RESUMO
Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon-oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.
Assuntos
Catepsinas/análise , Catepsinas/biossíntese , Catepsinas/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossínteseRESUMO
The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work, we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ-ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ-ymgABC promoter is recognised by σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC.
Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Complexos Multienzimáticos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Porinas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Complexos Multienzimáticos/genética , Fosfoproteínas Fosfatases/genética , Porinas/genética , Proteínas Quinases/genética , Fator sigma/genética , Transcrição Gênica/genéticaRESUMO
In Escherichia coli, the marRAB operon is a determinant for antibiotic resistance. Such phenotypes require the encoded transcription factor MarA that activates efflux pump expression. To better understand all genes controlled by MarA, we recently mapped binding of the regulator across the E. coli genome. As expected, many MarA targets were adjacent to genes encoding stress response systems. Surprisingly, one MarA-binding site overlapped the lac operon regulatory region. Here, we show that MarA specifically targets this locus and can block transcription of the lac genes. Repression is mediated by binding of MarA to a site overlapping the lacP1 promoter -35 element. Control of the lac operon by MarA does not impact antibiotic resistance.
Assuntos
Escherichia coli/genética , Óperon Lac/genética , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
The multiple antibiotic resistance (mar) operon of Escherichia coli is a paradigm for chromosomally encoded antibiotic resistance in enteric bacteria. The locus is recognised for its ability to modulate efflux pump and porin expression via two encoded transcription factors, MarR and MarA. Here we map binding of these regulators across the E. coli genome and identify an extensive mar regulon. Most notably, MarA activates expression of genes required for DNA repair and lipid trafficking. Consequently, the mar locus reduces quinolone-induced DNA damage and the ability of tetracyclines to traverse the outer membrane. These previously unrecognised mar pathways reside within a core regulon, shared by most enteric bacteria. Hence, we provide a framework for understanding multidrug resistance, mediated by analogous systems, across the Enterobacteriaceae. Transcription factors MarR and MarA confer multidrug resistance in enteric bacteria by modulating efflux pump and porin expression. Here, Sharma et al. show that MarA also upregulates genes required for lipid trafficking and DNA repair, thus reducing antibiotic entry and quinolone-induced DNA damage.
