RESUMO
The outer membrane protein PhoE of members of the family Enterobacteriaceae consists of conserved membrane-spanning segments and hypervariable surface-exposed regions. Two oligonucleotides based on DNA sequences encoding two different cell-surface-exposed regions of the Escherichia coli K12 PhoE protein were tested for their specificity in polymerase chain reactions. They reacted with all strains of the species E. coli/Shigella tested, except for strain S. boydii serovar 13, which is known to represent a different DNA-relatedness group. The probes did not react with any other Enterobacteriaceae tested, including strains of Escherichia blattae, Escherichia hermanii, Escherichia vulneris and Escherichia adecarboxylata, except for an Escherichia fergusonnii strain, which is most closely related to E. coli. Therefore, the primer couple showed a high degree of species-specificity. In addition, a second primer couple based on two conserved regions of the phoE genes was tested. This primer couple recognized a broad group of closely related enteric bacteria including Salmonella and Shigella.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/isolamento & purificação , Escherichia/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Shigella/isolamento & purificação , Sequência de Bases , Sondas de DNA/genética , Eletroforese em Gel de Ágar , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Escherichia/genética , Escherichia coli/genética , Técnicas In Vitro , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Shigella/genéticaRESUMO
PhoE protein is a major outer membrane protein of Escherichia coli. The polypeptide spans the membrane 16 times, thereby exposing 8 regions at the cell surface. Insertions in these regions did not affect the biogenesis of the protein. Therefore, we considered the possibility of using PhoE as a vector for the exposure of foreign antigenic determinants at the cell surface, with the ultimate goal of constructing new (live oral) vaccines. Via recombinant DNA techniques, B-cell epitopes of VP1 protein of foot-and-mouth-disease virus were inserted in the exposed regions of PhoE. The inserted epitopes were antigenic and immunogenic in the PhoE-associated conformation. Guinea pigs, immunized with such a hybrid protein were protected against viral challenge. Similarly, a T-cell epitope of the 65 kDa heat-shock protein of Mycobacterium tuberculosis remained antigenic and immunogenic in the PhoE-associated conformation, although recognition by the cells of the immune system was dependent on the amino acids, flanking the epitope. When the amino acid sequences of the PhoE proteins of different members of the family of Enterobacteriaceae are compared, the cell surface-exposed regions are hypervariable. Therefore, we considered the possibility that the DNA segments encoding these regions are species-specific. By using synthetic oligonucleotides corresponding to such DNA segments, primer couples for the specific detection and identification of different enterobacterial species, including Salmonella, by polymerase chain reactions have been developed.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Sondas de DNA , Escherichia coli/genética , Escherichia coli/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Linfócitos B/imunologia , Vacinas Bacterianas/isolamento & purificação , Sequência de Bases , DNA Bacteriano/genética , Epitopos/genética , Cobaias , Dados de Sequência Molecular , Porinas , Linfócitos T/imunologiaRESUMO
In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Sondas de DNA , Salmonella typhimurium/genética , Sequência de Aminoácidos , Autorradiografia , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Porinas , Mapeamento por Restrição , Salmonella/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Citrobacter freundii/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Porinas , Mapeamento por Restrição , Salmonella/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
On the basis of the idea that DNA sequences encoding cell surface-exposed regions of outer membrane proteins are genus or species specific, two oligonucleotide probes which were based on the PhoE protein of Klebsiella pneumoniae were evaluated. In slot blot hybridizations and in polymerase chain reactions, no cross-hybridizations were observed with non-Klebsiella strains. When the probes were tested on 75 different K-antigen reference Klebsiella strains, 16 strains were not recognized although they did produce PhoE protein under phosphate starvation. To determine whether these 16 strains belong to (a) different species, the reference strains were also tested for the ability to produce indole and to grow at 10 degrees C and their whole-cell fatty acid patterns were analyzed by gas chromatography. A strong correlation was observed among (i) reaction with the probes, (ii) the inability to produce indole, (iii) the inability to grow at 10 degrees C, and (iv) the presence of the hydroxylated fatty acid C14:0-2OH. From these results we conclude that the two oligonucleotides are specific for the species K. pneumoniae. Furthermore, analysis of fatty acid patterns appears to be a useful tool to distinguish K. pneumoniae from other Klebsiella species.
Assuntos
DNA Bacteriano , Ácidos Graxos/análise , Klebsiella pneumoniae/classificação , Sequência de Bases , Cromatografia Gasosa , Eletroforese em Gel de Poliacrilamida , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.
