Journal of General Microbiology (1982), 128,49-59.
Printed in Great Britain
The Role of Oxygen in the Regulation of Glucose Metabolism, Transport
and the Tticarboxylic Acid Cycle in Pseudomonas aeruginosa
By COLIN G. MITCHELL? AND EDWIN A. DAWES*
Department o f Biochemistry, University o f Hull, Hull HU6 7RX, U.K.
(Received 21 May 1981)
The effect of dissolved oxygen concentration on the metabolism of glucose in Pseudomonas
aeruginosa was studied with chemostat cultures using both single-step and gradual transitions
from either ammonium or glucose limitation to oxygen limitation and studying transient and
steady states. The pathway of glucose metabolism was regulated by the availability of
oxygen. The organism responded to oxygen limitation by adjusting its metabolism of glucose
from the extracellular direct oxidative pathway, which produces gluconate and 2-
oxogluconate, to the intracellular phosphorylative route. This change was a consequence of
decreased activities of glucose dehydrogenase and gluconate dehydrogenase and of the
transport systems for gluconate and 2-oxogluconate, and an increased activity of glucose
transport, while relatively high activities of hexokinase and glucose-6-phosphate dehydro-
genase were maintained. Citrate synthase, isocitrate dehydrogenase and malate dehydro-
genase activities responded to changes in dissolved oxygen concentration rather than to
changes i n the glucose or ammonium concentrations. The effect of oxygen limitation on the
0x0-acid dehydrogenases and aconitase was probably due, wholly or in part, to repression by
glucose consequent upon the increase in residual glucose concentration. Succinate
dehydrogenase was repressed by an increase in ammonium concentration under an oxygen
Studies on the effects of dissolved oxygen concentration on the enzymic activities and
metabolic versatility of obligate aerobes are relatively meagre compared with those on
facultative anaerobes. This is understandable in view of the diversity of fermentation patterns
encountered with the latter organisms on their transition from aerobic to anaerobic conditions
(for recent reviews, see Harrison, 1976; Stouthamer, 1978; Jones, 1979). However, in their
natural habitat obligate aerobes encounter a wide range of oxygen concentrations.
Nitrogen-fixing azotobacters exhibit the phenomenon of respiratory protection; in these
organisms high oxygen concentrations are toxic, inhibiting nitrogenase (Parker, 1954;
Phillips & Johnson, 1961; Dalton & Postgate, 1969). Studies on the effect of oxygen on the
enzymic complement of Azotobacter chroococcum and Azotobacter vinelandii have been
carried out (Drozd & Postgate, 1970; Haaker & Veeger, 1976). We have previously shown
that the energy-reserve polymer poly-/I-hydroxybutyrate (PHB) accumulates in Azotobacter
beijerinckii when cultures become oxygen-limited; the reductive stage of polymer synthesis
serves as an alternative electron acceptor when oxygen is no longer so readily available, and
thus permits the organism to grow under such conditions (Senior et al., 1972; Senior &
Dawes, 1973; Ward et al., 1977). The activities of key enzymes of PHB metabolism and of
certain enzymes of the tricarboxylic acid cycle responded to changes in oxygen concentration
Present address: Department of Biochemistry, University of Bath. Claverton Down, Bath BA2 7AY. U.K.
0022-1287/82/0000-9962 $02.00 O 1982 SGM
C . G . MITCHELL AND E. A. DAWES
(Jackson & Dawes, 1976) but the Entner-Doudoroff enzymes were not affected (Stephenson
et al., 1978; Carter & Dawes, 1979).
The observed regulation of tricarboxylic acid cycle activity by oxygen concentration in A.
beijerinckii posed the question of whether this behaviour was representative of obligate
aerobes in general or whether it was a manifestation of respiratory protection and therefore
characteristic only of nitrogen-fixing organisms. As we have previously studied the aerobe
Pseudomonas aeruginosa, which is a denitrifier that utilizes glucose via the Entner-
Doudoroff and tricarboxylic acid cycle pathways, we chose this organism for a comparative
study of oxygen effects. Pseudomonas aeruginosa also metabolizes glucose via an
extracellular direct oxidative pathway to gluconate and 2-oxogluconate (Ng & Dawes, 1973;
Midgley & Dawes, 1973; Roberts et al., 1973; Whiting et al., 1976a, b). Transport of
glucose, gluconate and 2-oxogluconate occurs by independently regulated systems (Whiting
et al., 1976 a) and the extracellular oxidative enzymes and associated transport systems are
repressed when the organism is transferred from ammonium to glucose limitation (Whiting et
al., 1976b). Hunt & Phibbs (1977) observed that the extracellular route was also repressed
when P. aeruginosa was grown anaerobically with nitrate as electron acceptor in batch
culture. It was of interest to examine the effect of varying oxygen concentration on the
pathways of glucose metabolism in P. aeruginosa under the controlled conditions of the
chemostat. We have thus examined the effect of transitions from animonium or glucose
limitation to oxygen limitation on various enzymes of glucose metabolism and the
tricarboxylic acid cycle, and also on the transport systems for glucose, gluconate and
Organism and growth. Pseudomonas aeruginosa PA0 1 was kindly provided by Professor B. W. Holloway.
Routine maintenance, batch growth of the organism and harvesting procedures were as previously described (Ng
& Dawes, 1973; Midgley & Dawes, 1973). For chemostat inocula, the organism was subcultured at least ten times
in the appropriate medium. All cultures were grown at 37 "C.
The chemostat vessel (2.5 1 volume) was built in this Department. It was fitted with automatic pH control
(E.I.L. Instruments, Richmond, Surrey; with pH electrodes supplied by Activion, Kinglassie, Fife), temperature
control (Fielden Electronics, Manchester) and oxygen control (Leeds & Northrup, Birmingham; Precision
Products and Controls, Tulsa, U.S.A.). The oxygen electrodes were made in this Department. COz was measured
with an infrared analyser (Mine Safety Appliances, Glasgow) and oxygen with a paramagnetic oxygen analyser
(Servomex OA 13 7; Servomex Controls, Crowborough, Sussex). Continuous readout of gas composition was
obtained with a Kent chart recorder (George Kent, Luton, Beds). The total gas flow was 11 min-' and the re-
quired dissolved oxygen tension (d.0.t.) was secured by adjusting the proportion of oxygen in the inflowing oxygen/
nitrogen gas mixture. Oxygen limitation (undetectable d.0.t.) occurred at 4.5 % (v/v) oxygen in the inflowing gas.
Medium for continuous cultivation was prepared in 40 1 batches which were sterilized by filtration through a
Sartorius filter (142 mm diam., pore size 0.25 pm) at 292 kPa. The medium contained (per litre): KH,PO,, 5.4 g;
nitrilotriacetic acid, 0.286 g; (NH,),SO,, 0.9 g (for ammonium-limited growth) or 1-8 g (for glucose- and
oxygen-limited growth); trace metal solutions 1 (5 ml), 2 (5.25 ml) and 3 (0.1 ml) (Ng & Dawes, 1973); glucose,
4.0 g (for carbon-limited growth) or 8.11 g (for ammonium-limited growth). For most of the single-step transitions
from ammonium- or glucose-limited growth to oxygen limitation, the inflowing medium was simultaneously
changed to furnish excess ammonium or glucose. Initial studies were carried out to ascertain the effect of such
additions of ammonium or glucose, as noted in the Results. In the gradual transition experiments with
ammonium-limited cultures, the inflowing medium was changed to provide excess ammonium as soon as
ammonium ions could be detected in the medium supernatant; this occurred at approximately 9% oxygen in the
inflowing gas. In similar experiments with glucose-limited Cultures, glucose was detected in the supernatant at
between 6 and 8 % oxygen in the inflowing gas and the medium was changed at 4 % oxygen to ensure an excess of
Six to ten vessel volumes were allowed to pass through the fermenter between each steady state before sampling
except in experiments to study transient responses.
Transport studies. These were performed by the methods of Midgley & D8wes (1973) using the substrate
concentrations and specific radioactivities specified by Whiting et al. (1976 6). The radioactivity was assayed as
described by Midgley & Dawes (1973).
EfSect o f oxygen on glucose metabolism
glutarate. Metabolic and taxonomic significance. WHITING, P. H., MIDGLEY, M. & DAWES, E. A.
FEBS Letters 3, 265-267.
WHITING, P. H., MIDGLEY, M. & DAWES, E. A.
( 1 976 a). The regulation of transport of glucose,
gluconate and 2-oxogluconate and of glucose
catabolism in Pseudomonas aeruginosa. Bio-
chemical Journal 154,659-668.
(1976b). The role of glucose limitation in the
regulation of the transport of glucose, gluconate and
2-oxogluconate, and of glucose metabolism in
Pseudomonas aeruginosa. Journal o f General
Microbiology 92, 304-3 10.