Construction and characterization of a cyanobacterial bioreporter capable of assessing nitrate assimilatory capacity in freshwaters

Abstract

The use of cyanobacterial whole-cell luminescent bioreporters has enhanced our ability to monitor nutrient availability in aquatic ecosystems. We have constructed a Synechocystis sp. strain PCC6803 bioluminescent reporter for the assessment of nitrate bioavailability. Specifically, a 380-base pair DNA fragment containing the NtcA/B-dependent nitrate/nitrite-activated nirA promoter (regulating expression of genes encoding nitrite reductase) was fused to the bacterial luciferase genes, luxAB, and introduced into Synechocystis by genetic transformation. Characterization of this strain, designated AND100, yielded dose-dependent increased bioluminescence coincident with increased nitrate added to the growth medium from 1 to 100 µM. Bioluminescence in response to nitrate addition was light dependent up to 50 µmol quanta m -2 s -1

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

“…Whole water was collected as described above and drawn through a 0.2-m polycarbonate filter, which was immediately placed onto a BG-11 agarose plate (1) containing 250 g ml Ϫ1 cycloheximide, and the plates were stored in the dark until the end of the cruise. All BG-11 media were modified with reduced nitrate as described previously (16). In the laboratory, plates were incubated at 18°C in 10-mol quanta m Ϫ2 s Ϫ1 .…”

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

“…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).…”

Cyanobacterial Bioreporters as Sensors of Nutrient Availability

“…In our laboratory, we have constructed both types and successfully used them to assess Fe (Class II; [17][18][19]) and nitrate bioavailability (Class I; [22]). In this section we discuss strategies for bioreporter construction, taking into consideration the host strain and gene transfer protocols.…”

“…Also developed has been a Synechocystis sp. PCC 6803 Class I reporter employing the NtcA/NtcB-regulated nirA (nitrite reductase) promoter [22]. Such strains have been employed to assess Fe and nitrate bioavailability in environmental samples (see Sect.…”