INTRODUCTION

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Adaptation of many facultative anaerobic and some obligate aerobic bacteria to oxygen limitation is mediated by a family of transcriptional regulators related to the FNR (fumarate and nitrate reductase regulation) protein of Escherichia coli (43, 48, 49). In Pseudomonas aeruginosa and other Pseudomonas species belonging to rRNA homology group I, the FNR homolog ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways) is a major regulator of genes involved in anaerobic metabolism, e.g., the genes required for anaerobic respiration (1, 55) and the arcDABC (anaerobic arginine catabolism) operon of P. aeruginosa (10, 12, 41, 54, 56). When oxygen levels decrease, the ANR protein binds to a conserved sequence, the ANR box, located about 40 bp upstream of the transcription initiation site and thereby activates transcription, as shown for the arcDABC operon (10, 12, 28a, 30).

FNR of E. coli and ANR of P. aeruginosa and Pseudomonas fluorescens share essential features in all domains that are important for sensing oxygen tension and for transcriptional activation (13, 25, 38, 53, 54, 56). The sensory domain of FNR contains four cysteine residues, Cys-20, Cys-23, Cys-29, and Cys-122, serving to bind a [4Fe-4S]²⁺ cluster at a ratio of one cluster per FNR monomer (20, 22, 26). This type of iron-sulfur cluster has a redox potential that can be as low as 700 mV and therefore acts as a strong reductant (39). In its reduced state, the [4Fe-4S]²⁺ cluster appears to mediate dimerization and thus activation of FNR (20, 47, 49). By virtue of its C-terminal helix-turn-helix motif, activated FNR recognizes specific binding sites (FNR boxes) in anaerobically inducible or repressible promoters (43, 48) and modulates transcription by interaction with RNA polymerase (38, 53). Upon exposure of the cells to oxygen, the [4Fe-4S]²⁺ cluster is oxidized to a [2Fe-2S]²⁺ cluster, which remains bound to the FNR monomers but is unable to support the dimeric structure. Prolonged oxidation may eventually lead to disassembly of the iron-sulfur cluster, leaving FNR as a monomeric apoprotein (20, 22, 26, 48). Oxygen is considered to be the direct effector of FNR by oxidation of the [4Fe-4S]²⁺ cluster (6). It has been calculated that the diffusion of oxygen into the cytoplasm is rapid enough to maintain ambient oxygen concentrations despite respiratory consumption, as long as the concentration of oxygen in the extracellular environment is above 10² Pa (2, 48).

Genetically engineered bacteria that can be introduced and report the physicochemical conditions in natural environments have proved very useful in recent years (28, 36). In the present study, we have developed an oxygen-sensitive biosensor based on a transcriptional fusion of a modified, ANR-dependent arcDABC promoter of P. aeruginosa PAO1 to the lacZ structural gene of E. coli. The reporter system was cloned into a mini-Tn7 transposon (3), which can be transposed into a specific and unique target site (attTn7) found in a noncoding chromosomal region of many bacteria, including P. fluorescens (4, 8, 9). Although nonspecific Tn7 insertion has been reported for some P. putida strains (44), the Tn7 transposon system generally provides a system for stable insertion with a minimal risk of affecting cell functions. We inserted the oxygen reporter construct into the obligate aerobe P. fluorescens CHA0, a strain used widely in biological control studies (21, 52). In this organism, ANR has been identified as a transcriptional activator of the hcnABC operon, encoding HCN synthase, under low-oxygen conditions (25, 56). The biosensor constructed, P. fluorescens CHA900, was used to evaluate oxygen concentrations in a plant-soil microcosm, previously developed for studying nutrient starvation responses of another root-colonizing P. fluorescens strain (24).

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. The bacterial strains and plasmids used are listed in Table 1. Bacterial cultures were pregrown in Luria-Bertani (LB) broth (40) or a defined Na-citrate (0.7%)-asparagine (0.2%) medium (CAMM; modified from reference 7) containing the appropriate antibiotic(s) (ampicillin or kanamycin). The oxygen response of the constructs was monitored in batch cultures grown in OS minimal medium (33) containing 0.1% (NH₄)₂SO₄ and supplemented with either glucose (0.5%) or yeast extract (0.4%) and glutamate (25 mM) as carbon sources. Oxystat experiments were carried out with the yeast extract-glutamate OS medium.

Construction of plasmids. A plasmid (pME3535) suitable for the construction of transcriptional lacZ fusions was constructed as follows. The 8.6-kb pUC8-based vector pNM480, which contains the lacZ and lacY genes allowing the construction of translational fusions (31), was opened with PstI and HindIII, and a linker coding for the authentic 5' end of lacZ mRNA was inserted. This linker reconstituted the lacZ gene with its own ribosome binding site and consisted of the annealed primers 5'-G AATTGT GAGCGG ATAACA ATTTCA CACAGG AAACAGCT ATG ACC ATG ATT CA-3' and 3'-ACGTC TTAACA CTCGCC TATTGT TAAAGT GTGTCC TTTGTCGA TAC TGG TAC TAA GTTCGA-5'. For the insertion of reporter constructs into the P. fluorescens chromosome, plasmid pTJ1R containing a mini-Tn7-Km transposon (Table 1) was used as the carrier. The arcDABC-derived FNR consensus promoter carried by a 56-bp BamHI-PstI fragment of pME3781 (54) was fused to the 3.2-kb PstI-DraI fragment of pME3535 carrying the lacZ gene. The resulting transcriptional fusion was inserted into pME6000 (29) cleaved with BamHI and PstI (with removal of the 3' overhang by Klenow enzyme), allowing the recovery of the fusion as a 3.2-kb XbaI-XhoI fragment, which was treated with Klenow enzyme to fill in the sticky ends and ligated into the 6.3-kb plasmid pTJ1R opened at its unique HincII site. This produced plasmid pME6502 (Fig. 1). The ANR-independent, arcDABC-derived promoter of pME3771-1 (54) fused to the lacZ gene of pME3535 was recruited similarly as a BamHI-DraI fragment. After treatment with Klenow enzyme, the 3.2-kb promoter-lacZ fragment was purified and ligated into the 6.3-kb HincII fragment of pTJ1R to give plasmid pME6504 (Fig. 1). Plasmids pME6502 and pME6504 were introduced into E. coli CC118/pir by transformation and selection on LB agar plates containing 25 µg of kanamycin per ml and 200 µg of X-Gal (5-chloro-4-bromo-3-indolyl--D-galactopyranoside) per ml. The carrier plasmids pME6502 and pME6504, as well as the helper plasmid pUX-BF13, were purified from E. coli by using the Qiagen midi plasmid purification kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Construction of reporter plasmids. pME6502 and pME6504 were constructed by insertion of PANR-lacZ and PConst.-lacZ, respectively, into a mini-Tn7-Km transposon in the carrier plasmid pTJ1R. PANR, ANR- and FNR-dependent promoter; PConst., constitutive promoter; oriColE1, origin of replication; ApR, ampicillin resistance gene; KmR, kanamycin resistance gene; mob, mobilization region; Tn7L and Tn7R, left and right Tn7 ends, respectively; , polylinker missing from pME6504.

Preparation of competent cells of P. fluorescens. Strain CHA0 was grown in LB broth at 30°C. After incubation overnight, 0.25 ml of the cell suspension was shifted to 25 ml of fresh LB broth in a 100-ml Erlenmeyer flask and grown at 35°C (to inactivate the restriction system) until the culture reached an optical density at 600 nm (OD₆₀₀) of 0.5 to 1.0. The cells were harvested by centrifugation and washed twice with 10 ml of an ice-cold solution of 15% (wt/vol) glycerol, 1 mM MOPS (3-morpholinopropanesulfonic acid). The cells were resuspended in 200 µl of the glycerol-MOPS solution, stored on ice, and used within 2 to 3 h.

Chromosomal insertion of reporter constructs by electroporation. Competent cells (40-µl suspension) were mixed in an Eppendorf tube with ~0.5 µg of plasmid DNA (pME6502 or pME6504) in 5 µl of TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA) and 0.5 µg of pUX-BF13 DNA in 5 µl of TE buffer. Plasmid pUX-BF13 carries the genes encoding the transposition proteins necessary for insertion of the Tn7 cassette into the genomic target site (3). The mixture was transferred to an ice-cold electroporation cuvette and treated in a Bio-Rad electroporator (25 µF, 200 , 5 ms, 2.5 kV/cm). Immediately thereafter, 1 ml of prewarmed (35°C) SOC medium (2% [wt/vol] Bacto tryptone, 0.5% [wt/vol] yeast extract, 10 mM NaCl, 10 mM MgCl₂, 10 mM MgSO₄, 2.5 mM KCl, 20 mM glucose) was added to the cuvette. The cell suspension was transferred to an Eppendorf tube and incubated at 35°C for 3 h, followed by spread plating of the entire culture on five selective plates (LB agar with 25 µg of kanamycin per ml). The plates were incubated overnight at 35°C, transferred to 30°C, and incubated for 48 h until Km^r colonies appeared. The incubation at 35°C after electroporation was done to keep the CHA0 restriction system inactive. This procedure enhanced the recovery of Km^r transformants. Typically, about 200 transformants were obtained. Integration of mini-Tn7-Km from pME6502 and pME6504 produced strains CHA900 and CHA901, respectively.

Southern blot analysis. Chromosomal DNA was purified from mini-Tn7-Km-transformed P. fluorescens strains by phenol extraction (11). DNA samples (~2 µg) were digested with SmaI or HindIII, separated electrophoretically on a 0.7% agarose gel, and transferred to a Hybond N membrane (Amersham) according to the instructions of the supplier. The 1.37-kb PstI fragment of pTJ1R constituting the Km^r gene was labeled with digoxigenin-11-dUTP (DIG) (Boehringer Mannheim) and used as a probe. After hybridization at 60°C for 12 h and being washed twice in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (40) with 0.2% sodium dodecyl sulfate (SDS) at room temperature, the DIG-labeled fragments were detected by reaction with anti-DIG antibodies coupled to alkaline phosphatase, according to a protocol supplied by the manufacturer (Boehringer Mannheim). Nylon membranes were exposed to an X-ray film for 3 min.

TSO test. The genetic instability of strain CHA0 can be monitored by using tryptophan side chain oxidase (TSO) as a marker (52). Km^r transformants were streaked on King's B agar (23) and incubated at 30°C for 3 days. The colonies were then overlaid with 20 ml of TSO agar and incubated at room temperature for 4 h, after which time, TSO-positive and TSO-negative colonies could be observed as black and white colonies, respectively. TSO agar consisted of 1.2 g of agar melted in 54 ml of water and mixed at 50°C with 6 ml of 10% (wt/vol) SDS and 60 ml of 20 mM L-tryptophan in 1 M glycine-HCl buffer (pH 3.0) (45).

Growth measurements. P. fluorescens CHA0 (wild type), CHA900 (oxygen reporter), and CHA901 (constitutive control) grown in LB broth were used to inoculate CAMM (100 ml) in 500-ml baffled Erlenmeyer flasks. Cultures were incubated on a rotary shaker (300 rpm [IKA KS250 Basic; Janke and Kunkel, Staufen, Germany]) at 30°C, and the OD₆₀₀ was monitored for 15 h.

Determination of -galactosidase activities. For pure cultures, -galactosidase activity was measured in toluenized cells with o-nitrophenol--D-galactoside (ONPG) as a substrate and expressed in Miller kilounits (40). For soil experiments, samples of 0.5 g of soil were suspended in 5 ml of phosphate-buffered saline (PBS) (40), and 2 ml of this was then centrifuged (14,000 rpm, 3 min, model 5415C; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) and resuspended in 200 µl of PBS. Toluene (10 µl) was added, and -galactosidase activities were determined by addition of ONPG, as described above. Soil particles were removed by centrifugation (14,000 rpm, 3 min). Enzyme activities (A₄₂₀ units) were related to the number of P. fluorescens CHA900 or CHA901 cells in the soil slurries determined as CFU on LB agar containing kanamycin (25 µg per ml) by the drop plate method (16). An OD₆₀₀ value of 1 equals ~2 × 10⁹ cells per ml.

Oxygen response measurements of reporter constructs. In preliminary experiments, E. coli CC118/pir carrying pME6502 or pME6504 and P. fluorescens CHA900 and CHA901 were grown in LB broth overnight. These cultures were used to inoculate 60 ml of OS minimal medium (in 125-ml serum bottles) or 100 ml of OS minimal medium (in 500-ml Erlenmeyer flasks). The serum bottles were sealed with Teflon-coated rubber stoppers. Bacterial cultures in these bottles consumed the oxygen initially present. P. fluorescens CHA0 cells, being strictly aerobic, stopped growth at ca. 5 × 10⁸ cells per ml. Bottles and flasks were incubated on a rotary shaker (200 rpm) at 30°C. Cells from both the oxygen-limited and the well-aerated cultures thus obtained were harvested at an OD₆₀₀ of ~0.5 for determination of -galactosidase activity.

Soil and plant experiments. A sandy loam soil (coarse sand, 26.9%; fine sand, 39.9%; silt, 15.4%; clay, 15.3%; humic particles, 2.5%) was sampled from an agricultural field (Højbakkegaard, Taastrup, Denmark), passed through a 2-mm-pore-diameter sieve, and stored at 4°C until use. Barley seeds (Hordeum vulgare, var. Lamba) were allowed to germinate for 48 h on wetted filter paper, resulting in ~1-cm-long roots. Precultures of P. fluorescens CHA900 and CHA901 were grown in LB broth, shifted to 100 ml of CAMM (OD₆₀₀ of ~0.005) in baffled 500-ml Erlenmeyer flasks, and incubated on a rotary shaker (300 rpm) at 30°C. The cells were harvested from early-exponential-phase cells (OD₆₀₀ of between 0.25 and 0.50), washed once, and resuspended in PBS to a final cell density of ~2 × 10¹⁰ cells per ml. The barley seedlings were placed in the cell suspension for 30 min. Bulk soil was inoculated by spraying the cell suspension onto the soil, reaching a final concentration of ~1 × 10⁸ to 5 × 10⁸ cells per g of soil and a water content of 15% (wt/wt), i.e., 60% of the field capacity of this soil. The soil was packed loosely in polyvinyl chloride (PVC) tubes (2.8 × 11.5 cm) to a final density of ~1.1 g of soil per cm³. Two bacterium-coated barley seedlings were planted in each tube. The tubes were placed in sealed transparent plastic bags containing wetted soil to avoid desiccation and incubated at 20°C with 12 h of light and 12 h of dark. Throughout the experiments, the water content of the soil was monitored by weighing of the tubes and was adjusted when needed.

	RESULTS

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Construction of reporter strains. An artificial promoter (P_ANR), which was derived from the arcD promoter of P. aeruginosa and contains the consensus FNR box (TTGAT...ATCAA) as well as an optimal 10 hexamer (TATAAT) for ⁷⁰ RNA polymerase (Fig. 1), was recognized equally well by FNR in E. coli and ANR in P. aeruginosa during oxygen limitation (54). This promoter was used to construct a transcriptional lacZ fusion, which was inserted into a mini-Tn7 transposon carried by the suicide plasmid pME6502 (Fig. 1). Chromosomal integration of mini-Tn7-P_ANR-lacZ from pME6502 was achieved in P. fluorescens CHA0 by coelectroporation with a nonreplicating helper plasmid expressing transiently the Tn7 transposition functions (see Materials and Methods). The reporter strain obtained was designated CHA900. In parallel, a constitutive promoter (P_Const.) previously characterized in E. coli and P. aeruginosa (54) was used for the construction of a control strain, CHA901, via the suicide plasmid pME6504 (Fig. 1).

Oxygen response of reporter constructs in batch experiments. The P_ANR-lacZ reporter was strongly induced in E. coli CC118/pir carrying pME6502 and in P. fluorescens CHA900 during oxygen limitation, compared to the basal expression in well-aerated cultures (Table 2), indicating that the construct could indeed be used as an oxygen reporter. The control strains E. coli CC118/pir carrying pME6504 and P. fluorescens CHA901 showed little if any response to oxygen limitation (Table 2). The response of the P_ANR-lacZ reporter to oxygen limitation was enhanced in E. coli and P. fluorescens when glucose was replaced by yeast extract and glutamate in the growth medium (Table 2), probably reflecting faster growth of the bacteria with the latter two substrates. In a control experiment designed to demonstrate regulation by ANR, the P_ANR-lacZ cassette was cloned into the high-copy-number shuttle vector pME6000. The recombinant plasmid obtained gave anaerobically inducible -galactosidase activities in the wild-type CHA0: i.e., oxygen limitation caused a shift from 8.7 to 118.8 Miller kilounits in glucose medium. In the anr-negative mutant CHA21, anaerobic induction was essentially prevented; i.e., expression amounted to 11.6 Miller kilounits under oxygen limitation.

Oxygen response of reporter constructs in oxystat experiments. P. fluorescens CHA900 and E. coli CC118/pir(pME6502) were grown at defined oxygen tensions in an oxystat. A fully aerated growth medium (OS medium supplemented with yeast extract and glutamate) was inoculated at 1% with the reporter strain, and exponential growth was allowed to be established before the oxygen tension was decreased to a defined constant level. At intervals, -galactosidase activities in samples were determined. In the representative experiment shown (Fig. 2), the oxygen tension was reduced from 210 × 10² Pa to 50 × 10² Pa (10² Pa corresponds to ~1 µM dissolved O₂). The reporter strain CHA900, initially adapted to optimal aeration (210 × 10² Pa) by a decrease in -galactosidase activity, reflecting a dilution of the -galactosidase present in the stationary-phase, partially-oxygen-limited inoculum. Strain CHA900 then responded rapidly to the decrease in the oxygen tension to 50 × 10² Pa by an increase in -galactosidase activity. The activity reached a steady-state level within a couple of hours while the culture was still growing exponentially (Fig. 2). Similar results were obtained with E. coli harboring pME6502 (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Oxygen response of P. fluorescens CHA900 in an oxystat experiment. OS medium supplemented with yeast extract and glutamate was inoculated with 1% of an overnight culture. The oxygen concentration (solid line) was initially kept close to 210 mbar (210 × 102 Pa) by flushing the medium with atmospheric air. After 4 h of growth, the oxygen concentration was decreased to 50 mbar (50 × 102 Pa) by flushing with a mixture of nitrogen gas and atmospheric air. The -galactosidase activity () then increased to a steady-state level in the growing culture. Growth was measured as OD600 units ().

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Steady-state levels of -galactosidase activities () and generation times () of P. fluorescens CHA900 (A) and E. coli CC118/pir(pME6502) (B) grown in the oxystat at different oxygen concentrations. Triplicate experiments were run for some of the oxygen concentrations (data points with standard error bars). The other points represent single oxystat experiments. In panel A, the highest -galactosidase values were obtained at 2 mbar (2 × 102 Pa) of O2; in panel B, the highest values correspond to 0 mbar (0 × 102 Pa) of O2.

Expression of the reporter construct in carbon-free medium. The oxygen response of P. fluorescens CHA900 was examined after shifting exponentially growing, well-aerated cells to OS medium without a carbon source under three different oxygen tensions (7 × 10², 70 × 10², and 210 × 10² Pa of O₂). This shift resulted in cessation of growth at an OD₆₀₀ of ~0.13 in the three cultures. Nevertheless, the expression of the reporter increased with decreasing oxygen tension (Fig. 4), although a steady-state level was reached only after 10 to 12 h of incubation. Incubation at 7 × 10² Pa of oxygen resulted in a -galactosidase activity of 0.9 to 1.0 Miller kilounits, a value which is considerably lower than that (15 to 20 Miller kilounits) obtained in an exponentially growing culture at the same oxygen level in the oxystat experiment (Fig. 3A). Under conditions of carbon starvation, the anaerobic induction factor was below 10 (Fig. 4), i.e., much smaller than the corresponding value determined during growth in the oxystat (Fig. 3A).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Oxygen response of P. fluorescens CHA900 shifted from an exponentially growing culture to 25 ml of carbon-free OS medium in sealed 100-ml serum bottles. At time zero, the headspace was flushed with nitrogen gas and the oxygen concentration was adjusted to 7 (), 70 (), or 210 () mbar (7 × 102, 70 × 102, or 210 × 102 Pa, respectively) of O2 by injection of atmospheric air. All results are given as means ± standard error (n = 3).

Induction factor in plant-soil experiments. P. fluorescens CHA900 (oxygen reporter) and CHA901 (constitutive control strain) were introduced into soil microcosms with and without barley. At intervals, expression of -galactosidase was analyzed in harvested microcosms. The ability of the reporter bacteria to respond to decreased oxygen tensions was tested by incubating some of the microcosms in an anaerobic jar at ~5 × 10² Pa of O₂ for 12 h; the other microcosms remained exposed to atmospheric air.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Oxygen response of P. fluorescens CHA900 (oxygen reporter) retrieved from bulk soil (A) and the rhizosphere (B) of plant-soil microcosms. For each sampling time, samples were taken from parallel sets of microcosms, half of them previously incubated at 5 mbar (5 × 102 Pa) of O2 for 12 h () and half of them incubated at atmospheric air (210 mbar [210 × 102 Pa] of O2) (). -Galactosidase activities were calculated as 1,000 × A420 × (t)1 × (108 CFU)1, where t is the assay time (in minutes). All results are given as means ± standard error (n = 6, two microcosms with 3 samples from each).

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Oxygen response of P. fluorescens CHA900 retrieved from bulk soil (A) and rhizosphere soil (B and C) of plant-soil microcosms. The water content of the soil was initially adjusted to 60% of field capacity. After 24 h, the water content was increased to 85% of field capacity in half of the plant microcosms. For each sampling time, half of the microcosms were incubated at 5 mbar (5 × 102 Pa) of O2 for 12 h (gray bars) and the other half were kept at atmospheric air (210 mbar [210 × 102 Pa] of O2) (black bars). All results represent mean ± standard error (n = 6, three microcosms with two samples from each).

View larger version (15K): [in this window] [in a new window]   FIG. 7.   Oxygen response of P. fluorescens CHA900 retrieved from bulk soil packed at five different densities. The water content was adjusted to 60% () or 85% () of field capacity, and the soil was incubated at 20°C for 24 h. Two sets of samples were previously incubated at 2 mbar (2 × 102 Pa) () and 5 mbar (5 × 102 Pa) () of O2, respectively, for 12 h. All results are given as means ± standard error (n = 3).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

It may seem surprising, at first sight, that an obligate aerobe such as P. fluorescens CHA0 should have an anaerobic regulator, ANR, which is very similar (53% identical) to the anaerobic regulator FNR of E. coli, a facultative anaerobe. However, as we have found here, strain CHA0 grows quite well with as little as 2 × 10² Pa of O₂ (Fig. 3). This level of O₂ would not stop growth of, e.g., Clostridium haemolyticum, an organism usually considered to be an oxygen-sensitive, strict anaerobe (27). Thus, P. fluorescens CHA0 is well adapted to ecological niches containing little O₂, and it is only in environments containing less than 2 × 10² Pa of O₂ that growth of strain CHA0 is handicapped. The oxygen biosensor construct P_ANR-lacZ was regulated by FNR in E. coli and by ANR in P. fluorescens in virtually the same way (Fig. 3A). This implies that FNR and ANR are also very similar in terms of mechanisms. All essential features of FNR (i.e., the cysteine residues involved in [4Fe-4S]²⁺ binding, the RNA polymerase  subunit interacting sites, the dimerization domain, the contacts with the ⁷⁰ factor, and the helix-turn-helix motif) are highly conserved in ANR (25, 38).

Electrochemical microsensors have been widely used to study distribution of oxygen and oxygen consumption in biofilms and sediments (37). Such microsensors have also been used in artificial soil systems like waterlogged aggregates (18, 42) and gel-stabilized soil systems (19, 34). For optimal utilization of these microsensors, however, it is crucial to determine the diffusion characteristics, e.g., porosity and diffusion coefficients. However, this is difficult in natural soil environments because of heterogeneity in texture and distribution of water (18). Here the use of oxygen reporter bacteria is an improvement, because this technology meets the criteria of in situ detection and high sensitivity (28). Recently, a technique has been developed allowing quantification of -galactosidase in single cells (35). Combination of this technique with the biosensor strain CHA900 may further extend the usefulness of the biosensor.

The rhizosphere is a habitat in which living roots release a range of low-molecular-weight substrates, including sugars, oligosaccharides, and amino acids. These compounds allow microbial maintenance or growth (5). Oxygen consumption by microorganisms or by the root cells themselves can lead to low oxygen levels in the rhizosphere (17, 19). However, the oxygen status in the rhizosphere depends on the texture, structure, and water content of the soil (46). In the present study, this tenet has been verified experimentally, either by an increase of soil water or by soil compaction (Fig. 6 and 7). The prevalence of microaerobic conditions in the lower part of wheat roots has been previously observed by the use of a nifH-gusA reporter construct in Azospirillum brasilense (50). In further agreement, it is known that ANR-dependent HCN production by P. fluorescens CHA0 occurs predominantly in poorly aerated, wet soils, as judged from a strong suppressive effect on Thielaviopsis basicola-induced black root rot of tobacco, whereas in more aerated soil, the cyanogenic capacity of strain CHA0 does not appear to be expressed (25). In conclusion, the oxygen-sensing reporter strain presented here seems a promising tool for in situ studies of spatial and temporal variations in bioavailability of oxygen in natural habitats.