1 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117 543
2 Pacific Northwest National Laboratory, Richland, WA, USA
Correspondence
Sanjay Swarup
dbsss{at}nus.edu.sg
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ABSTRACT |
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The GenBank accession numbers for the P. putida PNL-MK25 gdhA, nql and cyoD genes reported in this paper are AF321093, AF321092, and AF321090, respectively.
Present address: Centre for Forensic Science, Health Sciences Authority, 11 Outram Road, 169078, Singapore.
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INTRODUCTION |
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Bioavailability of organic nutrients is inherently low in both bulk and vegetated soils (Williams, 1985; van Overbeek & van Elsas, 1997
). In bulk soils, polymeric organic nutrients are readily adsorbed to negatively charged clay particles or humic material, rendering them unavailable (Greenland, 1971
; Dashman & Stotzky, 1986
), while other studies have shown that bacterial populations in the rhizosphere are actually starved (Meikle et al., 1995
; Marschner & Crowley, 1996a
, b
; Normander et al., 1999
). Hence, bacteria introduced into either soil type are likely to face conditions of carbon/energy shortage.
Poor conditions in the soil environment extend beyond organic nutrients to include inorganic nutrients and oxygen limitations as well. Instances of phosphate and nitrate limitation have been widely reported (Kragelund et al., 1997; Jensen & Nybroe, 1999
). Due to the low diffusion coefficients of most gases, the static nature of soil, and respiration by plant roots and soil microfauna and microflora, oxygen in the soil environment becomes depleted while carbon dioxide accumulates (Kozlowski et al., 1991
; Vande Broek et al., 1993
; Hojberg et al., 1999
). Additionally, waterlogging leads to oxygen deficiency. Consequently, many genes that are normally active under aerobic laboratory conditions become downregulated (Lin & Iuchi, 1991
; Spiro & Guest, 1991
).
Traditional use of indigenous micro-organisms in environmental bioremediation involved expression of the biodegradation genes under the regulation of their native promoters, which may not be most suitable in certain field applications. Aromatic catabolic operons are generally controlled by transcriptional activators that interact with the aromatic substrates or with pathway intermediates, which serve as inducer molecules and provide regulatory specificity (Diaz & Prieto, 2000). Many such regulators have a narrow inducer specificity and/or have a minimum threshold concentration for their activation (Timmis & Pieper, 1999
). Several studies have also demonstrated that the catabolic pathways of Pseudomonas spp. involved in the biodegradation of aromatic xenobiotics are regulated by carbon catabolite repression (CCR) (Hartline & Gunsalus, 1971
; O'Connor et al., 1996
; Duetz et al., 1997
).
The above observations suggested a need to perform a broad-spectrum screening for Pseudomonas spp. gene promoters that are highly active under low-nutrient and low-oxygen conditions typical of soil environments. De novo identification of Pseudomonas genes and subsequent isolation of their promoters may be necessary since regulatory mechanisms in the well-studied Escherichia coli are not always transferable to soil pseudomonads. Hence, genes and promoters from heterologous species are not likely to express suitably in Pseudomonas spp. Promoters from pseudomonads would allow the expression of biodegradation and biocontrol genes to be uncoupled from the signals that ordinarily control or limit their expression and would thus offer greater flexibility in their application. We describe here three genes, gdhA, nql and cyoD, from a plant-growth-promoting Pseudomonas putida strain, which show strong expression under conditions of low nutrient availability, anoxia, or both. Genes with desired expression characteristics were identified using Tn5-gus transcriptional fusions and screening the mutant strains for -glucuronidase activity under different nutritional and environmental conditions using reporter gene assays. Selected strains were used for further studies and gene isolation.
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METHODS |
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The following five classes of culture conditions were used for the detailed expression studies.
(1) Minimal-nutrient studies: half-strength Stanier's medium (Stanier et al., 1966) supplemented with 0·5 % (v/v) glycerol (SG), 0·5 % (w/v) glucose (GLC) or 0·5 % (w/v) monosodium glutamate (MSG).
(2) Low-carbon studies: carbon content was reduced by either 20- or 50-fold fold by supplementing half-strength Stanier's medium with 0·025 % (w/v) glucose (LGLC), 0·025 % (w/v) monosodium glutamate (LMSG) or 0·01 % (v/v) glycerol (LGL).
(3) Phosphate- and nitrate-limitation studies were performed in a similar way to CCR studies of aromatic catabolic operons (O'Connor et al., 1996; Duetz et al., 1997
). The phosphate and nitrate concentrations in SG and GLC media were reduced from the original 20 mM to 2 mM (LP, LPGLC) and 15·12 mM to 1·51 mM (LN, LNGLC), respectively. Ionic strength was maintained by addition of an equimolar concentration of NaCl.
(4) Anoxic studies: cells were cultured in (capped) tubes filled to the brim with SG medium (SGA), supplemented with 10 mM KNO3 as previously described (Sawers, 1991; Zimmerman et al., 1991
).
(5) Root exudate studies were done with half-strength Stanier's medium supplemented with 40 p.p.m. total organic carbon of wheat root exudates (RE).
Preliminary screening for nutrient-responsive genes.
Tn5 with a promoterless -glucuronidase reporter gene (gus) (Sharma & Signer, 1990
) was used to generate insertion mutants in PNL-MK25 and these were screened for enhanced or consistently strong
-glucuronidase (GUS) expression in low-nutrient media (SG-based) as compared to rich medium (LB). Twelve NRM (nutrient-responsive mutant) strains were selected for GUS expression studies, from which three were selected for molecular characterization.
Preparation of wheat root exudates.
Wheat seeds (Carolina Biological Supply) were surface-sterilized and germinated for 4 days as previously described (van Overbeek & van Elsas, 1995). Seedlings were then grown for 6 weeks (16 h light : 8 h dark) either in solid substrate or hydroponically.
Solid substrate growth.
Fifteen seedlings were planted per Phytacon (Sigma-Aldrich Chemicals) containing 350 ml sand/vermiculite (1 : 1) mix. The containers were opened weekly to allow exchange of gases and for addition of 0·1x Stanier's medium supplemented with 0·25 mg CaCl2 ml1 (SC).
Hydroponic growth.
Three hundred seedlings were transferred onto a support mesh placed in a plastic tank filled with 3·5 l SC. An air-stone (of the type used in fish tanks) was placed under the mesh and connected to an air-pump through a 0·22 µm syringe filter. The tank was sealed. SC medium was added by injecting through the 0·22 µm syringe filter. All sterilization, transfer, germination and growth procedures were performed aseptically.
Root exudates were collected from the solid substrate by washing the sand/vermiculite mix twice with three volumes of water. For hydroponic growth, the growth medium in the tank was collected and filtered sequentially through 30 µm, 20 µm, 8 µm and 2·7 µm filter paper to remove particulate matter. Root exudates were concentrated by rotary evaporation, filtered through a 0·22 µm filter, and stored at 20 °C. Total organic carbon content in the root exudates was determined with an O.I. Analytical model 1020A Combustion TOC Analyser at the Wastewater Biotreatment Group, Department of Civil Engineering, National University of Singapore.
Analysis of gus-tagged gene expression.
Tn5-gus-tagged NRM strains were cultured in different test media for pre-conditioning, inoculated at a dilution of 1 : 1000 (v/v) in fresh media, and grown to mid-exponential phase (OD600 0·2). Bacterial cultures were harvested, washed once with 0·1x Stanier's medium, and resuspended in GEB (50 mM sodium phosphate, pH 7·0; 10 mM EDTA; 0·3 % SDS; 0·1 % Triton X-100; 1 mM DTT; 1 mM PMSF). Aliquots were used for both protein and GUS activity determination. Protein quantity was determined using the Bio-Rad Protein Assay Dye Reagent Concentrate with BSA as standard.
GUS activity was determined fluorometrically based on previously described methods (Gallagher, 1992), using a Perkin Elmer LS50B Luminescence Spectrometer. Enzyme activity was expressed as nmol 4-methylumbelliferone formed h1 (µg total protein)1. Mean and standard error values were determined from at least two independent assays with five replicates each. One-way ANOVA followed by Tukey's test were performed to determine significance of difference in gus reporter gene expression between strains.
DNA gel blot analysis of NRM strains.
Genomic DNA from the 12 NRM strains was isolated using a previously described method (Syn & Swarup, 2000). EcoRI- and SalI-digested DNA fragments were separated by electrophoresis and transferred onto Hybond-N (Amersham Pharmacia Biotech) nylon membranes using standard protocols (Sambrook et al., 1989
). aph2 (GenBank accession. no. AF061930) and tetA (GenBank accession. no. J01830) gene fragments were generated with PCR using NRM17 genomic DNA as the template. The gel-purified PCR products were used as templates for 32P-labelled DNA probe synthesis using the Amersham Rediprime II Random Prime Labelling System.
Cloning and sequencing of chromosomal DNA flanking transposon-insertion sites.
Genomic DNAs of NRM5, NRM7 and NRM17 were digested with EcoRI for cloning of the fragment upstream of the transposon; digestion with SalI was used for cloning the downstream fragment. These enzymes were chosen since they cut only once within the transposon (Fig. 1), such that they preserve either an intact gusAaph2 (conferring Km resistance in the upstream region) or a tetAIS50R (conferring Tc resistance in the downstream region). Digested genomic DNA was ligated to pBluescriptII-SK+ and used to transform E. coli JM109 cells. LB agar supplemented with either Km and Amp or Tc and Amp was used to select for clones containing sequences upstream or downstream of the Tn5-gusA transposon, respectively. Plasmids from selected positive clones were isolated and digested with either EcoRI or SalI.
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RESULTS |
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The P. putida gdhA gene
Sequencing of cloned chromosomal DNA flanking the Tn5-gus showed that P. putida NRM5 carried the insertion in the 3' region (position 1285) of the gdhA (glutamate dehydrogenase) gene (Fig. 2a). The partial nucleotide sequence (1095 nt) and deduced amino acid sequence showed 84 % and 82 % identity, respectively, to the Pseudomonas aeruginosa PAO1 gdhA gene. CLUSTALW pairwise alignment analysis (Fig. 2a
) showed that the P. putida Gdh was well conserved, and showed >63 % sequence identity with GdhA of enterobacteria (E. coli, Klebsiella pneumoniae and Salmonella typhimurium); similarity with GdhA from other species was lower (non-enterobacteria, 62 %; fungi, 57 %; archaea, 26 %; mammals, 28 %). A rho-dependent terminator was identified 70 bp downstream of the gdhA stop codon (Fig. 2a
).
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Analysis of the amino acid sequence indicated that the protein possessed a QOR/-crystallin motif (Gonzalez et al., 1993
) in the central region of the protein, with a key difference: the pair of glycine residues conserved in all QOR/
-crystallins is replaced by histidine-cysteine in Nql (Fig. 2b
). Interestingly, the protein also possessed a zinc-ADH signature motif (Jornvall et al., 1987
) in the N-terminus region, which was absent in other QOR/
-crystallins. Phylogenetic relationship analysis suggested that the protein was not closely related to QOR/
-crystallins (data not shown). This ORF was, therefore, named nql (NADPH : quinone oxidoreductase like).
The promoter of the nql gene was predicted to be localized to within a 140 nt region upstream of the initiation codon. A 14 bp partially dyadic sequence (TG-N10-CA) was found centred at the 65 position (Fig. 2b), which closely matched the consensus LysR-type transcriptional regulator binding motif (TG-N9-CA; Schell, 1993
). An ANR box (TTGAT-N5-ATCAA) identical to the E. coli FNR and P. aeruginosa ANR binding motifs (Ye et al., 1995
) was also located immediately preceding the 35 promoter sequence. The region further upstream of the nql promoter was sequenced and found to contain the first 336 nt of an ORF transcribed divergently from nql (Fig. 2b
). The deduced amino acid sequence of this ORF showed strong sequence identity to two efflux pump regulatory proteins, P. putida DOT-T1E TtgS (93 %) and P. aeruginosa PAO1 MexT (91 %). An ORF with no matches to GenBank database entries was located downstream of nql.
The P. putida cyoD gene
In the mutant strain NRM17, Tn5-gus was inserted at position 80 of the 336 nt cyoD (subunit IV of the cytochrome o ubiquinol oxidase complex) gene (Fig. 2c). Sequencing of the region flanking cyoD identified the other members of the five-subunit cytochrome o ubiquinol oxidase complex. The predicted amino acid sequences of all five subunits showed strong similarity to corresponding subunits in P. putida IH-2000, P. aeruginosa PAO1 and E. coli cytochrome ubiquinol oxidases. A KyteDoolittle hydropathy plot (Kyte & Doolittle, 1982
) of CyoD indicated three putative transmembrane regions (data not shown). These transmembrane regions are similar to those of E. coli CyoA determined experimentally using phoA-fusion protein studies (Chepuri & Gennis, 1990
; Chepuri et al., 1990
).
Gene expression under various nutrient-limiting conditions
Detailed analyses of the expression profiles of the three genes under various nutrient conditions (Table 1) were performed. The presence of two other carbon sources, viz. 0·5 % glucose (GLC) or 0·5 % glutamate (MSG), resulted in increased gdhA and cyoD expression compared to that observed in SG. This increased gene expression by a readily metabolized carbon source coupled with previous observations that growth of the other NRM strains in mineral medium was fastest when the culture was supplemented with glutamate (data not shown) suggested that these two genes disrupted by the transposon were associated with growth and metabolism. In contrast, expression of nql was reduced 1·31·6-fold, which suggested possible regulation by a carbon catabolite repression (CCR) mechanism (Table 1
). Under carbon-limiting conditions (0·01 % glycerol, 0·025 % glucose, 0·025 % glutamate, or wheat root exudates), expression of these three genes was comparable to that observed during growth in SG. These data further supported the potential use of the three gene promoters in driving expression of foreign genes in the nutrient-limited soil environment.
The possible effects of CCR were also examined under low phosphate and low nitrate levels. In the presence of glucose or glutamate (LPGLC), expression of gdhA and cyoD was increased 2·6- and 3·9-fold, respectively, while that of nql was reduced 3·1-fold when compared with LP medium (Table 1). When the glycerol in low-nitrate medium (LN) was replaced with glucose (LNGLC), expression of gdhA and nql was reduced by 2·8- and 11·3-fold, respectively, whereas that of cyoD was increased by 2·9-fold. The marked reduction in nql gene expression in the presence of glucose, a preferred carbon source, further supports the hypothesis that this gene is regulated by a CCR mechanism.
Low-oxygen-responsive gene expression
During growth under low-oxygen conditions the expression of nql was highest amongst the genes screened (Table 1), and was upregulated 3·4-fold (Table 1
, SGA compared to SG). In contrast, only slight changes in the expression of gdhA and cyoD were observed expression of gdhA decreased 1·4-fold while expression of cyoD increased by 1·2-fold.
Root-exudate-responsive gene expression
Bacteria were grown in half-strength Stanier's medium supplemented with wheat root exudates to simulate growth in the rhizosphere. The quantity of total organic carbon in eight different root exudate preparations ranged from 0·15 to 0·49 mg per plant. Root exudate was added to the medium at 40 p.p.m. total organic carbon concentration, similar to levels detected in the sand/vermiculite substrate after the seedlings had been grown for 6 weeks. No qualitative differences were observed between the different batches of root exudates with respect to induction of reporter gene expression in the three NRM strains (data not shown). Expression of nql was the highest, followed by cyoD.
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DISCUSSION |
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gdh expression differs in various bacteria
The partial sequence of the P. putida PNL-MK25 gdhA gene showed strong identity to other bacterial glutamate dehydrogenase genes. The expression of the P. putida gdhA gene was carbon quality- and quantity-dependent but was nitrate-independent; expression of gdhA was highest in glutamate-containing, followed by glucose- and glycerol-containing mineral media. Such regulation is similar to that observed in S. typhimurium and Corynebacterium callunae (Brenchley et al., 1975; Ertan, 1992
). The absolute expression of gdhA was lower than that of many of the other genes studied, but it was increased 526-fold, relative to rich medium (LB), under all low-nutrient conditions studied. The increased gdh expression observed during carbon-limiting conditions suggested that P. putida, like E. coli, may also use Gdh for glutamate synthesis when the cell is limited for carbon (Helling, 1994
). The Gdh pathway may also be the preferred pathway in energy-limiting conditions since the GOGAT (glutamine synthetaseglutamate synthase) system requires 20 % higher ATP levels than the Gdh pathway. However, in the case of E. coli, utilization of Gdh occurred when ammonium and phosphate were not limiting. In contrast, the P. putida gdh promoter-probe construct showed strong increase in expression even when the carbon-limited cells were further deprived of phosphate or nitrate. These results suggest that the P. putida gdhA promoter might be suitable for regulating expression of foreign genes in oligotrophic bulk soil.
Regulation of nql gene expression
The observation that nql expression was repressed in the presence of glucose and glutamate coupled with the further decline in expression when glycerol in the low-phosphate (LP) or low-nitrate (LN) media was replaced with glucose (LPGLC or LNGLC) suggested a CCR-like regulation of gene expression. However, it should be noted that this result was based on studies using synthetic media designed to evoke a CCR-type response. nql expression during growth under low-nutrient conditions was comparable to that observed in the presence of root exudates. This suggested that nql was unlikely to be affected by CCR-linked repression during expression in the soil environment. This is unlike several aromatic xenobiotic degradation pathways that are CCR-regulated (Hartline & Gunsalus, 1971; O'Connor et al., 1996
; Duetz et al., 1997
).
Bacteria respond to low oxygen availability by downregulating the expression of aerobic genes and up-regulating anaerobic genes (Lin & Iuchi, 1991; Spiro & Guest, 1991
) as well as slowing or stopping growth (Stretton & Goodman, 1998
). nql was, however, found to be synergistically upregulated by low-carbon and low-oxygen conditions: expression of nql was increased 4·5-fold in SG (compared to LB) and further increased 3·4-fold under anoxic conditions (SGA compared to SG; Table 1
), yielding a total induction of 15·3-fold in the presence of both conditions relative to rich media (LB). As in the case of Gdh, Nql may represent a potential parallel pathway for balancing growth efficiency during nutrient-limited growth as suggested by Helling (1994)
.
The 4·5-fold increase in nql gene expression under minimal conditions is similar to that observed for the E. coli qor gene (Tao et al., 1999). The further increase of nql during anoxic growth suggested a synergistic interaction between the two conditions. An ANR box, similar to the E. coli FNR and P. aeruginosa ANR binding motifs (Ye et al., 1995
), was located immediately preceding the 35 promoter sequence, which suggested that the anaerobic induction of nql was mediated by ANR (anaerobic transcriptional regulator). The region upstream of the nql gene showed strong sequence similarity to P. aeruginosa MexT, which is an efflux pump regulatory protein. MexT is a member of the LTTR family (LysR-type transcriptional regulator; Kohler et al., 1999
) and genes controlled by LTTRs are often located adjacent to their activators, which are transcribed in the opposite direction (Schell, 1993
). Given these observations, it is possible that this upstream regulatory protein also regulates the nql promoter. This hypothesis is supported by the presence of the consensus LTTR binding sequence centred at the 65 position of the nql gene.
cyo expression is nutrient-responsive and oxygen-independent
The cytochrome o oxidase complex has been extensively studied in E. coli (Chepuri et al., 1990) but similar studies in other bacteria have been very limited. Among the pseudomonads, the cyo operon has only recently been cloned from another strain of P. putida, IH-2000 (Hirayama et al., 1998
). Our cyoDgus transcriptional fusion studies indicated that expression of the cyo operon in P. putida was carbon-quality-dependent: expression was highest when the cells were grown in glutamate, followed by LB broth, glucose and glycerol, consistent with a CCR-like regulation. This contrasts with previous reports that cytochrome o levels in E. coli are regulated by cAMP, which is indicative of a global transcriptional regulation mechanism (Minagawa et al., 1990
; Tao et al., 1999
). Interestingly, cyo expression in P. putida was oxygen-independent (only 1·2-fold greater for SGA versus SG; Table 1
), which again contrasts with E. coli, where expression of the cyo operon is derepressed under high oxygen (Kranz & Gennis, 1983
; Minagawa et al., 1990
). Our results suggest that expression of cyo in P. putida may be influenced more by nutrient conditions than by oxygen tension, somewhat like regulation in Bacillus subtilis, where expression of the qoxABCD operon is highest during growth in rich medium (Liu & Taber, 1998
).
In the present study, the nql and cyoD genes were consistently amongst the most highly expressed under the variety of low-nutrient conditions tested. Their promoters are thus of potential use in driving the expression of foreign genes in various soil conditions where carbon, nitrogen, phosphorus or a combination of these nutrients is typically scarce. Additionally, the strong inducibility of the nql promoter under anoxic conditions makes it particularly suited for driving the expression of foreign genes in water-saturated soils and other conditions where oxygen is scarce.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atlas, R. M. (1991). Bioremediation of fossil fuel contaminated soils. In In Situ Bioreclamation: Applications and Investigations for Hydrocarbon and Contaminated Site Remediation, pp. 1432. Edited by R. E. Hinchee & E. F. Olfenbuttel. Boston, MA: Butterworth-Heinemann.
Brenchley, J. E., Baker, C. A. & Patil, L. G. (1975). Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium. J Bacteriol 124, 182189.[Medline]
Cases, I. & de Lorenzo, V. (1998). Expression systems and physiological control of promoter activity in bacteria. Curr Opin Microbiol 1, 303310.[CrossRef][Medline]
Chepuri, V. & Gennis, R. B. (1990). The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli. J Biol Chem 265, 1297812986.
Chepuri, V., Lemieux, L., Au, D. C. T. & Gennis, R. B. (1990). The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases. J Biol Chem 265, 1118511192.
Dashman, T. & Stotzky, G. (1986). Microbial utilization of amino acids and a peptide bound on homoionic montmorillonite and kaolinite. Soil Biol Biochem 18, 514.
Diaz, E. & Prieto, M. A. (2000). Bacterial promoters triggering biodegradation of aromatic pollutants. Curr Opin Biotechnol 11, 467475.[CrossRef][Medline]
Dowling, D. N. & O'Gara, F. (1994). Metabolites of Pseudomonas involved in the biocontrol of plant disease. Trends Biotechnol 12, 133140.
Duetz, W. A., Wind, B., Kamp, M. & van Andel, J. G. (1997). Effect of growth rate, nutrient limitation and succinate on expression of TOL pathway enzymes in response to m-xylene in chemostat cultures of Pseudomonas putida (pWWO). Microbiology 143, 23312338.
Edwards, K. J., Barton, J. D., Rossjohn, J., Thorn, J. M., Taylor, G. L. & Ollis, D. L. (1996). Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases. Arch Biochem Biophys 328, 173183.[CrossRef][Medline]
Ertan, H. (1992). The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae. Arch Microbiol 158, 4247.[Medline]
Ferenci, T. (1999). Regulation by nutrient limitation. Curr Opin Microbiol 2, 208213.[CrossRef][Medline]
Gallagher, S. R. (1992). GUS Protocols: Using the gus Gene as a Reporter of Gene Expression. San Diego, CA: Academic Press.
Gonzalez, P., Rao, P. V. & Zigler Jr J. S. (1993). Molecular cloning and sequencing of zeta-crystallin/quinone reductase cDNA from human liver. Biochem Biophys Res Commun 191, 902907.[CrossRef][Medline]
Greenland, D. J. (1971). Interactions between humic and fulvic acids and clays. Soil Science 111, 3441.
Hartline, R. A. & Gunsalus, I. C. (1971). Induction specificity and catabolite repression of the early enzymes in camphor degradation by Pseudomonas putida. J Bacteriol 106, 468478.[Medline]
Helling, R. B. (1994). Why does Escherichia coli have two primary pathways for synthesis of glutamate? Microbiology 176, 46644668.
Hirayama, H., Takami, H., Inoue, A. & Horikoshi, K. (1998). Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000. FEMS Microbiol Lett 169, 219225.[CrossRef][Medline]
Hojberg, O., Schnider, U., Winteler, H. V., Sorensen, J. & Haas, D. (1999). Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil. Appl Environ Microbiol 65, 40854093.
Jensen, L. E. & Nybroe, O. (1999). Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere. Appl Environ Microbiol 65, 43204328.
Jornvall, H., Persson, B. & Jeffrey, J. (1987). Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167, 195201.[Abstract]
Kohler, T., Epp, S. F., Curty, L. K. & Pechere, J. C. (1999). Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. J Bacteriol 181, 63006305.
Kozlowski, T. T., Kramer, P. J. & Pallardy, S. G. (1991). The Physiological Ecology of Woody Plants. San Diego: Academic Press.
Kragelund, L., Hosbond, C. & Nybroe, O. (1997). Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere. Appl Environ Microbiol 63, 49204928.[Abstract]
Kranz, R. G. & Gennis, R. B. (1983). Immunological characterization of the cytochrome o terminal oxidase from Escherichia coli. J Biol Chem 258, 1061410621.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Leahy, J. G. & Colwell, R. R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiol Rev 54, 305315.[Medline]
Lin, E. C. C. & Iuchi, S. (1991). Regulation of gene expression in fermentative and respiratory systems in Escherichia coli and related bacteria. Annu Rev Genet 25, 361387.[CrossRef][Medline]
Liu, X. & Taber, H. W. (1998). Catabolite regulation of the Bacillus subtilis ctaBCDEF gene cluster. J Bacteriol 180, 61546163.
Marschner, P. & Crowley, D. E. (1996a). Physiological activity of a bioluminescent Pseudomonas fluorescens (strain 2-79) in the rhizosphere of mycorrhizal and non-mycorrhizal pepper (Capsicum annuum L.). Soil Biol Biochem 28, 869876.[CrossRef]
Marschner, P. & Crowley, D. E. (1996b). Root colonization of mycorrhizal and non-mycorrhizal pepper (Capsicum annuum) by Pseudomonas fluorescens 2-79RL. New Phytol 134, 115122.
Meikle, A., Amin-Hanjani, S., Glover, A., Killham, K. & Prosser, J. I. (1995). Matric potential and the survival and activity of a Pseudomonas fluorescens inoculum in soil. Soil Biol Biochem 27, 881892.[CrossRef]
Minagawa, J., Nakamura, H., Yamato, I., Mogi, T. & Anraku, Y. (1990). Transcriptional regulation of the cytochrome b562-o complex in Escherichia coli: gene expression and molecular characterization of the promoter. J Biol Chem 265, 1119811203.
Normander, B., Hendriksen, N. B. & Nybroe, O. (1999). Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere. Appl Environ Microbiol 65, 46464651.
O'Connor, K., Duetz, W., Wind, B. & Dobson, A. D. (1996). The effect of nutrient limitation on styrene metabolism in Pseudomonas putida CA-3. Appl Environ Microbiol 62, 35943599.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sawers, R. G. (1991). Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli. Mol Microbiol 5, 14691481.[Medline]
Schell, M. A. (1993). Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47, 597626.[CrossRef][Medline]
Sharma, S. B. & Signer, E. R. (1990). Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA. Genes Dev 4, 344356.[Abstract]
Spiro, S. & Guest, J. R. (1991). Adaptive responses to oxygen limitation in Escherichia coli. Trends Biochem Sci 16, 310314.[CrossRef][Medline]
Stanier, R. Y., Palleroni, N. J. & Doudoroff, M. (1966). The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43, 159271.[Medline]
Stretton, S. & Goodman, A. E. (1998). Carbon dioxide as a regulator of gene expression in microorganisms. Antonie van Leeuwenhoek 73, 7985.[CrossRef][Medline]
Syn, C. K. C. & Swarup, S. (2000). A scalable protocol for the isolation of large-sized genomic DNA within an hour from several bacteria. Anal Biochem 278, 8690.[CrossRef][Medline]
Tao, H., Bausch, C., Richmond, C., Blattner, F. R. & Conway, T. (1999). Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J Bacteriol 181, 64256440.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Timmis, K. N. & Pieper, D. H. (1999). Bacteria designed for bioremediation. Trends Biotechnol 17, 200204.[CrossRef][Medline]
Vande Broek, A., Michiels, J., van Gool, A. & Vanderleyden, J. (1993). Spatial-temporal colonization patterns of Azospirillum brasilense on the wheat root surface and expression of bacterial nifH gene during association. Mol PlantMicrobe Interact 6, 592600.
van Overbeek, L. S. & van Elsas, J. D. (1995). Root exudates-induced promoter activity in Pseudomonas fluorescens mutants in the wheat rhizosphere. Appl Environ Microbiol 61, 890898.[Abstract]
van Overbeek, L. S. & van Elsas, J. D. (1997). Adaptation of bacteria to soil conditions: applications of molecular physiology in soil microbiology. In Modern Soil Microbiology, pp. 441477. Edited by E. M. H. Wellington, J. T. Trevors & J. D. van Elsas. New York: Marcel Dekker.
Williams, S. T. (1985). Oligotrophy in soil: fact or fiction? In Bacteria in the Natural Environment: the Effect of Nutrient Conditions, pp. 81110. Edited by M. Fletcher & G. Floodgate. London: Academic Press.
Ye, R. W., Haas, D., Ka, J. O., Krishnapillai, V., Zimmerman, A., Baird, C. & Tiedje, J. M. (1995). Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J Bacteriol 177, 36063609.[Abstract]
Zimmerman, A., Reimann, C., Galimand, M. & Haas, D. (1991). Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depends on anr, a regulatory gene homologous with fnr of Escherichia coli. Mol Microbiol 5, 14831490.[Medline]
Received 27 November 2003;
accepted 15 January 2004.
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