Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli

Gene 186 (1997) 113–118

Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli Hideto Tsujimoto a, Naomasa Gotoh a,*, Jun-ichi Yamagishi b, Yoshihiro Oyamada b, Takeshi Nishino a a Department of Microbiology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan b Discovery Research Laboratories II, Dainippon Pharmaceutical Co., Suita, Osaka 564, Japan Received 24 May 1996; revised 30 August 1996; accepted 31 August 1996; Received by A. Nakazawa

Abstract The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF ), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of M 35 696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A r and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl b--thiogalactopyranoside (IPTG). Keywords: Hydropathy plot; trc promoter; Homology

1. Introduction Burkholderia (formerly Pseudomonas) cepacia is recognized as a pathogen in nosocomial infections in immunocompromised patients and severe pulmonary infections in patients with cystic fibrosis ( Ederer and Matsen, 1972; Gilligan, 1991). One of the clinically important characteristics of B. cepacia is its intrinsically high resistance to structurally unrelated antimicrobial agents and disinfectants (Fass and Barnishan, 1980; Gilligan, 1991). Hydrophilic agents such as b-lactams diffuse via water-filled channels, called porin pores, through the * Corresponding author. Tel. +81 75 5954642; Fax +81 75 5832230; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); IPTG, isopropyl b--thiogalactopyranoside; kb, kilobase pair(s) or 1000 bp; kDa, kilodalton; MCS, multiple cloning site(s); M , relative r molecular mass (dimensionless); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PCR, polymerase chain reaction; Ptrc, trc promoter; OpcPO, the porin oligomer of B. cepacia; OpcP1, the major subunit protein of OpcPO; OpcP2, the minor subunit protein of OpcPO; opcP, OpcP1-encoding gene; RBS, ribosome-binding site; SDS, sodium dodecyl sulfate.

outer membranes of Gram-negative bacteria to access their targets (Nakae, 1976; Nikaido and Rosenberg, 1983; Yoshimura and Nikaido, 1985; Nikaido and Vaara, 1985; Hancock, 1987). Therefore, the characteristics of the porin pore constitute one of the significant determinants of bacterial susceptibilities to b-lactams (Nikaido, 1989). Two species of porin proteins of B. cepacia have been reported. The 81-kDa OpcPO is a porin oligomer composed of two distinct component proteins, 36-kDa OpcP1 and 27-kDa OpcP2 (Parr et al., 1987; Gotoh et al., 1994b). The other porin is the 40-kDa OpcS, which allows the penetration of chloramphenicol, but not penicillin G (Burns and Clark, 1992). The structure of OpcPO, which is composed of two distinguishable subunit proteins, is unique as compared with the ordinary porin oligomer in Gram-negative bacteria; the latter consists of a trimer of a single species of monomer protein (Nakae, 1986; Benz, 1988). Identification and characterization of genes encoding the component proteins, OpcP1 and OpcP2, of OpcPO might provide information on the structure of OpcPO. Here, we report the cloning and sequencing of the OpcP1-encoding gene (opcP) and its expression in Escherichia coli.

0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0 3 7 8- 1 1 19 ( 96 ) 0 06 9 2 -0

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2. Experimental and discussion 2.1. Isolation and sequencing of an OpcP1-encoding DNA fragment Attempts to determine the N-terminal aa sequence of the purified OpcP1 (Gotoh et al., 1994b) were unsuccessful due probably to blocking of its N-terminus by an unidentified residue. Therefore, purified OpcP1 protein was treated with 10 mg/ml of lysyl endopeptidase at 23°C for 21 h and fractionated by reversed phase liquid chromatography. N-terminal aa analysis of the digested fragments revealed the following sequences: fragment A, VNXGSSXWNQFGVQAXYXL, fragment B, LGNXGGMFNRQAFVGLXXQY, fragment C, YNLXPALGLGAAY and fragment D, QYDAXQDYLAPLXA (where X indicates an unidentified aa). Four mixed oligo primers were synthesized, based on

the aa sequences of fragments A and B. PCR using the primers II-R, 5∞-GCYTGNACNCCRAAYTGRTT3∞, and III-F, 5∞-ATGTTYAAYTAYCARGCNTT-3∞ (where Y, R and N indicate C/T, A/G and A/G/C/T, respectively) led to amplification of an approximately 650-bp fragment of the B. cepacia chromosome. The fragment was blunt-ended (to yield F650), cloned at the SmaI site on pUC18 ( Yanisch-Perron et al., 1985), and sequenced. The translated sequence of F650 contained the aa sequences of the OpcP1 lysyl endopeptidase fragments (data not shown), indicating that a portion of the opcP gene had been amplified. Additional primers V, 5∞-ACGTACTTCGCGCACCCGAT-3∞ and VI, 5∞-CGGCGTCAGGTTGTACTTTGCG-3∞ were subsequently synthesized from the nt sequence of F650, and used to amplify an approximately 400-bp internal fragment of F650. This newly amplified fragment was used to investigate the

Fig. 1. The nt sequence of the 1.64-kb chromosomal DNA containing opcP. The predicted aa sequence of OpcP1 is shown in a single letter code above the nt sequence. Some restriction sites are indicated with heavy lines below the nt sequence. Putative RBS and promoter sequences are double- and single-underlined, respectively. Convergent arrows indicate the region of the putative terminator. The dashed arrows under the nt sequence indicate both termini of F650. The dashed line under the aa sequence shows the deduced signal peptide. The arrowhead indicates the consensus signal peptidase recognition site. Four aa sequences determined from purified OpcP1 are shaded. An asterisk indicates stop codons. These sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number D63823. Methods: The nt sequence was determined by the dideoxy chain-termination method (Sanger et al., 1977) of both strands using the BcaBEST@ Dideoxy Sequencing Kit ( Takara, Kyoto, Japan).

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size of the chromosomal EcoRI fragment of B. cepacia. Southern hybridization experiment showed that a 10-kb EcoRI fragment contained the 400-bp fragment. Subsequently, the 10-kb EcoRI fragment (F10000) was cloned into a low-copy number plasmid, pMW118 (Bernardi and Bernardi, 1984) to yield pKMC004. Sequencing of the upstream and downstream regions of the F650 sequence within F10 000 revealed one complete ORF of 1086 bp, which contained the nt sequence of F650 and encoded 361 aa (Fig. 1). The putative promoter and terminator sequence were located upstream and downstream of the ORF, respectively. A consensus potential ribosome-binding site (RBS ) was located at a site 12 nt upstream from the predicted translation start codon. The translated sequence of the ORF possessed a signal-like sequence as well as a consensus signal peptidase cleavage site at its N-terminus ( Fig. 1). The M of the predicted mature protein was 35 696, which r is in agreement with the size of 36 kDa (Gotoh et al., 1994b; refer to lanes 1 in Fig. 4(A) and 4(B)) that was estimated by SDS-PAGE. Thus, the ORF was predicted to be the opcP gene. Moreover, hydropathy analysis of the predicted OpcP1 aa sequence indicated a 16-membrane-spanning structure (Fig. 2), similar to that of the E. coli OmpF porin (Cowan et al., 1992), suggesting that OpcP1 forms the diffusion pore in the

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oligomer OpcPO. Indeed, we observed that the purified OpcP1 forms a pore for several small M neutral sacchar rides, such as ribose and glucose, in the reconstituted proteoliposome (Gotoh et al., unpublished results). Furthermore, supplementation of purified OpcP2 into the reconstitution assay system reduced the permeability of the pore, suggesting that addition of OpcP2 to OpcP1 gives specificity to the pore of OpcP1. For detailed characterization, cloning of the OpcP2-encoding gene might be essential. We have been trying unsuccessfully to search for the OpcP2 on the F10 000 fragment. At present, an attempt to clone the gene is in progress, based on the determined partial aa sequences of this protein. 2.2. Sequence homology The predicted aa sequence of OpcP1 was used to conduct a homology search of the SWISS-PROT database. OpcP1 showed some homology to several other Gram-negative bacterial porins as follows: Bordetella pertussis (identity, 33%), Neisseria gonorrhoeae PI (32%), N. meningitidis class 1 (27%), class 2 (28%), class 3 (28%), N. lactamica (27%), and Comamonas acidovorans (26%). The alignment analysis of OpcP1 and B. pertussis porin showed some similar regions at the

Fig. 2. Hydropathy profiles of the predicted aa sequence of OpcP1 and that of OmpF. The Analysis Prot program included in the GeneWorks software (Intelligenetics) employs the Kyte and Doolittle algorithm ( Kyte and Doolittle, 1982) and was used to assess the hydropathy (vertical axis) of an 11-aa interval as a function of the position in the protein (N- to C-terminus; horizontal axis). Bars indicate the signal sequence (ss) and predicted membrane-spanning region of OpcP1 and the reported data of OmpF (Cowan et al., 1992).

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N-terminal half of both sequences (Fig. 3), supporting the notion that the OpcP1 precursor contains a putative signal sequence consisting of 20 aa, and that ATG at nt 361 was a potential start codon of the opcP gene. Homology between families of enteric-bacterial porins and neisserial porins shows only about 20% identity in spite of high homology (about 60–70% identity) within each family (Jeanteur et al., 1991). Thus, the low homology between OpcP1 and other porins suggests that OpcP1 is a member of an unknown family, which is different from that of enteric and neisserial porins. 2.3. Expression of opcP gene in E. coli To identify a product encoded by the predicted opcP gene, attempts were made to produce OpcP1 in E. coli cells. Immunoblot analysis using murine antiserum specific to OpcP1 (Gotoh et al., 1994b) could not detect

any proteins in E. coli JM109 [pKMC004] cells (data not shown). Therefore, a portion of F10 000 including the ORF, predicted to be the opcP gene, was subcloned downstream of the hyper-expression promoter, trc, on pTrc99A (Amann et al., 1988). Plasmid pKMC004 was partially digested with SacI, blunt-ended with T4 DNA polymerase and digested with EcoRI. The resulting 1.5-kb fragment was ligated into pTrc99A, which was digested with NcoI, blunted with T4 DNA polymerase, then digested with EcoRI. The plasmid pKMC014 thus obtained was transformed into E. coli JM109 cells. PCR screening using a set of primers V and VI identified an E. coli clone bearing pKMC014. E. coli JM109 [pKMC014] cells grown to mid-logarithmic phase were further incubated in the presence of IPTG and used for the preparation of outer membrane proteins. In immunoblot analysis of the outer membrane proteins using the mouse anti-OpcP1 antiserum, a 36-kDa protein,

Fig. 3. Alignment of predicted aa sequences of B. cepacia OpcP1 and B. pertussis porin protein (BpPor) (Li et al., 1991) using the Protein Alignment program included in the GeneWorks software (Intelligenetics). Identical residues are shown by asterisks. The protein sequences were aligned by insertion of gaps (-) to obtain maximum sequence identity.

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Fig. 4. Expression of the opcP gene in E. coli. (A) Coomassie-stained SDS-PA gel. (B) Immunoblot analysis using anti-OpcP1 murine antiserum (Gotoh et al., 1994b). Lanes: 1, B. cepacia ATCC25416; 2, IPTG-induced E. coli JM109 [pTrc99A]; 3, E. coli JM109 [pKMC014] without IPTG; 4, IPTG-induced E. coli JM109 [pKMC014]. Mobility of standard proteins is shown on the left of (A). Open and closed triangles show B. cepacia OpcP1 (36 kDa) and E. coli OmpF (37 kDa), respectively. OpcP1 detected by using anti-OpcP1 antiserum is shown by an arrow. Methods: For cultivation of the E. coli JM109 cells bearing pKMC014, Ap was added to L broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl ) at the final concentration of 100 mg/ml. Derepression of lacI q repressor on the plasmid pKMC014 was performed by addition of IPTG (to the final concentration of 10 mM ) to mid-log phase cultures. Outer membrane proteins were prepared by extraction of cell envelopes with sodium N-lauroyl sarkosinate and subjected to immunoblot assay followed by SDS-PAGE analysis as described previously (Gotoh et al., 1994a,b).

which comigrated with OpcP1 from B. cepacia ATCC25416 cells, was detected at the same position as that of OmpF in E. coli JM109 [pKMC014] cells only when IPTG was added ( Fig. 4). This finding suggested that the outer membrane porin protein OpcP1 of B. cepacia is encoded by the opcP gene.

of proteolytic fragments and in synthesizing oligo for PCR. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

References 3. Conclusions (1) The predicted OpcP1 protein precursor consisted of 361 aa containing a 20-aa signal sequence and the M of the mature protein was 35 696, which was in r agreement with the protein band of 36 kDa on SDS-PAGE. (2) Homology of the aa sequences between OpcP1 and several other Gram-negative bacterial porins suggested that OpcP1 is a pore-forming component of OpcPO. This notion was supported by similarity of the hydropathy plots of OpcP1 and OmpF. (3) The trc promoter-carrying plasmid, pTrc99A was used to express the opcP gene in E. coli. E. coli harboring pKMC014 produced a 36-kDa protein after induction with IPTG, which was recognized specifically by anti-OpcP1 antiserum. Acknowledgement The authors thank Kazutoshi Fukui and Hideko Yokoyama for their assistance in N-terminal sequencing

Amann, E., Ochs, B. and Abel, K.J. (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69, 301–315. Benz, R. (1988) Structure and function of porins from Gram-negative bacteria. Annu. Rev. Microbiol. 42, 359–393. Bernardi, A. and Bernardi, F. (1984) Complete sequence of pSC101. Nucleic Acids Res. 12, 9415–9426. Burns, J. and Clark, D.K. (1992) Salicilate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a poreforming outer membrane protein. Antimicrob. Agents Chemother. 36, 2280–2285. Cowan, S.W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R.A., Jansonius, J.N. and Rosenbusch, J.P. (1992) Crystal structures explain functional properties of two E. coli porins. Nature 358, 727–733. Ederer, G.M. and Matsen, J.M. (1972) Colonization and infection with Pseudomonas cepacia. J. Infect. Dis. 125, 613–618. Fass, R.J. and Barnishan, J. (1980) In vitro susceptibilities of nonfermentative Gram-negative bacilli other than Pseudomonas aeruginosa to 32 antimicrobial agents. Rev. Infect. Dis. 2, 841–853. Gilligan, P.H. (1991) Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4, 35–51. Gotoh, N., Itoh, N., Tsujimoto, H., Yamagishi, J., Oyamada, Y. and Nishino, T. (1994a) Isolation of OprM-deficient mutants of Pseudomonas aeruginosa by transposon insertion mutagenesis: evidence

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H. Tsujimoto et al. / Gene 186 (1997) 113–118

of involvement in multiple antibiotic resistance. FEMS Microbiol. Lett. 122, 267–274. Gotoh, N., Nagino, K., Wada, K., Tsujimoto, H. and Nishino, T. (1994b) Burkholderia (formerly Pseudomonas) cepacia porin is an oligomer composed of two component proteins. Microbiology 140, 3285–3291. Hancock, R.E.W. (1987) Role of porins in outer membrane permeability. J. Bacteriol. 169, 929–933. Jeanteur, D., Lakey, J.H. and Pattus, F. (1991) The bacterial porin superfamily: sequence alignment and structure prediction. Mol. Microbiol. 5, 2153–2164. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. Li, Z.M., Hannah, J.H., Stibitz, S., Nguyen, N.Y., Manclark, C.R. and Brennan, M.J. (1991) Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis. Mol. Microbiol. 5, 1649–1656. Nakae, T. Outer membrane of Salmonella. (1976) Isolation of protein complex that produces transmembrane channels. J. Biol. Chem. 251, 2176–2178.

Nakae, T. (1986) Outer-membrane permeability of bacteria. Crit. Rev. Microbiol. 13, 1–62. Nikaido, H. (1989) Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob. Agents Chemother. 33, 1831-1836. Nikaido, H. and Rosenberg, E.Y. (1983) Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J. Bacteriol. 153, 241–252. Nikaido, H. and Vaara, M. (1985) Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49, 1–32. Parr, T.R., Moore, R.A., Moore, L.V. and Hancock, R.E.W. (1987) Role of porins in intrinsic antibiotic resistance of Pseudomonas cepacia. Antimicrob. Agents Chemother. 31, 121–123. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119. Yoshimura, F. and Nikaido, H. (1985) The porin channels of Escherichia coli K-12. Antimicrob. Agents Chemother. 27, 84–92.