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Applied and Environmental Microbiology, September 1999, p. 4085-4093, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Oxygen-Sensing Reporter Strain of Pseudomonas fluorescens for Monitoring the Distribution of Low-Oxygen Habitats in Soil

Ole Højberg,1,2,* Ursula Schnider,1 Harald V. Winteler,1,dagger Jan Sørensen,2 and Dieter Haas1

Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland,1 and Section of Genetics and Microbiology, Department of Ecology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark2

Received 16 April 1999/Accepted 9 July 1999

The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 × 102 Pa to 2 × 102 Pa of O2 by a nearly 100-fold increase in beta -galactosidase activity. Half-maximal induction of the reporter occurred at about 5 × 102 Pa. This dose response closely resembles that found for E. coli 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 concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was 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 compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.

* Corresponding author. Present address: Microbiology Section, Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark. Phone: 45 8999 1183. Fax: 45 8999 1378. E-mail: ole.hojberg{at}agrsci.dk.

dagger Present address: ARPIDA, CH-4142 Münchenstein, Switzerland.

Applied and Environmental Microbiology, September 1999, p. 4085-4093, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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