ABSTRACT
Integrative and replicative plasmids for the expression driven by the P(43) promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5±0.2mgl(-1) and the replicative system produced 20.3±0.8mgl(-1) of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.
Subject(s)
Bacillus subtilis/genetics , DNA Replication/genetics , Interferon-gamma/genetics , Plasmids/genetics , Bacillus subtilis/metabolism , Base Sequence , Blotting, Western , DNA/biosynthesis , DNA/genetics , Enzyme-Linked Immunosorbent Assay , Genes, Synthetic , Humans , Interferon-gamma/biosynthesis , Molecular Sequence Data , Plasmids/metabolism , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Proteins , Sequence AlignmentABSTRACT
The so-called quorum sensing (QS) response is a bacterial genetic reply to a chemical signal, called autoinducer, produced by the same cells. In this way bacteria modulate the transcription of genes important for their survival at high densities. In this paper we review the different elements involved in P. aeruginosa QS response, showing that it is a genetic regulatory network that not only responds to high bacterial densities, but to other environmental signals as well. We propose that QS in P. aeruginosa constitutes a novel genetic regulon that integrates and responds to nutritional factors and stress conditions in addition to bacterial density.
Subject(s)
Gene Expression Regulation, Bacterial/physiology , Pseudomonas aeruginosa/physiology , Biofilms , Environment , Pseudomonas aeruginosa/genetics , Signal Transduction , Transcription, Genetic/physiologyABSTRACT
Under heat-stress conditions bacteria induce, among other heat-shock proteins, the Hsp70 molecular chaperone (DnaK), which is involved in protein stabilization. It has been shown in Escherichia coli that an Hsp70 homologue called Hsc66, which is widespread in bacteria, functions as a chaperone in vitro. This paper reports the isolation of a Pseudomonas aeruginosa W51D mutant (W51M22) by insertion of the mini-Tn5-Hg transposon, which was unable to grow on ethanol and other short-chain alcohols as sole source of carbon. The transposon insertion in this mutant was shown to be located in the hscA gene encoding Hsc66. The inability of mutant W51M22 to use ethanol was complemented by the E. coli hscBA-fdx operon. The authors characterized the transcriptional arrangement of hscA, showing that it forms part of an operon with the upstream hscB gene, and that it is also expressed from its own promoter. These results are compatible with the P. aeruginosa Hsc66 protein being a functional molecular chaperone involved in the stabilization, in the presence of ethanol, of some proteins required for bacterial growth on short-chain alcohols.
