially detected by SDS-PAGE of total cell lysates. To determine the cellular location of the mutant proteins, outer membrane vesicles were prepared, run on polyacrylamide gels and stained with Coomassie Blue (Fig. 4). Deletions in loop 3 (Fig. 4, lanes 6 and 7) or C3 epitope insertion in loop 3 of Hib porin (Fig. 4, lane 10) altered the mobility of the mutant porins when compared with the migration of wild type Hib porin. Deletions in loop 4 (Fig. 4, lanes 8 and 9) or C3 insertion in loop 4 (Fig. 4, lane 11) did not alter the mobility of the mutant porins when compared with the wild type Hib porin. Three anti-Hib porin mAbs (POR.1, POR.4, and POR.6; Ref. 14Srikumar R. Chin A.C. Vachon V. Richardson C.D. Ratcliffe M.J.H. Saarinen L. Käyhty H. Mäkelä P.H. Coulton J.W. Mol. Microbiol. 1992; 6: 665-676Google Scholar) and a polyclonal antibody against the C3 epitope of poliovirus designated 928 (18Moeck G.S. Fazly Bazzaz B.S. Gras" @default.
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