Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

Abstract

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5␣ and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 M, while GFP fluorescence was detected in cells harboring pNgfp at about 10 M. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E. cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice-and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.

Selective progressive response of soil microbial community to wild oat roots

“…The dynamic subset is not only increasing in relative abundance, but taxa in a diverse, yet-defined, subset differentially respond to the characteristics of specific root zones as the root moves through soil. Compositional differences in the rhizosphere community compared with bulk soil and between different root zones point to a fraction of the soil microbial community that is especially rhizosphere competent (Folman et al, 2001;DeAngelis et al, 2005;Nunan et al, 2005;DeAngelis, 2006). These compositional changes accompany large changes in soil function associated with rhizosphere N cycling (DeAngelis, 2006;DeAngelis et al, 2008).…”

“…Root movement through soil creates dynamic environmental gradients that are constantly reiterated with new root growth. A root moving through 'bulk soil' introduces labile carbon and nutrients, creates water conduits, and deposits antimicrobial compounds and hormones (Hawes et al, 1998;Brimecombe et al, 2001;Bringhurst et al, 2001;DeAngelis et al, 2005;Hawkes et al, 2007) across temporal scales of hours to days (Jaeger et al, 1999;Lubeck et al, 2000). As many soil microbes are carbon limited (Paul and Clark, 1996), they may be expected to respond quickly to root-induced changes in soil chemistry and nutrient status by reproducing and increasing in activity (Heijnen et al, 1995;Jaeger et al, 1999;Herman et al, 2006).…”

Challenges in Using Precision Agriculture to Optimize Symbiotic Nitrogen Fixation in Legumes: Progress, Limitations, and Future Improvements Needed in Diagnostic Testing

“…However, this method has limitations for use under field conditions in the short-medium term, as the GlnLux method cannot distinguish N derived from SNF versus soil-uptake N. This method has potential for precision agriculture if translated into a field-based technology, by conversion into an amperometric biosensor specific for gln [72] that can be linked to variable rate fertilization. There is also potential to engineer additional amperometric biosensors that can detect other fixed-N metabolites [73], as well as inorganic N [74]. Chemical-based tests are currently available (e.g., Merkoquant test strips, Reflectoquant strips) to measure plant nitrate concentration from plant sap at the field level [39].…”

Challenges in imaging and predictive modeling of rhizosphere processes

“…More recently optode foils combined with high resolution optical systems have been used for measurement of pH gradients and CO 2 release (Blossfeld and Gansert 2007;Rudolph-Mohr et al 2015). Bioreporters (i.e., bacteria tagged with fluorescent proteins to report a specific activity) have been used to study the release of AsIII (Kuppardt et al 2010), available nitrate (DeAngelis et al 2005) and the communication of root colonizing rhizobacteria (Gantner et al 2006). However, there is clearly merit in doing more work to correlate these bioreporter findings with structural and chemical mapping of the rhizosphere.…”