Characterization of Stress Responses of Heavy Metal and Metalloid Inducible Promoters in Synechocystis PCC6803

Abstract

In several biotechnological applications of living bacterial cells with inducible gene expression systems, the extent of overexpression and the specificity to the inducer are key elements. In the present study, we established the concentration ranges of Zn 2+ , Ni 2+ , Co 2+ , AsO 2-, and Cd 2+ ions that caused significant activation of the respective promoters of Synechocystis sp. without concomitant unspecific stress responses. The low expression levels can be increased up to 10-100-fold upon treatments with Cd 2+ , AsO 2-, Zn 2+ , and Co 2+ ions and up to 800-fold upon Ni 2+ treatment. These results facilitate the development of conditional gene expression systems in cyanobacteria.

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

“…The concentrations of metal ions used to induce the promoters were chosen based on a balance between having high enough concentration to get a high induction and low enough to not stress the cells. For Synechocystis , the half growth-inhibitory concentration (IC 50 ) of Ni 2+ was previously reported to be 27 μM, 8 μM for Co 2+ , between 8 and16 μM for Zn 2+ , and 2 μM for Cu 2+  27. Our initial designation of standard metal inducer concentration was 5 μM for Ni 2+ and 4 μM for Zn 2+ , as per the recommendations of Blasi et al 27…”

“…We did not aim to investigate the functional elements or initiation dynamics of each promoter. Earlier studies have examined the transcriptional activation of some of the metal inducible promoters2627. Here we expand on that work by providing systematic, useful data quantitatively comparing these promoters to each other and to some commonly used promoters, with fluorescent protein levels as well as formation of a model product, ethanol, as reporter.…”

Synthetic biology of cyanobacteria: unique challenges and opportunities

“…These have often been the basis of metal detection systems (Erbe et al, 1996; Boyanapalli et al, 2007; Peca et al, 2007, 2008; Blasi et al, 2012). Cyanobacteria balance metal intake for the organisms' needs against potential oxidative stress and protein denaturation (Michel et al, 2001; Peca et al, 2008) via tightly regulated systems.…”

Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment

“…strain PCC 6803 was studied as a model for photosynthesis and circadian rhythm and several light-responsive promoters were identified, including the light-responsive (LR) promoter1 (Marraccini et al, 1993) and the promoter of preprotein translocase subunit (secA; Mazouni et al, 1998), as well as the light-repressible promoter of PSI reaction center subunits (psaAB; Muramatsu and Hihara, 2006), and the promoter of light-repressed protein A homolog (lrtA; Imamura et al, 2004). More recently, cyanobacterial studies have turned their focus to biotechnological applications and numerous heavy metal-inducible promoters have been characterized (Peca et al, 2008;Blasi et al, 2012) as well as the copper-inducible promoter of plastocyanin (petE; Briggs et al, 1990;Ghassemian et al, 1994;Buikema and Haselkorn, 2001) and the copper-repressible promoter of cytochrome c553 (petJ; Ghassemian et al, 1994). Furthermore, promoters tightly regulated by nutrient availability have been characterized, including the promoter of the sodium-dependent bicarbonate transporter (sbtA) regulated by inorganic carbon availability (Wang et al, 2004) and the promoter of ferredoxin-nitrite reductase (nirA) regulated by nitrogen source (Ivanikova et al, 2005;Qi et al, 2005).…”

“…A range of different vectors and reporters have been used to test promoter activity in cyanobacteria (Marraccini et al, 1993;Ivanikova et al, 2005;Peca et al, 2008;Huang et al, 2010;Xu et al, 2011;Blasi et al, 2012). In an attempt to standardize the characterization of promoter activity for synthetic biology applications, a method was developed in E. coli whereby promoter activity could be measured relative to an in vivo reference promoter based on the fluorescence intensities of GFP as a reporter (Kelly et al, 2009).…”