RESUMO
Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.
Assuntos
Elastase Pancreática/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Elastase Pancreática/química , SulfetosRESUMO
Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.
Assuntos
Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Lipase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Acinetobacter calcoaceticus/genética , Acinetobacter calcoaceticus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/genética , Burkholderia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Lipase/genética , Plasmídeos , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especificidade por SubstratoRESUMO
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
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.