Journal of General Microbiology (I977), 99, 383-388
Printed in Great Brirain
383
Respiratory Properties of Synchronous Cultures of AlcaZigenes
eutrophus ~ 1 Prepared
6
by a Continuous-flow Size Selection Method
By C. E D W A R D S A N D C . W. JONES
Department of Biochemistry, School of Biological Sciences,
University of Leicester, Leicester LEI 7RH
(Received I 5 November I 976)
SUMMARY
Synchronous cultures of Alcaligenes eutrophus (Hydrogenomonas eutropha HI 6 )
were prepared by a size selection method which gave high synchrony indices
(0.70 to 0.85). Unlike the smooth exponential increase which was observed in
exponentially growing cultures, the respiration rate kg-atom 0 min-l (ml
culture)-l] of synchronous cultures exhibited a periodic increase which was composed of two ‘steps’ with rises centred at approximately 0.4 and 0.9 of the cellcycle. The respiration of exponentially growing and synchronous cultures was
stimulated by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone;
the degree of stimulation varied during the cell-cycle and was inversely related to
the respiration rate. The observed discontinuous patterns of respiration are discussed with reference to the reported respiratorypropertiesof other micro-organisms
in synchronous cultures.
INTRODUCTION
Techniques for the preparation of synchronous cultures of eukaryotic and prokaryotic
organisms have been broadly divided into induction and selection methods (James, 1964).
Detailed descriptionsof these experimentalapproaches, together with the inherent advantages
of selection synchrony, have been presented by Helmstetter (1969) and Mitchison (1971).
Selection of the smallest cells from an exponentially growing culture either by filtration
(Maruyama & Yanagita, 1956) or by velocity-sedimentation (Mitchison & Vincent, 1965)
has been widely used for preparing synchronous cultures. Such methods are not applicable
to many species of bacteria because of their short cell-cycle times relative to the duration of
the selection procedure. However, a recent method based on selection of t4e smallest cells
from an exponentially growing culture using continuous-flowcentrifugation overcomes these
difficulties (Lloyd et al., 1975).
We now report the use of a modification of this continuous-flow size selection technique
to prepare large-scale synchronous cultures of the Gram-negative bacterium Alcaligenes
eutrophus. Measurements of respiration rate with and without an uncoupling agent revealed
a periodicity of respiratory events during the cell-cycle.
METHODS
Maintenance and growth of the organism. Alcaligenes eutrophus ~ 1 6ATCCI7699
;
(formerly
Hydrogenomonas eutropha 1-116)was maintained on plates of nutrient agar (Oxoid). Starter
cultures (10m1) were grown in nutrient broth no. 2 (Oxoid). For batch cultures, 0.1 to
0.3 mi of a starter culture was used to inoculate 600 ml minimal salts medium (Schlegel,
384
C. E D W A R D S A N D C. W. JONES
Kaltwasser & Gottschalk, I 961) supplemented with DL-lactate (50 mM); cultures were
incubated at 30 "C for 15 to 16h in a rotary orbital incubator (Gallenkamp).
Preparation of synchronous cultures. Organisms from the late-exponential phase of batch
growth were used to inoculate three prewarmed 2 1 conical flasks, each containing 600 ml
minimal salts medium, such that the initial absorbance (at 680 nm) of each culture was
0.25 to 0.30; these were then incubated for 4 h, by which time the bacteria were growing
exponentially. The procedure for continuous-flow size selection was essentially that of
Lloyd et al. (1975)but we used a Sharples continuowflow centrifuge with a variable-speed
control box. The rotor was prewarmed by passing water through at 30 "C for 10min. and
then the exponentially growing culture was introduced at a flow rate of 300 ml min-l and
a rotor speed of 17000rev. min-l. Under these conditions the rotor effluent (500 ml) contained the smallest size class of bacteria, which corresponded to 3 to 4 % of the original
culture. These were collected in a prewarmed sterile flask, which was immediately returned
to the incubator and used as the source of the synchronous culture. The whole procedure,
from the removal of the exponentially growing culture until the return of the rotor effluent
to the incubator, routinely took about 5 min. All procedures, except passage of the cells
through the rotor, were carried out aseptically.
Assessment of synchrony. The degree of synchrony was assessed by the synchrony index
(F) of Blumenthal & Zahler (1962),calculated from the equation
F
= (NINO)- 2'"
in which F has a maximum value of I -0in a culture exhibiting perfect synchrony, N is the
number of organisms at time t, No is the number of organisms at zero time, and g is the
mean generation time.
Analytical methods. Growth was followed by measuring the absorbance of the culture at
680 nm or by determining cell numbers in a standard counting chamber slide (Hawksley,
Lancing, Sussex) after fixing the organisms in formaldehyde (4%, wlv).
Size distributions were determined from a culture sample diluted in Isoton (Coulter
Electronics, Harpenden, Hertfordshire) to approximately 2 x 105organisms ml-l and then
counted at increasing threshold settings using a model B Coultercounter (Coulter Electronics,
Hialeah, Florida, U.S.A.) with a 30pm orifice. All counts were corrected for background.
Oxygen uptake was measured polarographically at 30 "C using a 4004 Clark electrode
(Yellow Springs Instrument Co., Yellow Springs, Ohio, U.S.A.); 2-4ml samples of undiluted
cell suspensions were rapidly transferred from growing cultures into the reaction chamber
and the reaction was monitored for approximately 5 min. The uncoupling agent carbonyl
cyanide rn-chlorophenylhydrazone(CCCP ; Calbiochem) was made up as a methanolic
solution (10mM) and the requisite amountswereadded to the reaction chamber after a linear
rate of oxygen uptake had been obtained; controls showed that methanol alone had no
deleterious effects.
RESULTS
Respiration in exponentially growing cultures and the effect of CCCP
and respiration rate
During growth of -A. eutrophus, cell numbers, absorbance
hg-atom 0 min-l (ml c~lture)-~]
increased in a smooth exponential fashion and doubled
every 75 min. The maximum variation about the average respiration rate was & o-oiopgatom 0 min-l (ml culture)-'. In contrast the specific respiration rate remained relatively
constant [o-04
pg-atom 0 rnin-l(1o7 cells)-l] over the same period and showed a maximum
variation of k 0.016 pg-atom 0 min-l (10' cells)-l about the mean value.
Synchronous cultures oj’A. eutrophus
d
2oc
10
Fig.
I
385
18
26
34
Threshold setting
42
Fig. 2
Fig. I . Wect of CCCP on the respiration rate of an exponentially growing culture of A. eutrophus
(0.29 mg dry wt d-l).
Oxygen uptake was measured using undiluted samples as described in
Methods.
Fig. 2. Distributionof cell sizes in (a) an exponentially growing culture of A. eutruphus and (b) in the
effluent after continuous-flow size selection. Cells were counted at increasing threshold settings
using a model B Coulter counter as described in Methods.
Addition of low concentrations of CCCP stimulated respiratory activity, but at higher
concentrations the uncoupling agent was inhibitory (Fig. I).
Size selection
To confirm that the selection procedure for the preparation of synchronous cultures
separated the smallest organisms from the original exponentially growing culture, the size
distribution of the bacteria in the rotor effluent after centrifugation was compared with that
of the exponentially growing culture. The largest bacteria in the exponentially growing
culture were eliminated by the selection procedure leaving a relatively homogeneous population of small ones (Fig. 2).
Respiration in synchronous cultures and the effect of CCCP
After a lag of approximately 44 rnin following the initiation of synchronous growth, the
cell numbers rapidly doubled, indicating a burst of synchronous division (Fl = 0.72);
a second synchronous division occurred after 120rnin (F2= 0.77). The cell-cycle, defined
as the time from the midpoint of the first division to the midpoint of the second division,
lasted 76 rnin (Fig. 3), in good agreement with the generation time of 75 min observed in
exponentially growing cultures. The absorbance (Eseo)of the culture increased in a smooth
exponential fashion over the entire time course of the experiment and doubled every 75 min.
In contrast, the respiration rate bg-atom 0 mjn-l (ml culture)-l] of undiluted culture
samples increased discontinuously during each cycle; two ‘steps’ (Mitchison, I 969) were
observed, the midpoint of the first occurring at approximately 0.4 of the cycle, the second
at approximately 0.9 (Fig. 3). The variation about the average respiration rate determined by
regression analysis was & 0.080 pg-atom 0 min-l (ml culture)-l, i.e. eight times greater
than that observed in exponentially growing cultures. Addition of 16 ~M-CCCP
to culture
samples at various stages of the cell-cycle elicited a variable stimulation of respiration (not
shown) but did not alter the pattern of two steps per cycle.
386
C. EDWARDS A N D C. W. JONES
0.48
0.14
X
<
0.1 3
0.06
c;o
a.
L-
60
120
60
Time (.inin)
l i m e (inin)
Fig. 3
Fig. 4
1'0
Fig. 3. The growth of A. eutrophus in synchronous culture: a, cell numbers; 0,
EBW;m, oxygen
uptake rate bg-atom 0 min-' (ml ~ulture)-~].
Fl (0.72) and FZ(0.77)denote the synchrony indices
for the fist two doublings in cell numbers.
Fig. 4. Respiration of A. eurropiucs during growth in synchronous culture. Oxygen uptake was
measured using undiluted culture samples as described in Methods. m, Specific oxygen uptake
rate bg-atom 0 min-l (10' cells)-1] with no additions; 0, percentage stimulation of specific
oxygen uptake rate following the addition of I 6 phi-CCCP.
When the rate of respiration was expressed in terms of cell numbers bg-atom 0 min-l
cells)-l], the first step of respiration (midpoint centred at 0.40 of the cycle) was
retained; the' second step, due to the concomitant rise in cell numbers, became a peak at
0.90 of the cycle (Fig. 4). The variation about the average specific respiration rate was
0.140pug-atom 0 min-I (10' cells)-l compared with k 0.016pg-atom 0 mi+ (10'cells)-l
for exponentially growing cultures, i.e. approximately ninefold higher. The effect on the
specific respiration rate of adding I ~ ~ M - C C C
toPthe culture samples varied during the
cell-cycle: minimum stimulation (9 to 16 %) by CCCP occurred at 0-48 and 0.81 of the
cycle, whereas maximum stirnulation (38 to 50 %) was observed at 0.16 and 0.67 of the
cycle.
(10'
D I S C U SS I O N
Continuous-flow size selection using a Sharples centrifuge provides a simple and rapid
technique for establishing synchronous cultures of A. eutrophus with high synchrony indices
Synchronous cultures of A . eutrophus
387
wv
(a)
0
0.2
0.4
0.6
Cellcycle
0.8
1.0
Fig. 5. Cellcycle maps of A . eutrophus showing patterns of respiratory events and the effect of CCCP
on these events: (a) midpoints of step increases in oxygen uptake rates bg-atom 0 min-’
(ml culture)-l]; (b) points of maximum (V)and minimum (v) stimulation by CCCP. Each map
shows the pooled data from four separate experiments.
(0.70 to 0.85). In these cultures, absorbance and respiration rate doubled over each cellcycle; the duration of the latter was about the same as the mean generation time of
exponentially growing cultures (approximately 75 min), suggesting that the synchronous
cultures prepared by this method were exhibiting ‘balanced growth’ (Campbell, 1957).
Unlike the smooth exponential increase in respiration observed in exponentially growing
cultures of A. eutrophus, the respiration of synchronous cultures displayed a regular periodicity, expressed as two steps during the cell-cycle. This result contrasts with that observed
in synchronous cultures of Escherichia coli (Evans, 1975) and some eukaryotic microorganisms (Poole, Lloyd & Kemp, 1973; Edwards, Statham & Lloyd, 1975), in which the
respiration rate showed a number of ‘peaks’per cycle, but is similar to that observed in
Schizusuccharomycespombe 972h- grown on glycerol (Poole & Lloyd, I 974). The possibility
that the observed periodicity of respiratory rate wasa result of the selectionprocedurecannot
be discounted but Seems unlikely since the cells were never removed from the growth
medium, and were subjected to centrifugal forces for only a few seconds. The differing
values for variation about the mean respiration rate (per ml culture and per 10’cells) betNeenexponentially growing cultures and synchronous cultures, together with the persistence
of the periodicity over two cell-cycles, strongly indicates that this is a naturally occurring
event during the cell-cycle.
Stimulation of respiration of exponentially growitg cultures of A. eutrophus by CCCP
has been reported previously by Beatrice & Chappell (1974), who also observed that this
uncoupling agent increased the rate of decay of the respiration-driven proton pulse. Since
our results also show that low concentrations of CCCP stimulated the oxygen uptake rate
of exponentially growing cultures by up to 50 %, it seems that respiration and energy conservation are relatively tightly coupled in this organism (see also Probst & Schlegel, 1976).
However, the discontinuous pattern of stimulation by CCCP observed during synchronous
growth indicates that the extent of respiratory control varied during the cell-cycle. The
periodicity of respiratory activity, together with the variable stimulation of this activity by
CCCP, are shown in the cell-cycle maps in Fig. 5. An approximately inverse relationship
exists between the oxygen uptake rate (averaged midpoints of rises at 0.48 and 0.88 of the
cycle; Fig. gafand the stimulation of this rate by CCCP (averaged maxima at 0.14and 0.61
of a cycle; averaged minima at 0.43 and 0430 of a cycle; Fig. 5b). Poole et al. (1973) studied
25
MIC
99
388
C . E D W A R D S A N D C. W. JONES
the effect of CCCP on oxygen uptake during the cell-cycle of Schizosaccharomyces pombe
and, in contrast to our results, observed maximum stimulation when the oxgyen uptake rate
was highest and very little stimulation when respiration was low. They concluded that transitions between maximum and minimum respiratory activity could be associated with
glycolytic reactions via a Pasteur effect. Clearly the transitions of respiratory activity which
we observed during the cell-cycle of A. eutrophus must have a different basis. Given the
presence of a tightly-coupled respiratory system, it is likely that these transitions reflect the
different requirements of the cell for energy at different stages of the cell-cycle; in this nonfermentative organism these would be expressed solely in terms of respiratory activity.
The respiratory system is being studied in detail to elucidate the respiratory changes occurring during synchronous growth of A. eutrophus.
We are indebted to Mrs D. Everitt for expert technical assistance and the Science Research
Council for financial support (grant no. B/SR/591oz).
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