Two novel bacterial biosensors for detection of nitrate availability in the rhizosphere.

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. A major part of the nitrogen cycle results from interactions between plants and soil microorganisms in the rhizosphere. As a critical compound in many transfers and transformations of nitrogen, nitrate is a labile, dynamic pool in rhizosphere N cycling. Nitrate is a significant source of plant N, and it can also be incorporated by a variety of microorganisms through assimilatory nitrate reduction (15) and is subject to denitrification in the absence of oxygen (13). Roots are successful competitors with microorganisms for limited NO3 -N (40). However, depletion of nitrate in various root zones may be different, as in the case of rice and maize, where uptake of nitrate reaches a maximum in the zone where root hairs emerge (9). Since the spatial patterns of nitrate concentration in the rhizosphere may correspond to patterns of root ion uptake as well as reflect microbial N transformations, a clear understanding of nitrate availability in the root zone is essential to understanding the mechanisms of rhizosphere N cycling. It has been difficult to characterize nitrogen cycling processes in the rhizosphere because of the dynamic and heterogeneous nature of the intact root-soil-microbe system. A finescale study of nitrate is even more complicated because nitrate pools are quite transient due to the ion’s great mobility; it is rapidly produced through nitrification and utilized by plants