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1.
Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b. 总被引:9,自引:13,他引:9
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E J Hansen F R Gonzales N R Chamberlain M V Norgard E E Miller L D Cope S E Pelzel B Gaddy A Clausell 《Infection and immunity》1988,56(10):2709-2716
The major outer membrane protein (P2) of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 37,000 to 40,000 has been previously shown to function as a porin and also as a target for antibodies protective against experimental Hib disease. The gene encoding the Hib P2 protein was cloned by using a shuttle vector capable of replication in both Escherichia coli and H. influenzae. The amino acid sequence of the amino terminus of the Hib P2 protein was determined and used to design an oligonucleotide probe corresponding to the first 20 amino acids of this protein. This oligonucleotide probe was used to identify Hib chromosomal DNA fragments containing the Hib P2 gene. These DNA fragments were ligated into the plasmid vector pGJB103 and then used to transform a rec-1 mutant of H. influenzae Rd. Recombinant clones expressing the Hib P2 protein were identified in a colony blot-radioimmunoassay by using a monoclonal antibody specific for a surface epitope of the Hib P2 protein. The gene encoding this Hib protein was present on a 10-kilobase Hib DNA insert in the recombinant plasmid. Transformation experiments involving the recombinant plasmid suggested that unregulated synthesis of Hib P2 is a lethal event in E. coli. The recombinant Hib P2 protein was exposed on the surface of the recombinant H. influenzae strain. This recombinant strain was used to develop a system for detecting polyclonal serum antibodies directed against surface determinants of the Hib P2 protein. The availability of the gene encoding the Hib P2 protein should facilitate investigation of both the immunogenicity and the structure-function relationship(s) of this major outer membrane protein. 相似文献
2.
The gene encoding protein D (hpd) is highly conserved among Haemophilus influenzae type b and nontypeable strains. 总被引:1,自引:0,他引:1
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The molecular conservation of a surface-exposed lipoprotein, protein D, of Haemophilus influenzae was studied by cloning and sequencing of the gene encoding protein D from three encapsulated type b strains and three nontypeable strains of H. influenzae. These nucleotide sequences were analyzed with previously reported sequences from one type b strain and one nontypeable strain. The nucleotide sequences and the deduced amino acid sequences for protein D were highly conserved. The deduced amino acid sequence (364 amino acids) of protein D from six strains differed only in two amino acids near the C-terminal end. The remaining two strains, one type b and one nontypeable, differed from the consensus sequence in 7 amino acids each. Protein D is 64 and 36% identical and 77 and 56% similar to the glycerophosphodiester phosphodiesterases (GlpQ) of Escherichia coli and Bacillus subtilis. 相似文献
3.
Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b. 总被引:11,自引:16,他引:11
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The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
4.
Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene. 总被引:5,自引:13,他引:5
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By using antiserum against Haemophilus influenzae type b (Hib) strain M43p+ denatured pilin, we screened a genomic library of Hib strain M43p+ and identified a clone that expressed pilin, but not assembled pili, on its surface. Southern blot analysis revealed the presence of one structural gene, which was also present in strain M42p-, a nonpiliated variant. Five exonuclease III deletion mutants, two of which had deletions that extended into the structural gene and failed to express pilin, were used to obtain the nucleotide sequence of the structural gene. The amino acid sequence of the open reading frame agrees with 38 of 40 amino acids from the published sequence of purified Hib M43p+ pilin. The pilin gene coded for a mature protein of 193 amino acids, with a calculated molecular mass of 21,101 daltons. Comparison of the Hib M43p+ pilin amino acid sequence with those of pilins of other bacteria revealed strong conservation of amino- and carboxy-terminal regions in M43p+ and Escherichia coli F17, type 1C, and several members of the P pili family, as well as Klebsiella pneumoniae type 3 MR/K, Bordetella pertussis serotype 2, and Serratia marcescens US46 fimbriae. 相似文献
5.
Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum. 总被引:14,自引:9,他引:5
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A hemagglutinin with an M(r) of 67,000 (pMGA) from Mycoplasma gallisepticum S6 was purified by using monoclonal antibody affinity chromatography. Purified pMGA was treated with a number of enzymes, the resultant peptides were purified, and their amino acid sequence was determined by using an Applied Biosystems (model 471A) protein sequencer. The DNA sequence encoding two peptides was used to dictate the sequences of synthetic oligonucleotides which were used to screen a library of EcoRI-cut M. gallisepticum DNA in pUC18. A clone reactive to both probes was isolated and found to contain a recombinant insert of 10 kb. The clone was mapped by using restriction endonucleases and fragments subcloned into pUC18 for DNA sequencing. Analysis of part of the DNA sequence revealed an open reading frame containing 1,941 nucleotides which encoded 647 amino acids. The amino terminus was preceded by a putative leader sequence of 25 amino acids. A promoter region preceding the putative start codon GUG was also located. This gene would encode a mature protein of 67,660 Da. There were a number of differences between the predicted amino acid sequence and that determined by direct peptide sequencing. Also, two tryptic peptides of pMGA were not found in the DNA sequence. This suggested that the cloned gene did not encode pMGA but did encode a homolog (pMGA1.2). Furthermore, downstream of pMGA1.2 was a region of DNA encoding a leader sequence followed by an amino acid sequence with high homology to that encoded by the pMGA1.2 gene. The presence within M. gallisepticum of a family of pMGA genes is inferred from the DNA sequence and Southern transfer data. A possible role for this gene family in immune evasion is discussed. 相似文献
6.
Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen. 总被引:3,自引:1,他引:3
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R Ahrens M Ott A Ritter H Hoschützky T Bühler F Lottspeich G J Boulnois K Jann J Hacker 《Infection and immunity》1993,61(6):2505-2512
The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells. 相似文献
7.
The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli 总被引:11,自引:0,他引:11
A M Hamers H J Pel G A Willshaw J G Kusters B A van der Zeijst W Gaastra 《Microbial pathogenesis》1989,6(4):297-309
An oligonucleotide probe, derived from the N-terminal amino acid sequence of the CFA/I fimbrial subunit protein, was used to identify the gene encoding this protein within a cloned DNA fragment encoding CFA/I fimbriae. The gene (cfa b) was found and sequenced. Flanking it upstream was a gene (cfa a) encoding a protein of 206 amino acids and downstream a gene (cfa c) probably encoding an 85 kDa protein was found. This genetic organisation of the CFA/I operon differs from that of other fimbrial operons in Escherichia coli. All three proteins have signal peptides. The nucleotide sequence was analysed for homology with other sequences, secondary structure, ribosomal binding sites and possible promoter sequences. A region of dyad symmetry probably involved in the regulation of translation of the cfa c gene was found at the 5' end of this gene. A region of dyad symmetry was also observed within the cfa b gene. In front of the CFA/I operon part of insertion sequence IS2 was found. This IS2 sequence was found in a number of CFA/I plasmids, obtained from strains isolated from various geographic locations. The insertion of the IS2 element in the CFA/I operon therefore probably happened rather early during evolution of CFA/I producing Escherichia coli strains. 相似文献
8.
目的克隆和表达弓形虫虎源分离株(HT株)ROP5蛋白基因。方法运用RT—PCR技术从弓形虫HT株中扩增出ROP5基因,将其克隆人T载体中进行测序和分析.并将目的基因亚克隆到大肠杆菌表达载体pET28a中进行诱导表达。结果该基因全长1650bp,编码549个氨基酸。其中前24个氨基酸构成信号肽序列。与GenBank中报道的RH株相比,有12个核苷酸有差异,导致7个氨基酸发生改变,两者核苷酸和推导氨基酸序列的同源性分别为99.2%和98.9%。转化重组质粒pETROP5的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出相对分子质量为64800的重组蛋白,并且能与弓形虫抗体发生血清学反应,表达量占菌体蛋白的15.6%。结论成功克隆和表达了弓形虫HT株ROP5蛋白基因,表达的重组蛋白具有良好的反应原性。 相似文献
9.
Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli.
An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined. The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids. The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids. The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins. As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al. (1987). The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al. (1983). FtwA contains two deletions found in comparison to the other fimbrial subunits. 相似文献
10.
Immunological properties of recombinant porin of Haemophilus influenzae type b expressed in Bacillus subtilis. 总被引:5,自引:2,他引:3
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R Srikumar D Dahan M F Gras L Saarinen H Kyhty M Sarvas L Vogel J W Coulton 《Infection and immunity》1993,61(8):3334-3341
The major surface-located, channel-forming protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M(r), 37,782). In order to generate Hib porin that is devoid of lipooligosaccharides and capsular polysaccharide, the Hib porin gene ompP2 was subcloned into a plasmid vector and recombinant Hib porin was expressed in Bacillus subtilis. Recombinant porin was produced in large quantities in B. subtilis and formed intracellular inclusion bodies. Recombinant porin was extracted from inclusion bodies and shown to be active in forming pores in synthetic black lipid membranes. However, these pores demonstrated different pore characteristics than wild-type Hib porin. Mouse hyperimmune sera against recombinant porin were generated and subjected to epitope scanning with a library of 336 overlapping synthetic hexapeptides that corresponded to the entire sequence of Hib porin. The epitope specificities of the anti-recombinant porin antibodies were similar to those of antibodies against Hib porin: selected regions near the amino terminus which include a buried loop in the native structure of Hib porin were more immunogenic than regions at the carboxy terminus. Although some mouse anti-recombinant porin antibodies mediated complement-dependent binding to Hib by polymorphonuclear leucocytes in opsonophagocytosis assays, the antibodies were not bactericidal, nor did they abrogate bacteremia in the infant rat model of infection. It was concluded that the native state of Hib porin is required for the generation of a protective immune response against the bacterium. 相似文献
11.
A Martin-Gallardo K A Fien B T Hu J F Farley R Seid P L Collins S W Hildreth P R Paradiso 《Virology》1991,184(1):428-432
A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter. Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein. Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide. The region of the F protein which reacted with a virus-neutralizing and fusion-inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E. coli and testing antibody reactivity by Western blot analysis. Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298. Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A. 相似文献
12.
The basic DNA-binding protein of the Bombyx mori nuclear polyhedrosis virus (BmNPV) was purified by HPLC and a sequence of 45 amino acids from the N-terminus was determined. There were no detectable modifications such as N-terminal blockage, glycosylation, or phosphorylation. The amino acid sequence showed high homology to the predicted amino acid sequences of the basic proteins of Autographa californica NPV (AcNPV) and Orgyia pseudotsugata NPV (OpNPV) (90 and 76%, respectively), however, the BmNPV basic protein possessed an additional sequence of 10 amino acids. A DNA fragment encoding the basic protein was identified in a BmNPV DNA library by screening for possible DNA sequences coding for the basic protein's amino acid sequence. The nucleotide sequence of the basic protein of BmNPV was more similar to that of AcNPV (97%) than to that of OpNPV (62%). Homology plot analysis of the nucleotide sequence indicates that the BmNPV basic protein internal repeat evolved very recently. 相似文献
13.
The infection of and interaction of human endothelial cells with Bartonella henselae is one of the most interesting aspects of Bartonella -associated disease. The gene encoding the 43 kDa B. henselae outer membrane protein (Omp43) that binds endothelial cells was cloned and sequenced. Sequence analysis revealed an open reading frame of 1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced amino acid sequence shows 38% identity over the entire sequence to the Brucella spp. In addition to this Omp2b porin also shows a signal sequence and peptidase cleavage site. Cleavage of the signal peptide results in a mature 380 amino acid polypeptide with a predicted molecular weight of 42 kDa. Omp43 was expressed in Escherichia coli as a fusion protein. Purified recombinant Omp43 at concentrations of 11 and 2.75 microg/ml bound to intact human umbilical vein endothelial cells. Membrane topology analysis predicts that Omp43 exists as a 16 stranded beta barrel protein, similar to that predicted for the Omp2b Brucella abortus porin. Characterization and expression of the gene encoding Omp43 should provide a tool for further investigation of the role of adherence to endothelial cells in the pathogenesis of B. henselae. 相似文献
14.
Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria. 总被引:8,自引:3,他引:5
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W J Watson J R Gilsdorf M A Tucci K W McCrea L J Forney C F Marrs 《Infection and immunity》1994,62(2):468-475
Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods. 相似文献
15.
Sequence determination and mutational analysis of the lly locus of Legionella pneumophila. 总被引:5,自引:1,他引:4
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E Wintermeyer M Flügel M Ott M Steinert U Rdest K H Mann J Hacker 《Infection and immunity》1994,62(3):1109-1117
The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus. 相似文献
16.
The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene. 相似文献
17.
Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2. 总被引:19,自引:0,他引:19
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We cloned and sequenced the gene encoding the muramidase-released protein (MRP) of a pathogenic Streptococcus suis type 2 strain to determine whether its amino acid sequence resembles that of proteins with known functions and to determine its function in virulence. The complete nucleotide sequence composing the gene and the regions flanking it was determined. The deduced amino acid sequence revealed the presence of a signal peptide at the N terminus and a cell envelope anchor at the C terminus, both of which resembled similar regions in several other surface proteins from gram-positive bacteria. The processed form of MRP has a length of 1,209 amino acids and a calculated molecular weight of 131,094. A highly repetitive region preceded the envelope anchor. The repeated units were preceded by a proline-rich stretch of amino acids (26 of 86). No overall homologies were observed between the amino acid sequence of MRP and protein sequences in the EMBL data bank. A particular region within the amino acid sequence, however, showed some similarity with the fibronectin-binding protein of Staphylococcus aureus. Binding of MRP to human fibronectin, however, could not be confirmed. 相似文献
18.
19.
The basic DNA-binding protein of the Bombyx mori nuclear polyhedrosis virus (BmNPV) was purified by HPLC and a sequence of 45 amino acids from the N-terminus was determined. There were no detectable modifications such as N-terminal blockage, glycosylation, or phosphorylation. The amino acid sequence showed high homology to the predicted amino acid sequences of the basic proteins of Autographa californica NPV (AcNPV) and Orgyia pseudotsugata NPV (OpNPV) (90 and 76%, respectively), however, the BmNPV basic protein possessed an additional sequence of 10 amino acids. A DNA fragment encoding the basic protein was identified in a BmNPV DNA library by screening for possible DNA sequences coding for the basic protein's amino acid sequence. The nucleotide sequence of the basic protein of BmNPV was more similar to that of AcNPV (97%) than to that of OpNPV (62%). Homology plot analysis of the nucleotide sequence indicates that the BmNPV basic protein internal repeat evolved very recently. 相似文献
20.
The sim gene of Escherichia coli phage P1: nucleotide sequence and purification of the processed protein 总被引:2,自引:0,他引:2
The sim gene of bacteriophage P1 causes exclusion of a superinfecting P1 phage. We determined the nucleotide sequence of a 1.9-kb DNA fragment that, in plasmids, causes Sim phenotype. There are two open reading frames within this region for proteins of 82 and 259 amino acids. A 1.3-kb fragment containing the larger open reading frame was inserted into an expression vector. Induced cells carrying the hybrid plasmid, termed pBD5, were not infected by phage P1 and produced a 24-kDa protein and, to a smaller extent, a 25-kDa protein. The 24-kDa protein was purified. Comparison of its amino-terminal amino acid sequence with the nucleotide sequence indicated that it is processed from a precursor protein by removal of a hydrophobic leader peptide of 20 amino acids. In vivo processing depends on secA gene function and is necessary for Sim interference with P1 infection. The data are discussed with respect to the function of the sim gene in superinfection exclusion. 相似文献