RESUMO
The pCloDF13 encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murecin lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF 13.
Assuntos
Proteínas de Bactérias/metabolismo , Cloacina/biossíntese , Proteínas de Escherichia coli , Sinais Direcionadores de Proteínas/metabolismo , Proteínas de Bactérias/genética , DNA Recombinante/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Isopropiltiogalactosídeo/farmacologia , Peptidoglicano/metabolismo , Plasmídeos/genética , Sinais Direcionadores de Proteínas/genéticaAssuntos
Bacteriocinas/biossíntese , Cloacina/biossíntese , Colicinas/biossíntese , Escherichia coli/metabolismo , Sequência de Aminoácidos , Cloacina/genética , Cloacina/metabolismo , Colicinas/genética , Colicinas/metabolismo , Meios de Cultura , Escherichia coli/genética , Óperon , Fosfolipases A/metabolismo , Relação Estrutura-AtividadeRESUMO
In gram-negative bacteria only few proteins are exported across both the cytoplasmic membrane and the outer membrane which forms an extra barrier for protein excretion. In this review we describe the mechanisms of production and export of two types of plasmid-encoded proteins in Escherichia coli. These proteins are the bacteriocin cloacin DF13 and the K88ab and K99 fimbrial subunits. Specific so-called helper proteins located at different positions in the cell envelope play an essential role in the export of these proteins. The genetic organization, subcellular location and functions of these helper proteins, as well as the effects of mutations and culture conditions on the export of the proteins are described. Models for the export mechanisms are presented and future application possibilities for engineering foreign protein excretion in E. coli with these export systems are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Parede Celular/metabolismo , Cloacina/biossíntese , Cloacina/genética , Cloacina/metabolismo , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Modelos Biológicos , Peso Molecular , Mutação , PlasmídeosAssuntos
Plasmídeos de Bacteriocinas , Replicação do DNA , Plasmídeos , Proteínas de Bactérias/genética , Sequência de Bases , Cloacina/biossíntese , Colicinas/biossíntese , Escherichia coli/genética , Regulação da Expressão Gênica , Genes , Mutação , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RepliconRESUMO
The nucleotide sequence of the Clo DF13 DNA region comprising the immunity gene has been determined. We also elucidated the aminoacid sequence of the 40 N-terminal and 7 C-terminal aminoacids of the purified immunity protein. From analysis of the data obtained we were able to locate the immunity gene between 11.7 and 14.5% on the Clo DF13 map, and to determine the complete aminoacid sequence of the immunity protein. It was observed that the Clo DF13 immunity gene encodes an 85 aminoacid protein and is transcribed in the same direction as the cloacin gene. These experimental data support our model, presented elsewhere, which implicates that the cloacin and immunity genes of Clo DF13 are coordinately transcribed from the cloacin promoter. We also present DNA sequence data indicating that an extra ribosome binding site precedes the immunity gene on the polycistronic mRNA. This ribosome binding site might explain the fact that in cloacinogenic cells more immunity protein than cloacin is synthesized. The comparison of the complete aminoacid sequence of the Clo DF13 immunity protein, with the aminoacid sequence data of the purified, comparable Col E3 immunity protein revealed that both proteins have extensive homologies in primary and secondary structure, although they are exchangeable only to a low extent in vivo and in vitro. It was also observed that a lysine residue was modified in immunity protein isolated from excreted bacteriocin complexes.
Assuntos
Proteínas de Bactérias/biossíntese , Bacteriocinas , Proteínas de Escherichia coli , Genes , Plasmídeos , Sequência de Aminoácidos , Sequência de Bases , Cloacina/biossíntese , Códon , Colicinas/biossíntese , Enzimas de Restrição do DNA , Escherichia coli/metabolismoRESUMO
Mitomycin C treatment of Escherichia coli K-12 cells containing the nonconjugative plasmid CloDF13 resulted in inhibition of host chromosome protein synthesis and a high rate of synthesis of two CloDF13-specified proteins whose molecular weights correspond to cloacin and immunity protein. Five molecules of immunity protein were synthesized for each cloacin DF13 molecule. Mitomycin C-treated cells containing a copy mutant of CloDF13 made three to four times as much of each protein as cells containing wild-type CloDF13. CloDF13 plasmids that contained the transposon Tn1 were isolated. Two did not induce after mitomycin C treatment, failing both to inhibit host cell synthesis and to produce the two new proteins. In minicells, they showed reduced CloDF13-specified protein synthesis and produced three Tn1-specified proteins.
