Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil

The root colonizing bacterium Pseudomonas fluorescens CHA0 was used to cons truct an oxygen-responsive biosensor. An anaerobically inducible promoter o f Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate re ductase regulation)-like transcriptional regulator ANR (anaerobic regulatio n of arginine deiminase and nitrate reductase pathways), was fused to the s tructural lacZ gene of Escherichia coil. By inserting the reporter fusion i nto the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-y east extract medium in an oxystat at defined oxygen levels, the biosensor C HA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa t o 2 x 10(2) Pa of O-2 by a nearly 100-fold increase in beta-galactosidase a ctivity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coil promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentr ation, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil mi crocosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6 day period. When the water content in the microcosm wa s raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compa ction of the soil was shown to have a faster and more dramatic effect on th e expression of the oxygen reporter than soil water content alone, indicati ng that factors other than the water-filled pore space influenced the oxyge n status of the soil. These experiments illustrate the utility of the biose nsor for detecting low oxygen concentrations in the rhizosphere and other s oil habitats.