U.S. Air Force Research Laboratory, Tyndall
Air Force Base, Florida 32403-5323
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INTRODUCTION |
p-Nitrophenol (PNP) is a
priority environmental pollutant (10), occurring in
industrial effluents (20) and in the soil as a hydrolytic
product of parathion (14) or methyl parathion (17,
21). Several aerobic pure cultures of bacteria belonging to
species of Flavobacterium, Pseudomonas,
Moraxella, Nocardia, and Arthrobacter
metabolize PNP with removal of the nitro group as nitrite (7, 8,
19, 22, 26). Two alternative pathways that convert PNP to
maleylacetate have been elucidated for aerobic PNP degradation
(24). The first pathway is more common in gram-negative isolates and results in the formation of hydroquinone from PNP, probably via 1,4-benzoquinone, with concomitant nitrite release. Hydroquinone is oxidized by a ring-cleaving dioxygenase to yield 
-hydroxymuconic semialdehyde, which is subsequently transformed to
maleylacetate (26). In the second catabolic pathway, an
Arthrobacter sp. hydroxylates PNP to produce either
4-nitrocatechol or 4-nitroresorcinol. Subsequent oxidative removal of
the nitro group yields 1,2,4-trihydroxybenzene (THB) with concomitant
release of nitrite. The THB is oxidized by a ring cleavage dioxygenase
to yield maleylacetate, which is converted enzymatically to
3-ketoadipate (8). While a complete pathway for PNP
degradation via hydroquinone has been described in detail, the initial
steps in the pathway involving conversion of PNP to THB are not fully
understood.
Oxidative removal of the nitro groups from nitroaromatic compounds has
been described for several degradative pathways (24). A
preliminary characterization of p-nitrophenol-2-hydroxylase, which catalyzes the conversion of PNP to 4-nitrocatechol, was reported
in cell extracts from a Nocardia sp. (13). A
particulate monooxygenase from a Moraxella sp. that releases
nitrite from PNP has been partially purified (26). Zeyer and
Kocher (32) purified a soluble nitrophenol oxygenase from
Pseudomonas putida B2 that converts
ortho-nitrophenol to catechol and nitrite. Ecker et al.
(4) suggested the involvement of a dioxygenase attack in the
displacement of a nitro group from 2,6-dinitrophenol. Recently, 4-methyl-5-nitrocatechol oxygenase has been purified from
Burkholderia sp. strain DNT (6).
4-Methyl-5-nitrocatechol oxygenase oxidizes 4-methyl-5-nitrocatechol to
a quinone with concomitant release of nitrite.
We report here a preliminary characterization of a novel monooxygenase
from Bacillus sphaericus JS905 that catalyzes the first two
steps in the degradation of PNP via 4-nitrocatechol and THB. The enzyme
consists of two components, a flavoprotein reductase and an oxygenase,
and catalyzes two sequential monooxygenation reactions that convert PNP
to THB. The first reaction converts PNP to 4-nitrocatechol, and the
second removes the nitro group. The reactions are very specific, and
the enzyme does not release nitrite directly from PNP.
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MATERIALS AND METHODS |
Organism and culture conditions.
A gram-positive, motile rod
with round terminal spores, lacking fluorescent pigments, was isolated
by selective enrichment with PNP from an agricultural soil in India
with a history of methyl parathion application. The strain, identified
as B. sphaericus JS905, based on morphological and
biochemical characteristics (Institute of Microbial Technology,
Chandigarh, India), was maintained on minimal salts medium (MSB)
(25) containing 15 mg of PNP, 200 mg of yeast extract, and
18 g of agar per liter. For induction with PNP, cells grown in
0.75% (wt/vol) tryptic soy broth (TSB) were harvested by filtration,
washed, and suspended in MSB containing PNP (150 µM) and yeast
extract (0.1%). The cultures were incubated at 37°C with shaking
(300 rpm), and the disappearance of PNP was monitored by
high-performance liquid chromatography (HPLC). For preparation of cell
extracts, cells were cultivated in 4 liters of TSB overnight, harvested
by centrifugation at 7,000 × g, and suspended in MSB
containing PNP (150 µM) to an A600 of 1.5. The cell suspension was incubated with shaking at 250 rpm. Additional PNP
(150 µM) was added when the yellow color of the PNP disappeared, and
the sequence was repeated six to eight times over 3 h. Ice was
added to the cell suspensions, and the culture was harvested by
centrifugation. The cell pellet was washed with Tris-HCl (50 mM, pH
7.6) and stored at 
70°C until further use.
Respirometry.
Cells were grown for induction as described
above, harvested by centrifugation, and suspended in MSB. Uninduced,
TSB-grown cells served as controls. Oxygen uptake was measured
polarographically at 25°C with a Clark-type oxygen electrode.
Preparation of cell extract.
Frozen cells (18 g [wet
weight]) were suspended in 2 volumes (wt/vol) of lysis buffer that
consisted of Tris-HCl (50 mM, pH 7.6), ethanol (2.5%, vol/vol),
glycerol (2.5%, vol/vol), and flavin adenine dinucleotide (FAD; 10 µM). Cells were broken by two passages through a French pressure cell
at 20,000 lb/in2. The resulting lysate was centrifuged at
100,000 × g for 1 h at 4°C, and the supernatant
was used immediately.
Partial purification of PNP monooxygenase.
All procedures
were carried out at 4°C unless otherwise specified. The clarified
cell extract was loaded onto a DEAE-Sepharose fast-flow column (2.5 by
14 cm; Pharmacia Biotech, Piscataway, N.J.) that had been equilibrated
with TEF buffer (50 mM Tris-HCl [pH 7.6], 0.25% [vol/vol] ethanol,
2 µM FAD). The column was washed with 150 mM NaCl in TEF buffer at a
flow rate of 1.5 ml/min. Bound proteins were eluted with a 270-ml
linear NaCl gradient (150 to 500 mM). Fractions (3 ml each) exhibiting
maximal nitrite release from PNP or 4-nitrocatechol were pooled and
concentrated over a Centriplus 100 (Amicon, Danvers, Mass.)
concentrator to a final volume of 3 ml. The protein solution was
diluted 1:3 in TEF buffer and applied to a Q-Sepharose fast-flow column
(1.0 by 10 cm; Pharmacia). The column was washed with 100 ml of 200 mM
NaCl in TEF buffer, and the adsorbed proteins were eluted with a linear
NaCl gradient (80 ml, 200 to 400 mM) at a flow rate of 1.0 ml/min. The
fractions containing the enzyme activity were pooled and concentrated
to 1.5 ml over a Centriplus 100 filter. Glycerol (10%, vol/vol) was added, and the sample was applied to a Sephacryl S-300 column (1.5 by
107 cm; Pharmacia) preequilibrated with 100 mM NaCl in TEF buffer. The
proteins were resolved by ascending chromatography at a flow rate of
1.0 ml/min with the same buffer.
Enzyme assays.
PNP monooxygenase activity was determined by
measuring the nitrite released from the substrate (PNP or
4-nitrocatechol) at 30°C. The standard reaction mixture contained in
1 ml of TE buffer (50 mM Tris-HCl, 0.25% ethanol, pH 8), 0.2 mM NADH,
0.02 mM FAD, 1 mM MgSO4, and various amounts of protein.
The reaction was initiated by addition of either PNP or 4-nitrocatechol
(0.08 mM). The substrates were omitted from the control reaction
mixtures. After 30 min, nitrite was determined by the method adapted by
Daniels et al. (3).
1,2,4-Trihydroxybenzene 1,2-dioxygenase activity was measured either
spectrophotometrically or polarographically at 25°C in reaction
mixtures containing phosphate buffer (20 mM, pH 6.8) and protein. The
reaction was started by addition of 100 µM THB.
NADH-2,6-dichlorophenolindophenol reduction, cytochrome c
reduction, and nitroblue tetrazolium reduction were measured
spectrophotometrically (5). The 1.0-ml reaction mixture
contained 50 mM TE buffer (pH 8.0), substrate (0.1 mM
2,6-dichlorophenolindophenol, 0.1 mM nitroblue tetrazolium, or 0.05 mM
cytochrome c) and 2 to 100 µg of protein. Concentrations
of reduced 2,6-dichlorophenolindophenol were calculated based on the
A600 using a molar extinction coefficient of
17,000 M
1 cm1.
Molecular weight determination.
The relative molecular
masses of the native proteins were determined by gel filtration on a
Sephacryl S-300 column (1.5 by 107 cm; Pharmacia) at a flow rate of 1.0 ml/min with 100 mM NaCl in TEF buffer. The calibration standards were
ferritin, catalase, aldolase, and ovalbumin (Pharmacia)
(12). Partially purified proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (11), and
the gels were stained with Coomassie brilliant blue to visualize
proteins.
Inhibition studies.
PNP-induced cells were suspended to an
A600 of 1.5 in 50 ml of MSB containing 0.1%
yeast extract, 0.2 mM PNP or 4-nitrocatechol, and 1 mM either
2,2'-dipyridyl or o-phenanthroline for inhibition of THB
oxidation. Nitrite release was determined, and the THB formed was
detected by HPLC. In a separate experiment, cell extracts prepared in
phosphate buffer (20 mM, pH 7.2) containing no added FAD were incubated
with an inhibitor at 30°C for 15 min prior to the addition of NADH,
FAD, MgSO4, and a substrate (PNP or 4-nitrocatechol) to the
reaction mixture.
Analytical techniques.
HPLC was performed on a Spherisorb
C8 column (5 µm; 250 by 4.6 mm; Altech, Deerfield, Ill.)
with an HP 1040A diode array detector (Hewlett-Packard Corp., Palo
Alto, Calif.) for detection of PNP or its metabolites.
Acetonitrile-water containing 13.5 mM trifluoroacetic acid (40:60) was
the mobile phase at a flow rate of 1.0 ml/min. Compounds were
identified by comparison of HPLC retention times and UV-visible spectra
to those of standards. Protein concentrations were determined by the
bicinchoninic acid protein assay (23).
Isolation and characterization of 4-nitrocatechol from PNP
hydroxylation.
4-Nitrocatechol was extracted from a reaction
mixture with ethyl acetate and characterized by HPLC-mass spectrometry
(MS) analysis (HP 1050 LC with an HP 5987 mass selective detector) using a particle beam interface (HP 59980A). All spectra were generated
by electron impact. HPLC conditions were as described above.
Chemicals.
Nitrophenols were purchased from Aldrich
(Milwaukee, Wis.). Methimazole, miconazole, metyrapone, and
-naphthoflavone were from Sigma. All other chemicals were of the
highest purity commercially available.
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RESULTS AND DISCUSSION |
Biodegradation of PNP.
A lag period of 3.5 h preceded
rapid degradation of PNP when TSB-grown cells of B. sphaericus JS905 were transferred to media containing PNP (Fig.
1). Nitrite was released in
stoichiometric amounts, and no other degradation products were detected
by HPLC. PNP-grown cells released stoichiometric amounts of nitrite and produced a purple compound when incubated in media containing PNP or
4-nitrocatechol and iron chelators such as 2,2'-dipyridyl or
o-phenanthroline. The color was identical to that produced when 2-hydroxy-1,4-benzoquinone was added to reaction mixtures (8), and HPLC analysis confirmed the presence of the quinone in the culture fluid. 2,2'-Dipyridyl is known to inhibit certain aromatic ring cleavage enzymes that require ferrous ions for their activities (1). It was also shown that PNP-grown
Moraxella sp. converted PNP stoichiometrically to
hydroquinone in the presence of 2,2'-dipyridyl (26),
presumably due to the inhibition of a dioxygenase that catalyzes ring
fission of hydroquinone. The reaction mixture was decolorized upon
addition of sodium dithionite, indicating the reduction of
2-hydroxy-1,4-benzoquinone to THB (2).
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FIG. 1.
Disappearance of PNP and release of nitrite during
induction. Cells were grown overnight in 0.75% TSB, washed, and
suspended in MSB supplemented with PNP (0.18 mM) to an
A600 of 0.87. The suspension was incubated at
37°C with shaking, and culture fluids were analyzed at intervals by
HPLC for the disappearance of PNP and by a colorimetric assay for
nitrite.
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PNP, 4-nitrocatechol, and THB stimulated oxygen consumption by PNP- and
4-nitrocatechol-grown cells (Table 1).
4-Nitroresorcinol stimulated oxygen uptake to a lesser extent.
Hydroquinone, 4-aminophenol, catechol, and resorcinol did not stimulate
oxygen uptake. Oxygen uptake was not detected with TSB-grown cells. The
results indicate that enzymes for PNP degradation are inducible and
strongly suggest that 4-nitrocatechol and THB are the intermediates in
the catabolic pathway for PNP in B. sphaericus JS905.
Enzyme activities in cell extracts.
Under aerobic conditions,
extracts of PNP-grown cells catalyzed the release of nitrite in
stoichiometric amounts from PNP or 4-nitrocatechol. The activity in
cell extracts was present in the soluble fraction and represented 75%
of the activity observed in intact cells. The rate of nitrite release
by cell extracts was linear for at least 30 min and was not directly
related to protein concentration (data not shown), which suggested that
the oxygenase might be a multicomponent enzyme. The enzymatic activity was dependent on the presence of NAD(P)H and FAD and was not stimulated by the addition of flavin mononucleotide. NADH was the preferred cofactor when residual cofactor was removed from cell extracts by
passage over a desalting PD-10 column (Pharmacia) or fractionation with
(NH4)2SO4. Magnesium ions enhanced
the enzyme activity. Extracts prepared without added FAD lost 52% of
their activity towards PNP and 4-nitrocatechol during storage on ice
for 48 h. The addition of FAD (2 µM) to chromatography buffers
was necessary to maintain full activity. The removal of the nitro group
from both substrates was maximum at pH 8 in 50 mM Tris-HCl buffer.
Addition of 0.25% ethanol to the buffer stabilized the activity during
desalting or dialysis. The presence of 
-mercaptoethanol was
inhibitory to the enzyme activity. The rate and extent of nitrite
release were the same in cell extracts whether PNP or 4-nitrocatechol was used as the substrate.
THB stimulated high rates of oxygen consumption by cell extracts, and
the stoichiometry was 0.93 ± 0.02 mol of O2 per mol of substrate. The enzyme catalyzing the oxidation of THB was stable during storage at 4°C. Spectrophotometric assays revealed the disappearance of THB and the appearance of a new compound with an
absorbance maximum at 243 nm identical to that of maleylacetate (2, 26). When NADH was included in the reaction mixture, the
absorbance peak at 243 nm disappeared, presumably due to the activity
of maleylacetate reductase. The results indicate that THB served as a
substrate for a ring cleavage dioxygenase, as evidenced by the
characteristic spectral changes and the rapid consumption of oxygen
with THB. The observed spectral changes suggest an ortho
ring fission of THB yielding 3-ketoadipate with maleylacetate as an
intermediate (2, 8).
Identification of the products of PNP oxidation.
Cell extracts
incubated in potassium phosphate buffer with NADPH converted PNP to a
yellow metabolite. Addition of 2.5 N NaOH to the reaction mixture after
incubation at 30°C gave a deep red solution, which is a
characteristic of 4-nitrocatechol (13). A large-scale
reaction was performed to confirm the identity of the product. The HPLC
retention time, UV spectrum, and MS fragmentation pattern of the
isolated compound were identical to those of authentic 4-nitrocatechol.
Both a Flavobacterium sp. (19) and a
Nocardia sp. (13) have been shown to convert PNP
to 4-nitrocatechol, but the details of the oxidative release of nitrite
were not presented. A recent study of the conversion of PNP to THB by
Arthrobacter sp. strain JS443 (8) did not clarify
whether 4-nitrocatechol or 4-nitroresorcinol was the product of the
initial reaction of PNP catabolism. The result presented here clearly
indicates that the initial reaction in the bacterial degradation of PNP
by B. sphaericus JS905 is hydroxylation of the ring at the 2 position to yield 4-nitrocatechol. PNP degradation in the reaction
mixtures containing cell extracts in 20 mM potassium phosphate buffer
and NADPH resulted in near-stoichiometric accumulation of both
4-nitrocatechol and nitrite (Fig. 2). No
other aromatic products accumulated, which indicates that some of the
4-nitrocatechol was further converted with release of nitrite. In
contrast, reactions carried out in Tris-HCl yielded negligible amounts
of 4-nitrocatechol in the presence of NADPH. When FAD was included in
the reaction mixtures, nitrite, but not 4-nitrocatechol, accumulated,
regardless of which buffer was used. The results suggest that in assays
with phosphate buffer, the rate of oxidation of 4-nitrocatechol from
PNP was so slow that 4-nitrocatechol accumulated in the reaction
mixture. There was no accumulation of 4-nitrocatechol or release of
nitrite from PNP when the reaction mixture was incubated under
anaerobic conditions, which confirmed that the hydroxylation of PNP is
an oxidation reaction requiring molecular oxygen.
Cell extracts incubated for 2 h with PNP or 4-nitrocatechol in the
presence of 2,2'-dipyridyl or o-phenanthroline accumulated THB or 2-hydroxy-1,4-benzoquinone. This supports earlier observations that intact cells contained oxygenases that converted PNP and/or 4-nitrocatechol to THB. However, it was not clear whether
2-hydroxy-1,4-benzoquinone or THB was the actual product of nitrite
release from 4-nitrocatechol. A quinone reductase may have catalyzed
the conversion of 2-hydroxy-1,4-benzoquinone to THB, as has been
suggested for PNP (27) or o-nitrophenol (32) biodegradation.
Separation of enzyme components.
The activities for oxidative
removal of the nitro group from PNP or 4-nitrocatechol coeluted during
ion-exchange chromatography of cell extract on a DEAE-Sepharose
fast-flow column, which suggested that the same enzyme catalyzes both
reactions. Further purification by Q-Sepharose chromatography enriched
for the enzyme (Table 2); however, size
exclusion chromatography on Sephacryl S-300 of Q-Sepharose fractions
led to substantial loss of enzymatic activity. The activity was
regained when two components that eluted separately (designated A and B
in the order of their elution) were combined (Fig.
3). This indicated that the enzyme was a
two-component system.
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FIG. 3.
Resolution of components A and B of PNP monooxygenase by
size exclusion chromatography on a Sephacryl S-300 column. Proteins
were eluted in 2-ml fractions with 100 mM NaCl in 50 mM TEF buffer.
Component A (reductase) was located by measuring the oxidative release
of nitrite from PNP in reaction mixtures containing component B (4 µg
of protein) (fraction 57). Component B (oxygenase) was located by
measuring nitrite release in assay mixtures containing component A (23 µg of protein) (fraction 52).  , A280.
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Fractions containing component A catalyzed NAD(P)H-dependent reduction
of 2,6-dichlorophenolindophenol or nitroblue tetrazolium but not
cytochrome c. NADPH was also an electron donor for the reductase activity. A flavoprotein and a second protein of the ferredoxin type are required for cytochrome c reductase
activity (9). The fact that the reductase component of the
monooxygenase reduces 2,6-dichlorophenolindophenol and nitroblue
tetrazolium but not cytochrome c and the lack of spectral
characteristics of an iron-sulfur center (30) suggest the
absence of a second prosthetic group (9). The
NADH-2,6-dichlorophenolindophenol reduction activity coeluted with
component A from the S-300 column. The fraction with an elution volume
of 97 ml which showed maximal nitrite release activity (when combined
with component B) also had maximum reductase activity (2.9 µmol/min/mg of protein). No absorption spectrum in the visible region
was observed for the dialyzed component A, suggesting that the flavin
cofactor was readily removed during dialysis. The addition of FAD, but
not flavin mononucleotide, was essential for the NADH-dependent
2,6-dichlorophenolindophenol reduction by the dialyzed component A. The
results strongly suggest that component A is a flavoprotein. Component
B appears to be the hydroxylase and showed no reductase activity with
the electron acceptors tested.
Cytochrome P-450 inhibitors (-naphthoflavone, miconazole, and
metyrapone) at 0.5 mM had very little effect (<5% inhibition) on
nitrite release from PNP or 4-nitrocatechol by the reconstituted mixture containing components A and B. Methimazole, a competitive inhibitor of flavin monooxygenases (28), at the same
concentration, greatly inhibited (58% inhibition) the enzymatic
release of nitrite from the substrates. The above observations argue
strongly that the enzyme system contains a flavoprotein reductase.
Based on the elution volumes of the active fractions containing
components A and B on the S-300 column, the molecular masses of the
native proteins were estimated to be 323 and 146 kDa, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the active components showed the presence of
several bands. Efforts to purify the components to homogeneity failed.
Both components A and B are essential for NADH-dependent oxidative
removal of the nitro group from PNP or 4-nitrocatechol (Table
3). THB was not detected in reaction
mixtures containing both protein components and PNP or 4-nitrocatechol,
even though the nitro group was removed from the substrates. The
failure of THB to accumulate can be explained by the presence of
THB-1,2-dioxygenase, which was detected in preparations of component A
(data not shown). In contrast to the results obtained with cell
extracts, NADPH did not support PNP or 4-nitrocatechol oxidation. Since
the enzyme system consisted of two components that catalyzed two
consecutive monooxygenation reactions, we designated it PNP
monooxygenase. The reductase component of PNP monooxygenase has a
loosely bound FAD that is readily lost during the purification process.
Like most of the hydroxylases (13, 15, 18, 29, 31), PNP
monooxygenase uses FAD as the redox chromophore and is NADH dependent.
In contrast, nitrophenol monooxygenases (26, 32) and
4-methyl-5-nitrocatechol oxygenase (6) use NADPH as the
preferred electron donor.
Experiments with the partially purified PNP monooxygenase revealed that
the enzyme has a narrow substrate range. 4-Nitroresorcinol also served
as a substrate with a rate of nitrite release similar to that observed
with the two physiological substrates, PNP and 4-nitrocatechol.
o-Nitrophenol, m-nitrophenol, 2-nitroresorcinol, and 2,4-dinitrophenol were not transformed.
Several lines of evidence indicate that a single monooxygenase system
catalyzes both the initial hydroxylation of PNP and subsequent
oxidative release of the nitro group from 4-nitrocatechol. (i) The
relative activities of the enzyme toward PNP and 4-nitrocatechol remained constant throughout several purification steps and during a
variety of manipulations, including inhibition. (ii) Both PNP and
4-nitrocatechol are converted to THB or 2-hydroxy-1,4-benzoquinone. (iii) Enzymes in cell extracts catalyze the conversion of PNP to
4-nitrocatechol in phosphate buffer. The fact that 4-nitrocatechol did
not accumulate in reaction mixtures containing partially purified enzyme and Tris buffer suggests that the 4-nitrocatechol is bound to
the enzyme until nitrite is released. Based on the accumulation of
2-hydroxy-1,4-benzoquinone during transformation of PNP or 4-nitrocatechol by cells and cell extracts incubated with iron chelators and on analogy with other systems (26, 32), we
suggest that 2-hydroxy-1,4-benzoquinone is an intermediate in the
pathway (Fig. 4).
Several bacterial monooxygenase enzymes that attack aromatic compounds
are multicomponent systems. 4-Hydroxyphenylacetate 3-hydroxylase, a
two-component, NADH-dependent flavomonooxygenase, has been isolated
from Escherichia coli W (18). Chlorophenol 4-monooxygenase from B. cepacia AC1100 is another
two-component enzyme system (31). A three-component enzyme,
toluene 2-monooxygenase, has been purified from B. cepacia
G4 (15). Toluene in P. mendocina KR1 is initially
hydroxylated by toluene-4-monooxygenase, a three-component enzyme
system, to form p-cresol (29). The conversion of
phenol to catechol by Pseudomonas strain CF600 is catalyzed
by a multicomponent phenol hydroxylase (16).
Sequential hydroxylation by a single enzyme system has been reported in
at least two instances. Toluene 2-monooxygenase oxidizes toluene to
o-cresol and then to 3-methylcatechol (15).
Chlorophenol 4-monooxygenase catalyzes the sequential hydroxylation of
2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and
then to 5-chlorohydroxyquinol (31). To our knowledge, PNP
monooxygenase from B. sphaericus JS905 is the only known
monooxygenase to sequentially hydroxylate a nitroaromatic compound. In
contrast to the chlorophenol-4-monooxygenase, the enzyme hydroxylates
the ring adjacent to the hydroxyl group first and then displaces the
nitro group.
The evolution of the pathway for PNP biodegradation by B. sphaericus JS905 would have been markedly simplified because the first two reactions are catalyzed by a single enzyme system. Such a
strategy would also minimize the accumulation of potentially toxic
4-nitrocatechol. The key to the reaction might be the ability of the
molecule to form the 1,4-quinone structure upon elimination of the
negatively charged leaving group.
We thank Mike Henley for help with HPLC-MS analysis and Billy E. Haigler, Charles C. Somerville, and Urs Lendenmann for useful discussions. Venkateswarlu Kadiyala thanks the Sri Krishnadevaraya University, Anantapur, India, for granting study leave.
This research was supported by the Air Force Office of Scientific
Research and the Strategic Environmental Research and Development Program. Venkateswarlu Kadiyala is grateful to the National Research Council for the award of a Senior Research Associateship.
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Abstract
Introduction
