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Journal of General Microbiology (1984), 130, 1097-1 105. Printed in Great Britain
1097
Isolation, Characterization and Autotrophic Metabolism
of a Moderately Thermophilic Carboxydobacterium,
Pseudomonas thermocarboxydovorans sp. nov.
By C A T H E R I N E M. LYONS,' P A U L I N E J U S T I N , , J O H N COLBY2
A N D E D W I N WILLIAMS'*
Microbiology Department, The Medical School, The University,
Newcastle-upon-Tyne NEl 7RU, UK
Biology Department, Sunderland Polytechnic, Sunderland SRl 3SD, UK
(Received 21 October 1983; revised 6 January 1984)
Three strains of Gram-negative, obligately aerobic, moderately thermophilic, CO-utilizing
bacteria have been isolated by enrichment at 45-55 "C in mineral medium under CO, H2 and
air. The properties of the strains, designated C2, S1 and FE, suggest that they should be included
in a single species that we have named Pseudomonas thermocarboxydovorans.The isolates can
grow on CO and a limited range of organic and amino acids; neither sugars nor H2/C02support
growth and €I2 is not oxidized. Strain C2 contains a soluble, inducible C0:phenazine ethosulphate oxidoreductase, lacks hydrogenase and assimilates CO via ribulose bisphosphate
carboxylase. The organism apparently contains a mechanism for assimilating phosphoglycollate.
INTRODUCTION
Carbon monoxide (CO) is a colourless, odourless, sparingly water-soluble gas. It is extremely
toxic to aerobes because of its affinity for the metal ions of respiratory chain components.
Although the atmospheric concentration of CO is low and constant (0.03-0.90 p.p.m.), it has
been estimated that anthropogenic and natural sources produce 12-14 x lo8 t per annum,
suggesting that CO is rapidly converted into other compounds (Robbins et al., 1968). Although a
variety of non-biological oxidation processes have been proposed, it is thought that CO-utilizing
bacteria play a significant role in soils and surface waters (Conrad & Seiler, 1980). The activity
of such bacteria, however, cannot alone account for the oxidation of atmospheric CO by soil
(Conrad & Seiler, 1982).
Some bacteria, although unable to grow on CO, perform a gratuitous oxidation by accepting
CO as a pseudosubstrate for an enzyme system evolved to catalyse another process (e.g. methane
monoxygenase; Colby et al., 1977). Other species designated carboxydobacteria and including
Pseudomonas carboxydovorans, P . carboxydoflava, P . carboxydohydrogena, P . gazotropha,
Cornomonas compransoris and Achromobacter carboxydus, can utilize CO as sole carbon and
energy source (Kim & Hegeman, 1983; Meyer & Schlegel, 1983). These bacteria are Gramnegative, mostly mesophilic and, with the exception of A . carboxydus, able to grow autotrophically with hydrogen (H,) and carbon dioxide (CO,). This paper describes the isolation,
characterization and growth of three strains of a novel thermophilic species of carboxydobacterium.
Abbreviations: API 20E, API ZYM and API 5OCH refer to the bacteriological test strips supplied by API
Laboratory Products, Grafton Way, Basingstoke, Hants, UK ; PES, phenazine ethosulphate; PHB, poly-Qhydroxybutyrate; DCPIP, 2,6-dichlorophenolindophenol.
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1098
C . M . LYONS A N D OTHERS
METHODS
Growth medium and conditions. The isolation medium consisted of mineral base E (Owens & Keddie, 1969)
supplemented with the following vitamins (1- l ) : thiamin hydrochloride, 0.5 mg ; calcium pantothenate, 0.5 mg ;
nicotinic acid, 0.5 mg; biotin, 0.1 pg; riboflavin, 0.5 mg; vitamin BIZ,2.5 pg; p-aminobenzoic acid, 10 pg; folic
acid, 1.0 pg; pyridoxal hydrochloride, 2 mg. Subsequently this 'vitamin cocktail' was replaced by 10 pg p-aminobenzoic acid I - ' only. Media were solidified with 1.5% (w/v) Oxoid purified agar. Cultures were grown on
solidified medium in stainless steel jars (Don Whitely Scientific, Baildon, Shipley, W. Yorks, UK) or in 50 ml
mineral medium contained in 250 ml conical flasks with Quickfit necks (B19/26) and sealed with 'Suba-seal'
stoppers. The jars were partly evacuated and refilled with the appropriate gases to give the concentrations
indicated in Results and Discussion. Gases were introduced into the flasks through the stoppers by syringe. CO
and Hz, both CP (certified-pure) grade, were obtained from BOC Special Gases, Deer Park Road, London, UK.
C 0 2 was obtained from Air Products, New Malden, Surrey, UK. Flasks were either incubated statically or at
200 r.p.m. on an orbital shaker. The initial isolations were done at a range of temperatures but subsequently 50 "C
was adopted as the standard growth temperature.
Isolations. Samples of sewage (0-1 ml) were added to 20 ml growth medium and incubated statically under an
atmosphere of approximately 25% CO, 25% H2 and 50% air (by vol.) at temperatures of 25 "C, 37 "C, 45 "C and
55 "C. Turbid cultures. (0.1 ml) were subcultured into 20 ml of the same medium and incubated under the same
conditions. Serial dilutions of 1 in 10 were made from these flasks and plated onto mineral medium solidified with
1-1.5% (w/v) Oxoid purified agar, Difco Bacto agar or Difco Noble agar. Plates were incubated for 1 week as
described, and single colonies were restreaked and purified.
Characterization ofpurijied isolates. Standard methods (Collins & Lyne, 1970; Norris & Swain, 1971)were used
for the Gram stain, spore stain, crystal violet, malachite green and Sudan black stains, and the oxidase, catalase
and motility tests. The cyst stain was as described by Vela & Wyss (1964) and the Nile blue A stain for poly-phydroxybutyrate (PHB) was the method of Ostle & Holt (1982). The size and shape of cells was determined by
examining phosphotungstic acid-stained Formvar films using an AEI 6B electron microscope. Nitrate reduction
was determined using 0.2% sodium pyruvate as carbon source. The API 20E galleries were used for some
biochemical tests; the recommended procedure was followed except that the incubation temperature was 50 "C.
The ability of the strains to produce certain enzymes was determined using the API ZYM test strips; again the
incubation temperature was 50 "C. DNA was isolated and purified by the method of Marmur (1961). The G + C
ratio was determined by the melting temperature method (Marmur & Doty, 1962) using a Beckmann DU-8
spectrophotometer ; Escherichia coli DNA was the standard. Potential carbon and nitrogen sources were tested by
adding them to mineral medium at concentrations between 0.05% and 0.2%. Carbon sources were tested in the
usual solidified mineral medium containing ammonium sulphate as nitrogen source. Nitrogen sources were tested
in liquid mineral medium lacking fixed nitrogen but containing 0.2% pyruvate as carbon source. A range of sugars
was also tested by using the API 50CH galleries and following the method described in the instructions, except
that the incubation temperature was 50 "C.The dinitrogen-fixing activity of cells removed from ammonia-limited
chemostat cultures grown on pyruvate was estimated as acetylene reduction (Postgate, 1972)using 50 mM-sodium
pyruvate as energy source.
Estimation of PHB. Strain C2 was grown in batch and chemostat culture as described below. Batch cultures
were harvested during the onset of stationary phase, chemostat cultures at steady-state. Cells were collected by
centrifugation, washed once with distilled water and freeze-dried. For the estimation of PHB, 20 mg freeze-dried
material was assayed by the spectrophotometric method of Law & Slepecky (Herbert et al., 1971). Alternatively,
PHB granules released by alkaline hypochlorite treatment of the freeze-dried material were hydrolysed by boiling
with 2 M-NaOH for 30 min and the samples neutralized with 2 M-HCl.The resulting crotonic acid was estimated
by HPLC on a column (25 cm x 0.5 cm) of Lichrosorb RP8 (LKB Instruments) equilibrated with 0.1 M
NaH2P04,pH 2.1, and eluted with the same solvent at 1 ml min- l . A UV monitor operated at 206 nm (LKB
Uvichord S) was used to detect the crotonic acid. This second method was quick, simple and reliable but routinely
gave values l0-15% lower than those given by the spectrophotometric method.
Isolation of virulent bacteriophage. A modification of the hydroxyapatite technique of Primrose & Day (1977)
was used. Sewage (4 1) from the primary (aerobic) settlement tank at Barker's Hough sewage works at Durham,
UK, was clarified by centrifugation at 18000g for 30 min. Hydroxyapatite slurry (10 ml) was added and the
sewage shaken slowly at 30 "Con an orbital shaker for 20 h. The hydroxyapatite was then collected by centrifugation at 1800g for 30min and the phage eluted by resuspending the hydroxyapatite in 10ml 0.4M-sodium
phosphate buffer, pH 7.2. The suspension was centrifuged at 6000g for 30 min and the supernatant retained.
Experiments with coliphage showed this method to concentrate phage from the sewage some 300-fold with 80%
yield. The concentrated suspension was tested for phage against strain C2 by a standard soft agar overlay method.
Phage suspension (0.1 ml) and exponential-phase C2 culture grown on 0.2% pyruvate (0.1 ml) were mixed with
3 ml molten top agar (0.6% purified agar in mineral medium) and poured onto a 0.2% pyruvate mineral agar plate
for incubation at 37 "C. This lower incubation temperature was used because it was found that phage could not be
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Physiology of therrnophilic carboxydobacteria
1099
recovered from cultures incubated at 50 "C. The presence of virulent phage in the suspension was detected by
plaque formation and the phage purified by repeated platings in the same way. Only one phage, designated phage
19, was isolated and it was subsequently tested against the other strains.
Determination of CO, C 0 2 ,H 2 and oxygen. The concentrations of H2,oxygen, and CO in gas samples withdrawn
from flasks were determined by gas chromatography at 35 "C on a 1.5 m column (internal diameter 4 mm) of
molecular sieve 13X (Phase Separations, Deeside Industrial Estate, Queensferry, Clwyd, UK). C 0 2 was
determined on a similar column of Porapak R. Nitrogen (30ml min- ') was used as carrier gas for the determination of hydrogen; otherwise helium (30ml min-I) was the carrier gas. The gas chromatograph (Pye-Unicam
model 104 with thermal conductivity detector) was calibrated by injecting samples of the pure gases and of dilutions in air.
Measurement ofgas utilization by cultures. Quickfit conical flasks (250ml nominal volume) capped with 'Subaseal' stoppers and containing 25 ml mineral medium were gassed with nitrogen and then additions of CO, O2and
H2 were made by injection through the stoppers. The flasks were placed on a shaking waterbath at 50 "C and the
experiment started by inoculating each flask with 1 ml of an exponential-phase culture grown under an
atmosphere of 25% CO, 25% H2 and 50% air (by vol.). Control flasks were inoculated with sterile medium only.
Samples of the gas phase (0-2ml) were removed at intervals and, at the same time, 1 ml samples of the culture were
removed to determine cell density by measuring OD540.
Bulk growth ofstrain C2for physiological studies. Cultures were grown in mineral medium containing twice the
usual ammonium sulphate concentration (except for ammonia-limited cultures, which contained half the usual
concentration) and with CO (usually 33%, v/v, in air) or sodium pyruvate (0.3%) as carbon and energy source.
Chemostat and batch cultures (3 1 or 2 1) were grown at 50 "C in a magnetically driven 4 1 fermenter (Ultroferm,
LKB Instruments) equipped with pH control and dissolved oxygen measurement (using a galvanic electrode) or in
a similar 2 1 fermenter (Bioengineering, Caterham, Surrey, UK). Both fermenters were housed in fume cupboards.
Cultures were inoculated with 100 ml exponential-phase culture grown on CO or on pyruvate. Except for oxygenlimited chemostat cultures, CO-grown cultures were gassed with 33% (v/v) CO in air at a flow rate of
240 ml min- The gases were supplied via separate flowmetersand allowed to mix in a 250 ml Buchner flask prior
to entering the culture via a stainless steel sparger located beneath the stirring blades. For oxygen-limited chemostat cultures, the proportion of CO in the input gas and the stirring-rate were adjusted as necessary to observe a
dissolved oxygen concentration of 0-2%. Batch cultures were harvested when OD540reached 1.2,chemostat
cultures at steady-state (dilution rate was normally 0.05h- '). Washed suspensions were prepared by centrifuging
the culture at 5400 g for 30 min, washing with 20 mM-sodium phosphate buffer, pH 7 (for oxygen electrode studies)
or ice-cold 20 mM-Tris/HCl buffer, pH 7 (cell-free studies) and resuspending in the same. Cell extracts were
prepared from washed suspensions by sonication (MSE 100 W ultrasonic disintegrator) in 10 ml amounts for ten
periods of 30 s separated by 30 s cooling periods. The crude extract was centrifuged at 10 000 g for 10 min to
remove unbroken cells and then separated into soluble and particulate fractions (S35 and P35, respectively) by
centrifuging at 35000g for 60 min.
Measurement of whole-cell oxidations. Oxygen uptakes were measured in a Rank oxygen electrode chamber
(Rank Bros, Bottisham, Cambs, UK) at 50 "C. Incubation mixtures (final vol. 3 ml) contained varying amounts of
washed bacterial suspension, phosphate buffer and substrate solutions (0.1 ml 100 mM-sodium pyruvate or 1.5 ml
water saturated with CO or H2). Reactions were started by injecting the washed suspension. The output from the
electrode was calibrated using gas mixtures of known oxygen partial pressure. The solubility of oxygen in water at
50 "C was taken to be 2.46 ml per 100 ml.
Enzyme assays. All assays were done at 50 "C. A Pye-Unicam SP8-100spectrophotometer and a Beckman
LS7500 liquid scintillation counter were used. Ribulose bisphosphate carboxylase activity was determined by
measuring the Dribulose 1,5-bisphosphate-dependentincorporation of NaHI4CO3 into acid-stable material
(Taylor, 1977). CO :PES oxidoreductase activity was determined spectrophotometrically under anaerobic
conditions using phenazine ethosulphate (PES) and 2,6-dichlorophenolindophenol(DCPIP) as primary and
secondary electron acceptors, respectively. Reaction mixtures (3 ml) in Teflon-stoppered cuvettes contained
0.15 mmol Tris/HCl buffer, pH 7.0,15 pmol PES, 0.26 pmol DCPIP. Reaction mixtures were bubbled with CO
and then the reaction started by the addition of degassed S35extract. Control reaction mixtures were gassed with
nitrogen instead of CO or contained boiled extract. NAD-linked hydrogenase was assayed similarly but with
NAD (0.1-5.0mM) in place of the dyes. Thionin-linked hydrogenase activity was determined as described by Kim
& Hegeman (1981).Hzwas also tested in the CO oxidoreductase assay in place of CO. Hydroxypyruvate reductase
and hexulosephosphate synthase were assayed spectrophotometrically (Colby & Zatman, 1975). Phosphoglycollate phosphatase was assayed as described by Anderson & Tolbert (1966)except that 50 mM-MES buffer,
pH 6.0, was used.
Protein estimation. The protein concentration of cell extracts and cell suspensions was estimated using a
modification of the Lowry method (Kennedy & Fewson, 1968).Cell suspensions were digested for 20 h at 30 "C in
0.66 M-NaOH prior to the determination.
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1100
C . M . LYONS A N D OTHERS
RESULTS A N D DISCUSSION
Isolation of CO-utilizing strains
Enrichment cultures of sewage, from a primary (aerobic) settlement tank or from final
effluent, became turbid within 7-10 d when statically incubated at 45 "C or 55 "C with a gas
atmosphere of 25 % CO, 25 % H2 and 50% air (by vol.). Turbid cultures from the primary enrichment flasks, when subcultured in the same mineral medium and incubated under the same
conditions, again became turbid within 2-3 d and utilized both the CO and H2 constituents of
the gas mixture (Fig. 1). Fourteen pure cultures were obtained from these secondary enrichments by diluting and plating on mineral medium solidified with Oxoid purified agar (1 -5%).
Three representative strains from different enrichments (designated C2, S 1 and FE) were
chosen for further study and 50 "C was adopted as the standard incubation temperature for all
three isolates. Yellow-brown, undulate and umbonate colonies of 3 mm diameter were produced
on well-dried plates after 7 d incubation at this temperature. Medium solidified with Difco
Bacto or Noble agar failed to support good growth of the strains.
Characterization of the pure cultures
The characteristics of pure cultures of C2, S1 and FE are summarized in Table 1. The three
strains were Gram-negative, oxidase positive and catalase positive, rod-shaped bacteria. Strains
C2 and S1 were motile and possessed a single polar flagellum; strain FE was non-motile. All
three strains had a G + C ratio of 72.0 f 0.5%. Older cultures consisted of slightly elongated,
spindle-shaped cells which stained unevenly with basic dyes and exhibited extensive metachromatic regions when stained with crystal violet or malachite green. The metachromatic
regions, which disappeared during storage of cultures at 4 "C,also stained with Sudan black and
-0.4
0.20
-0.4
h
z
x
U
2 0.16
-0.8
en
-0.8
3
L
g
2
0-12
a"
-1.2 0
W
-1.2
e
0"
U 0.08
L
0
-1.6
-1.6
0 0.04
u
-2.0
10
20
30
Time (h)
Fig. 1
0
9
4
2
2
0
0
v
-2.0
40
Time (h)
Fig. 2
Fig. 1. CO and H, utilization by primary enrichment culture C, which was obtained by incubating
sewage from the primary settlement tank at Barker's Hough sewage works at 45°C under an
atmosphere of CO, H2 and air, as described in Methods. A Quickfit flask (nominal volume 25 ml) fitted
with a test tube side-arm and containing 25 ml mineral medium (including vitamins) was gassed with
the mixture indicated and then inoculated with 1 ml primary enrichment culture. The flask was
incubated on an orbital shaker at 50 "C and 200 r.p.m. Growth was followed by measuring ODS4*and
gas samples removed for analysis of CO, H2 and CO,, as described in Methods. Similar results were
obtained with the other primary enrichment cultures. W, CO; A, H,; @, CO,;0 , OD,,,.
Fig. 2. Gas utilization by pure culture C2 isolated from primary enrichment C. The experimental
methods used and the symbolsare as described in the legend to Fig. 1 except that a pure culture of strain
C2 that had been grown under CO and H, was used as inoculum. Similar results were obtained with
strains FE and S1.
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Physiology of thermophilic carboxydobacteria
1101
Table 1. Some properties of pure cultures C2, S l and FE, designated
Pseudomonas thermocurboxydouorans
Morphology
Size
Oxidase
Catalase
Gram stain
Flagella
G + C ratio
Electron acceptors
Rods
1.1 x 0.5 pm
Positive
Positive
Negative
Single polar (except FE - none)
72 %
Oxygen; not nitrate, fumarate
or trimethylamine N-oxide
Storage material
PHB
API 20E TDA*
Positive
VP*
Positive
Others
Negative
API ZYM Esterase*
Positive
Lipase
Positive
Arylamidases
Positive
Phosphatase
Positive
Others
Negative
Phage 19 sensitivity
Positive
Autotrophic substrate
CO or CO
H2 (not C 0 2 + H2)
Heterotrophic substrate
Organic/amino acids, no sugars
(e.g. none of API 50CH)
N-sources
Nitrate, ammonia, urea, hippurate
various amino acids, not
methylamine, not dinitrogen
Growth temperature
37-65 "C
Temperature optimum
50 "C
Vitamin requirement
pAminobenzoic acid
* API 20E and APIZYM refer to the test strips supplied by API Laboratory Products; TDA, tyrosine
deaminase; VP, Voges-Proskauer.
+
Table 2. PHBproductWn by strain C2
Experimental details are given in Methods. Chemostat cultures were operated at a dilution rate of
0.05 h- * . The PHB contents of freeze-dried organisms grown under various conditions are expressed as
mg PHB per 100mg dry weight. The values quoted were obtained using the Law and Slepecky
spectrophotometric method (Herbert et al., 1971).
Carbon source
Growth mode
Limiting nutrient
PHB content
Pyruvate
Batch
Continuous
Continuous
Continuous
Batch
Continuous
Continuous
Continuous
-
0.4
0.1
co
pyruvate
oxygen
ammonia
co
-
oxygen
ammonia
9.1
31.9
0.0
0.2
0-9
20.9
appeared as fluorescent bodies when stained with Nile blue A and observed under light of wavelength 460 nm. This latter stain has been reported to be specific for PHB (Ostle & Holt, 1982).
No spores or cysts were produced by any of the strains.
The presence of PHB in cultures was confirmed by chemical assay (Table 2). PHB
accumulated to a significant extent in ammonia-limited chemostat cultures grown on pyruvate
or CO. Cells grown under oxygen-limitationcontained less and carbon-limited or batch cultures
contained very little PHB.
Growth studies with the pure cultures
All three strains were strictly aerobic with an obligate requirement for oxygen as the terminal
electron acceptor, The optimum temperature and pH for growth were 50 "C and 7, respectively.
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C. M. L Y O N S AND O T H E R S
Autotrophic growth occurred with CO alone or with a mixture of CO and H2, but not with C 0 2
and H2 mixtures or with the reduced C1 compounds methane, methanol, formate, methylamine,
dimethylamine, trimethylamine or trimethylamine N-oxide. The utilization of CO by cultures
was oxygen-sensitive. Shake-flask cultures supplied with gas mixtures containing up to 15%
(v/v) oxygen showed similar initial rates of growth and CO consumption whereas both these
rates decreased markedly in flasks containing higher oxygen concentrations. Cultures were
inhibited by concentrations of CO in excess of 80%(v/v). The doubling time for cultures grown
on CO was approximately 3 h at 50 "C.
H2 was not used by the pure cultures (Fig. 2): this result is supported by the whole-cell oxidation and cell-extract studies reported below. The utilization of hydrogen by the initial enrichment cultures could be attributed to the presence of H,-oxidizing bacteria in these mixed
cultures. Several strains able to grow on H2 and C 0 2 but not on CO were isolated from these
primary enrichments.
The three strains grew heterotrophically using a limited range of organic acids (lactate,
pyruvate, succinate, hippurate, fumarate, butyrate and propionate) and L-amino acids (proline,
glutamine, lysine, ornithine, asparagine and glutamate), but not with malate, malonate, tartrate,
acetate, citrate, oxalate, mandelate, hydroxybenzoate, gluconate, serine, threonine, methionine,
arginine, valine and histidine. The sugars and sugar alcohols (in all cases the usual natural
isomers) arabinose, glucose, fructose, ribose, rhamnose, xylose, sorbose, mannose, sucrose,
trehalose, galactose, lactose, dulcitol, inositol, adonitol, mannitol and sorbitol were not used
oxidatively or fermentatively and the API 50CH carbohydrate tests were all negative. Ethanol,
glycerol, glycine, ethylamine, cellulose, amylose, amylopectin and pectin were not utilized. The
API ZYM tests were positive for C4 and C8 esterase, alkaline phosphatase, C14 lipase, acid
phosphatase and leucine, valine and cysteine arylamidases. The API 20E tests were positive for
tyrosine deaminase and Voges-Proskauer.
Growth of colonies on plates containing 0.1 % (w/v) pyruvate was stimulated by the addition of
up to 10%(w/v) nutrient broth, 1% (w/v) yeast extract, 0.1 % (w/v) Casamino acids or 0.5 % (w/v)
peptone, but higher concentrations (25-50% nutrient broth, 1-5 % yeast extract, 0.5-1 %
Casamino acids, 1-5% peptone) inhibited growth. Doubling times in liquid media were also
reduced by 0.1% yeast extract (Table 3). The three strains produced colonies on Oxoid nutrient
agar, but not on Luria or Lemco agar. Strain C2 has a requirement for p-aminobenzoic acid
which was satisfied by supplementation with 50 pg 1- l . Ammonia, nitrate, urea, hippurate and
some amino acids were used as sources of nitrogen, but dinitrogen and methylamine were not
utilized. Ammonia-limited chemostat cultures of strain C2 grown with pyruvate as carbon and
energy source gave a negative reaction in the acetylene reduction test for nitrogenase activity.
The three strains were all sensitive to a lytic bacteriophage (phage 19) isolated on C2 from
sewage as described in Methods.
The three strains isolated from separate enrichments of different sewage samples show only
one minor difference: FE is non-motile whereas C2 and S1 are motile with a single polar
flagellum. The three strains are therefore considered to be a single species. It differs from
previously described species of carboxydobacteria (Kim & Hegeman, 1983; Meyer & Schlegel,
1983) in three ways: it is moderately thermophilic, has a high G + C ratio, and is unable to
utilize H2.It does, however, resemble other pseudomonad species of carboxydobacteria in many
of its basic characteristics and we therefore propose naming the species Pseudomonas thermocarboxydouorans.
Whole cell oxidation studies
CO-grown batch cultures of strain C2 oxidized CO but not H2 or pyruvate (Table 4). The COoxidizing activity was inducible, being absent from pyruvate grown cells. This inability to
oxidize H2 is a unique property of Pseudomonas thermocarboxydouorans : all previously described
species of carboxydobacteria, including Achromobacter carboxydus, oxidize H2 (Kim &
Hegeman, 1983) and, with the exception of A . carboxydus, can indeed grow as hydrogen
bacteria.
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1103
Physiology of t hermophilic carboxydobacteria
Table 3 . Doubling times ( t D )for strain C2 grown in various media
Batch cultures (50 ml in 250 ml conical flasks fitted with B19/26 Quickfit stoppers, 'Suba-seal' stoppers,
and test tube side-arms) were grown in mineral medium supplemented with p-aminobenzoic acid and
with the various additions indicated. The flasks were grown on an orbital shaker at 200 r.p.m. at the
temperatures indicated. Growth was measured using a nephelometer.
Carbon source
Addition
Temp. ("C)
tD (min)
0.2% Pyruvate
None
None
0.1 % yeast extract
0.2% yeast extract
None
None
42
50
50
50
42
50
120
60
24
30
300
180
CO :air (50 : 50)
Table 4. Effects of CO, hydrogen and pyruvate on oxygen consumption by CO-grown and
pyruvate-grown cultures of strain C2
See Methods for experimental details.
Oxygen consumption
[nmol min-' (mg protein)-']
Addition
r
1
CO-grown cells
Pyruvate-grown cells
298
0
0
12
0
59
co
H2
Pyruvate
Table 5 . Enzyme activities ofstrain C2 grown in chemostat culture
See Methods for experimental details. Activities are in nmol substrate transformed or product formed
min- (mg protein)- l . S35extract was used for all assays except for the dye-linked hydrogenase assay
where both S35and P35 were tried. All assays were done at 50°C.
Growth substrate
...
Nutrient limitation
...
co
7
Pyruvate
-
NH3
0 2
C
NH3
C
944
0
0
61
55
43
1293
0
0
68
81
I90
2349
0
0
34
64
120
0
720
0
0
8
Enzyme
CO oxidoreductase
Hydrogenase (NAD)*
Hydrogenase (dye)*
Ribulose bisphosphate carboxylase
Hydroxypyruvate reductase
Phosphoglycollate phosphatase
* Tested with up to 4 mg protein
ND,
not determined.
0
0
4
51
52
ND
47
of S35 or P35 cell extract.
Enzyme activities
Cell-free extracts of CO-grown C2 cultures, and to a lesser extent carbon-limited pyruvategrown cultures, contained a soluble CO oxidoreductase that was conveniently assayed by using
the artificial dye PES as primary electron acceptor (Table 5). This enzyme was not present in
oxygen-limited or nitrogen-limited pyruvate grown cultures. Hydrogenase activity (NADlinked or dye-linked) was not detected in S35or P35extracts of any of the cultures when 0.14.0 mg protein was tested in the assays described in Methods.
Ribulose bisphosphate carboxylase was present in CO-grown, but not in pyruvate-grown
cultures (Table 5), suggesting that the Calvin cycle operates during growth on CO. This
conclusion requires confirmation by rapid labelling experiments and the demonstration of the
other enzymes of the cycle. Specific activities in cells from CO-grown batch cultures [190200 nmol min- (mg protein)- '1 were much higher than those quoted in Table 5 for chemostatDownloaded from www.microbiologyresearch.org by
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1104
C . M . L Y O N S AND OTHERS
grown cells, perhaps reflecting the higher growth rate in the former case (0.15 h- compared
with 0.05 h- l ) . As expected, extracts lacked hexulose phosphate synthase activity, precluding
the use of the ribulose monophosphate pathway.
Hydroxypyruvate reductase was present at relatively high activity in both CO-grown and
pyruvate-grown cultures. This enzyme has previously been observed in a number of autotrophic
and methylotrophic bacteria that do not use the serine pathway (Bamforth & Quayle, 1977;
Taylor, 1977)and is thought to be involved in the assimilation of phosphoglycollate generated by
the ribulose bisphosphate oxygenase activity of ribulose bisphosphate carboxylase (Colby et al.,
1979; Taylor et al., 1981). The various pathways for glycollate metabolism in autotrophs have
been discussed by Beudeker et al., (1981). In view of the presence of relatively high levels of
hydroxypyruvate reductase, the glycine-serine pathway found in higher plants and algae and
possibly in Methylococcus capsulatus (Taylor et al., 1981), seems most likely to occur also in
Pseudomonas thermocarboxydovorans. The glycine-serine pathway is as follows :
Phosphoglycollate
3-phosphoglycerate
-
-
(1)
Glycollate
Glycerate
-
(2)
glyoxylate
Hydroxypyruvate
-
-
Glycine
Serine
where (1) is phosphoglycollate phosphatase and (2) is hydroxypyruvate reductase. The presence
of phosphoglycollate phosphatase activity in extracts of C2 (Table 5) supports this interpretation, although again this activity appears to be constitutive. The possibility that glycollate
assimilation occurs by an alternative route to that outlined above, for example the tartronic
aldehyde (Codd & Stewart, 1973) or /I-hydroxyaspartate (Kornberg & Morris, 1965) routes, has
not been excluded in the present study.
Description of Pseudomonas thermocarboxydovorans sp. nov.
Pseudomonas thermocarboxydovorans (ther.mo.car.box.y.do.vo.rans.Gr. n. therme heat; L. n.
carbo charcoal, carbon; Gr. adj. oxys sharp, acid; L. v. voro consume; M.L. part. adj.
thermocarboxydovorans thermophilic, carbon monoxide consuming).
Gram-negative rods 0.5 x 1.1 pm, motile (except strain FE) by a single polar flagellum. Older
cultures consist of slightly elongated, spindle shaped cells that stain unevenly with basic dyes
and exhibit extensive metachromatic regions when stained with crystal violet or malachite
green. Spores or cysts not formed. Colonies on agar plates incubated under CO for 7 d at 50 "C
are yellow-brown, 3 mm in diameter, undulate and umbonate.
DNA base composition 72.0 +_ 0.5% G + C. Oxidase and catalase positive. Nitrogenase is
not formed. Autotrophically grown cells contain ribulose-l,5-bisphosphatecarboxylase and
CO :phenazine ethosulphate oxidoreductase but lack hydrogenase. Poly-P-hydroxybutyrate is
formed as storage material.
Optimum temperature for growth is 50 "C, maximum c 65 "C and minimum > 37 "C.
Optimum pH for growth is 7. Obligate aerobe. Grows well in mineral medium under an
atmosphere of 50% (v/v) carbon monoxide in air. Growth is inhibited by high concentrations of
nutrient broth, yeast extract, Casamino acids or peptone (> lo%, 1%, 0.1% and 0.5%, w/v,
respectively).
Facultative chemoautotroph utilizing CO as carbon and energy source. Grows heterotrophically using a limited range of organic acids and amino acids as carbon and energy source.
Sugars not utilized as growth substrates. Reduced C1 compounds other than CO not utilized.
Does not oxidize H2. Nitrate not reduced. Gives positive reactions for Voges-Proskauer and
tyrosine deaminase with the API 20E test strip and for C4 and C3 esterases, alkaline and acid
phosphatases, C14 lipase, and leucine, valine and cysteine arylamidases with the API ZYM
tests; all other reactions negative. Ammonia, nitrate, urea, hippurate and some amino acids
used as nitrogen sources but dinitrogen and methylamine not used. Strain C2 requires p-aminobenzoic acid (10 yg 1-I) for growth. Sensitive to lytic phage 19.
The type strain (C2) is NCIB 11893. Source: raw sewage.
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Physiology of thermophilic carboxydobacteria
1105
Part of this work was supported by the Science and Engineering Research Council and by British Technology
Group.
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