INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

During aerobic growth, bacteria consume O₂ at high rates. The consumption of O₂ by oxidases takes place on the cytoplasmic side of the membrane. Since the diffusion of O₂ across the membrane is rapid, the supply of the oxidases with O₂ is guaranteed even at the very low O₂ tensions which are sufficient for aerobic growth (<1 mbar of O₂) (2, 4, 15, 16). Previously, the rate of O₂ diffusion into the cytoplasm of Escherichia coli was calculated from the cell dimensions and the diffusion coefficients and compared to the rates of O₂ consumption (2, 21, 22). It was estimated that at O₂ tensions as low as 0.2 mbar of O₂ (corresponding to 0.2 µM O₂), the supply of O₂ by diffusion exceeds the consumption by respiration. In agreement with this calculation, in E. coli the fermentation pathways were synthesized and used only at partial O₂ tension (pO₂) values well below 1 mbar of O₂ (3). Thus, O₂ is able to reach the active sites of the oxidases at rates sufficient to support aerobic respiration even at very low O₂ tensions.

The O₂ supply of the cytoplasmic space is not known and might be different from that of the membrane where the oxidases are located. From the diffusional parameters and the cell dimensions, it was calculated that the concentrations of O₂ should be the same within and outside the bacteria at O₂ tensions as low as 1 mbar of O₂ (21, 22). Therefore, we aimed for an experimental proof of the availability of O₂ in the bacterial cytoplasm under aerobic and microaerobic conditions.

For the degradation of aromatic compounds like benzoate, oxygenases are required for oxidative cleavage of the aromatic ring (7, 10). Due to the cytoplasmic location of the oxygenases and the need for molecular oxygen as a cosubstrate, the turnover of aromatic compounds depends on the availability of O₂ in the cytoplasm. The rate of metabolism of aromatic compounds therefore provides information on the minimal rate of O₂ diffusion into the cytoplasm. To this end, the relation of metabolism of various aromatic compounds to the pO₂ of the medium was studied. Pseudomonas putida KT2442 degrades benzoate by benzoate-1,2-dioxygenase and catechol-1,2-dioxygenase (ortho pathway), whereas 4-hydroxybenzoate is degraded via 4-hydroxybenzoate monooxygenase and protocatechuate-3,4-dioxygenase (ortho cleavage). 4-Methylbenzoate is metabolized by P. putida mt-2 by toluate-1,2-dioxygenase and catechol-2,3-dioxygenase (meta cleavage) (5, 8). The K_m values for O₂ of the oxygenases (7 µM) (1, 6, 12, 13) are much higher than those of the oxidases (<0.1 µM) (4, 15, 16). Therefore, limitation of growth or catabolism by O₂ must be due to the oxygenases, and information on O₂ diffusion into the cytoplasm and the O₂ concentration in the cytoplasm can be drawn from the growth-limiting pO₂ values. Here we report on experimental proof of the availability of O₂ in the cytoplasm. This finding also provides a basis for our understanding of the O₂ sensing by cytoplasmic O₂ sensor proteins like FNR (fumarate nitrate reductase regulator) from E. coli (9, 19, 22, 23) and the homologous proteins from Pseudomonas (17, 25) which are supposed to react directly with O₂ in the cytoplasm (2, 22, 23).

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Bacteria and media. P. putida KT2442 and P. putida mt-2(pWWO) were provided by I. Wagner-Döbler (Braunschweig, Germany) and M. Schlömann (Stuttgart-Hohenheim, Germany) (5, 24). P. putida KT2442 was grown in a modified M9 mineral medium (pH adjusted to 7.1) supplemented with a mineral salts solution and with glucose, succinate, benzoate, or 4-hydroxybenzoate (10 mM each) as sources of carbon and energy. The mineral salts solution was a combination of the following: solution 1, containing 25.39 g of MgCl₂, 2.0 g of CaCO₃, 4.5 g of FeSO₄ · 7H₂O, 0.85 g of MnSO₄ · H₂O, 1.44 g of ZnSO₄ · 7H₂O, 0.25 g of CuSO₄ · 5H₂O, 0.16 g of CaSO₄ · 0.5H₂O, and 0.02 g of H₃BO₃ dissolved in 51.3 ml of concentrated HCl and with water added to 100 ml; solution 2, containing 360 mM FeSO₄ · 7H₂O; and solution 3, containing 1 M MgSO₄. Solutions 1 and 2 were filter sterilized, and solution 3 was autoclaved. Then 50 ml of solution 1, 2.5 ml of solution 2, 25 ml of solution 3, and 22.5 ml of autoclaved H₂O were combined. The medium was supplemented with 0.25 ml of the resulting mineral salts solution per 100 ml. P. putida mt-2(pWWO) was grown in a phosphate-buffered medium (14.0 g of Na₂HPO₄ · 12H₂O, 2.0 g of KH₂PO₄ per liter) supplemented with a salts solution (20 ml/liter of medium) containing 2.5 g of Ca(NO₃) · 4H₂O (autoclaved separately) per liter, 0.5 g of Fe(III)NH₄-citrate per liter, 10 g of MgSO₄ · 7H₂O per liter, 50 g of (NH₄)₂SO₄ per liter, and 50 ml of the Pfennig SL6 metal salts solution (14) per liter. The C source for P. putida mt-2 was 4-methylbenzoate (10 mM). E. coli MC4100 (18) was grown in M9 medium (11) supplemented with an amino acid mixture (20) and glucose (10 mM) or succinate (10 mM).

Growth. P. putida was grown at 30°C. Growth under anaerobic conditions was performed in sealed bottles under an atmosphere of nitrogen (2, 3). For aerobic conditions, the bacteria were grown in Erlenmeyer flasks filled to within 10% of the maximal volume under vigorous shaking (3). The medium was inoculated from cultures grown overnight under aerobic conditions in the mineral medium (same C source as that in the main culture) to an A₅₇₈ not higher than 0.06.

Growth in an oxystat. Growth at defined O₂ tensions (pO₂) was performed in an oxystat (chemostat with constant pO₂) (Biostat MD; Braun, Melsungen, Germany) in batch culture (1.5 liters) with constant stirring (400 rpm) (2, 3). The pO₂ value of the medium was measured continuously with an O₂ electrode. The pO₂ was maintained at a constant level by supplying air (valve I) and N₂ (valve II) to the vessel. When the pO₂ fell below 98% of the set value, valve I opened and sterile air was supplied till the set value was reached. The flow of air was increased manually from about 0.16 to 1.6 liters min¹ during growth to compensate for the increasing O₂ consumption. The flow of N₂ (0.1 liters min¹) was decreased or switched off as required. E. coli was grown in the oxystat in the supplemented M9 medium as described previously (2, 3). Growth rates were calculated from µ = ln (A_578,t2/A_578,t1) · (t₂  t₁)¹, where t₂ and t₁ are the times of measurement and A_578,t1 and A_578,t2 are the absorbance values at 578 nm measured at t₁ and t₂, respectively.

Analytical procedures. Substrates (glucose, succinate, benzoate, 4-hydroxybenzoate, and 4-methylbenzoate) and products (catechol, protocatechuate) were determined from the supernatants of the cultures after removal of the bacteria by centrifugation. The substances were analyzed by high-performance liquid chromatography (HPLC) on an Aminex HPX87H column (300 by 7.8 mm; Bio-Rad) with 6.5 mM H₂SO₄ as the eluent (flow rate, 0.55 ml min¹) as described previously (20). The following substrates and products were determined and quantified with standard solutions by a refractive index and by a UV light detector (215 nm): glucose, glycerol, acetate, ethanol, formate, pyruvate, fumarate, succinate, and lactate. Benzoate (retention time [R_t] = 68 min), 4-hydroxybenzoate (R_t = 51 min), catechol (R_t = 32.0 min), and protocatechuate (R_t = 33.3 min) were identified by the R_t values of authentic substances, and the ratio of the refractive index/UV absorption at 215 nm was used to confirm the identities.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Growth of P. putida on aromatic compounds at limiting pO₂. P. putida was grown on nonaromatic and aromatic substrates like glucose, succinate, and benzoate in an oxystat at defined pO₂ values. In the oxystat, the set pO₂ values could be maintained constant for the duration of the growth experiment. With glucose or succinate as the substrate, the growth behavior changed only when the pO₂ fell below 10 mbar of O₂ (corresponding to about 10 µM O₂). At lower pO₂ values, the growth rate and final cell density decreased, and under anaerobic conditions, no growth was observed. With benzoate or 4-hydroxybenzoate as the substrate, under aerobic conditions (212 mbar of O₂; air saturation), growth of P. putida (Fig. 1A) was similar to that on glucose or succinate. However, with decreasing pO₂ values, growth rate and yield decreased significantly.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Growth (A) and growth rates (B) of P. putida KT2442 grown in an oxystat on aromatic and nonaromatic substrates as a function of pO2. (A) Growth with benzoate in the oxystat at different pO2 values. Growth was performed in the mineral medium with 10 mM benzoate at 212 (), 21 (), 8 (), 6 (), 4 (), and 0 () mbar of O2. (B) The rate constants for growth (µ) were determined from the growth curves shown in panel A. Substrates (10 mM each) for growth: benzoate (), 4-hydroxybenzoate (), glucose (), and succinate ().

1

1

pO_0.5 values for growth on aromatic substrates are higher than those for growth on nonaromatic substrates. For P. putida, from the relation of the growth rates to the pO₂ values, the pO_0.5 values for the substrates can be determined. The pO_0.5 value corresponds to the pO₂ value yielding half-maximal growth rates (2, 3). The measured pO_0.5 values can be classified into two groups. For growth of P. putida and E. coli on glucose and succinate, low values (pO_0.5  2 mbar of O₂) were found. For growth on aromatic compounds, the pO_0.5 values were distinctly higher and corresponded to about 8 mbar for growth on benzoate and 4-hydroxybenzoate and to 19 mbar for growth on 4-methylbenzoate.

Excretion of intermediates under O₂ limitation. The growth medium was analyzed for products or intermediates excreted by the bacteria during growth in the oxystat at different pO₂ values (Fig. 2). The medium was analyzed by HPLC for the presence of organic acids, alcohols, sugars, and aromatic compounds, in particular for intermediates of the respective metabolic routes. During growth at high oxygen tensions, all types of substrates were completely oxidized by P. putida and no organic end products were detected in significant amounts (>0.05 mol/mol of substrate). From glucose and succinate, no end products were excreted even at decreased oxygen tensions, indicating complete oxidation. When P. putida KT2442, however, was grown on benzoate, a product was found in the medium at oxygen tensions below 20 mbar which was identified as catechol. Catechol is an intermediate of the ortho cleavage pathway of benzoate. Up to 0.65 mol of catechol per mol of benzoate was measured, indicating a severe limitation in the ortho cleavage pathway resulting in the excretion of the intermediate without complete oxidation. During growth on 4-hydroxybenzoate, protocatechuate was excreted in large amounts (0.28 mol/mol of 4-hydroxybenzoate) at oxygen tensions below 20 mbar. Obviously, limitation of protocatechuate-3,4-dioxygenase activity (6) by low O₂ tensions causes accumulation and excretion of the intermediate protocatechuate. Therefore, in both pathways, central steps, i.e., the dioxygenases reacting on catechol and protocatechuate, are limiting under microaerobic conditions.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Products in the culture medium of P. putida KT2442 excreted after growth at various pO2 values in the oxystat: catechol () excreted during growth on benzoate (10 mM), protocatechuate () excreted during growth on 4-hydroxybenzoate, and end products () from succinate and glucose. For end products tested, see Materials and Methods.

Availability of O₂ as an intracellular substrate for aromatic substrate degradation. The data can be used to roughly estimate the rate of O₂ diffusion into the cells required for this process. The rate of O₂ consumption by the oxygenases in the cell interior (_O2in) is twice the rate of benzoate metabolism (_benzoate) (Table 1) corresponding to 0.22 mmol of benzoate · min¹ · g (dry weight)¹ and 0.44 mmol of O₂ · min¹ · g (dry weight)¹. The calculated rate of O₂ diffusion into the cells under aerobic conditions, 360 mmol of O₂ · min¹ · g (dry weight)¹ (Table 1), exceeds the rate of intracellular O₂ consumption by the oxygenases by 3 orders of magnitude.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Growth rate, O2 consumption by dioxygenases, and maximal rate of O2 diffusion into the bacteria (P. putida KT2442) as a function of the pO2 in the medium during growth on benzoate. The growth rate was determined as described in the legend to Fig. 1. The O2 consumption by the dioxygenases was calculated from µ and from the molar growth yield on benzoate (Ybenzoate) under the respective conditions as shown in Table 1. The rate of diffusion into the bacteria was calculated as a function of external oxygen concentration (with the internal concentration set at zero). For the calculation, the diffusion coefficients of O2 in water, phospholipid, and cytoplasm and the cell dimensions were used (2, 22).