RESUMEN
OprI, a small outer membrane lipoprotein from Pseudomonas aeruginosa, can be produced in large amounts and anchored at the surface on Escherichia coli cells. A four-time repeated (His-Cys) motif was fused to the C-terminal part of OprI. After induction, E. coli cells harbouring the recombinant oprI gene became more sensitive to Cd and Co. The same cells, after IPTG induction, bound four to eight times more Cd and Cr than control cells expressing oprI alone.
Asunto(s)
Secuencias de Aminoácidos/fisiología , Proteínas Bacterianas/metabolismo , Lipoproteínas/metabolismo , Metales Pesados/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencias de Aminoácidos/genética , Proteínas Bacterianas/genética , Medios de Cultivo , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoproteínas/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The pyrE gene of Lactobacillus plantarum CCM 1904, coding for the orotate phosphoribosyl transferase involved in the pyrimidine biosynthetic pathway, was cloned in Escherichia coli and sequenced. The predicted polypeptide sequence extending over 212 amino acids (MW 22,690) was compared to those of E. coli and to those of lower eukaryotes (Saccharomyces cerevisiae, Podospora anserina, Sordaria macrospora, Dictyostelium discoideum). Important conserved stretches were revealed, implying that these proteins are closely related.
Asunto(s)
Lactobacillus/genética , Orotato Fosforribosiltransferasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes Bacterianos , Lactobacillus/enzimología , Datos de Secuencia Molecular , Plásmidos , Homología de Secuencia de Ácido NucleicoRESUMEN
To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.
Asunto(s)
Lactobacillus/genética , Plásmidos , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia de Bases , Colifagos/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Vectores Genéticos , Sistemas de Información , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos , Proteínas/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transformación BacterianaRESUMEN
The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.
