FEMS MicrobiologyLetters 37 (1986) 317-320
Published by Elsevier
317
FEM 02613
Maintenance coefficients and rates of turnover of cell material
in Escherichia coli ML308 at different growth temperatures
(Escherichia coli; maintenance coefficient; turnover)
R. J o h n Wallace * a n d W. H a r r y H o l m s
Department of Biochemistry, Universityof Glasgow, GlasgowG12 8QQ, U.K.
Received 27 August 1986
Accepted 28 August 1986
1. S U M M A R Y
Maintenance coefficients and rates of turnover
of cell protein, cell walls and total cell phosphate
were determined during the growth of Escherichia
coli ML308 in fed-batch culture at different temperatures. The apparent Arrhenius activation energies (Ea) calculated for maintenance coefficients
at temperatures over 30°C were 212-515 kJtool-1, indicative of a process thermodynamically
similar to denaturation. The corresponding
Arrhenius values for turnover were of a different
magnitude, 44-115 kJ mo1-1, more typical of
enzyme-catalysed reactions. Addition of 0.5 M
NaCI had a minor effect on maintenance at both
temperatures. Turnover of cell material and salt
homeostasis were therefore concluded to be minor
components of the maintenance requirement of E.
coli.
might be required for processes other than net cell
growth, but it was not until the advent of continuous culture techniques that this rate of substrate
consumption for 'growth-unassociated functions'
[2] could be measured. Many estimates of the
magnitude of the maintenance coefficient have
since been made with different microorganisms,
mostly using the relationship derived by Pirt [3].
Despite earlier proposals that turnover of macromolecules like protein, cell walls and nucleic acids
might be the cause of much of the demand for
maintenance energy [4-7], Tempest and Neijssel
[2] focussed their review of maintenance on energetic inefficiencies such as solute leakage and
futile cycles, and assumed that energy demands of
turnover were of minor importance. The aim of
the present paper was to test this assumption, by
comparing the unusual sensitivity of maintenance
to growth temperature [4,8] with the corresponding influence of temperature on the turnover of
cell material.
2. I N T R O D U C T I O N
Monod [1] recognised that a proportion of the
energy-yielding substrate consumed by bacteria
3. MATERIALS A N D M E T H O D S
* Present address: Rowett Research Institute, Bucksburn,
Aberdeen AB2 9SB, U.K.
3.1. Organism and growth medium
E. coli ML308 (ATCC15224), constitutive for
0378-1097/86/$03.50 © 1986 Federation of European MicrobiologicalSocieties
318
the lac operon, was used throughout this work.
The basal growth medium contained 40 mM
K H 2 P O 4 (adjusted to p H 7.0 with NaOH), 40
m M (NH4)2SO4, 2 mM MgSO 4 and 0.04 mM
FeSO 4. Exceptions were the media used to label
cells with 32p, in which 1 mM K H 2 P O 4 and 40
m M Tris-HC1 replaced 40 mM KH2PO4, and that
used to inoculate high-salt cultures, which contained added 0.5 M NaC1. Inocula were adapted
to the required carbon source as described by
Holms and Bennett [9].
3.2. Fed-batch culture
Basal medium (1.6 1) was incubated in a flatbottomed Pyrex flask, maintained at 37°C and
stirred vigorously [10]. This was inoculated with 5
ml of a 16 h culture, or with washed, labelled cells
from 100 ml of a 16 h culture. Carbon source (0.32
M glucose, 0.64 M glycerol, 0.16 M lactose or 0.64
M malate) was supplied at 1 ml. h -1 by a peristaltic pump. Growth was followed turbidimetrically for 4 days, by removing samples (7 ml) from
the culture 4 times daily. There was no significant
change in the relationship between absorbance
and cell dry wt-m1-1 as growth progressed.
Nutrient flow rate was determined at each sampiing time. Respiration was measured by comparing the composition of air entering the flask (100
m l - r a i n -~) with the 0 2 and CO 2 content of the
effluent gas [11].
The rate of increase in cell density declined
with time, as a result of the increased maintenance
requirement of the growing biomass. This decline
enabled the determination of m, the maintenance
coefficient, and Y~ the extrapolated maximum
growth yield [3]. Full details of experimental protocol and calculation of results can be found
elsewhere [12].
3.3. Measurement of turnover
Turnover was measured in fed-batch cultures
at ¼ the scale of those used for maintenance
determinations. Bacteria were labelled with
L-[2,5-3H]histidine by the addition of 20 /~Ci (50
Ci. mmo1-1) to 100 ml of exponential-phase cells
during the final subculture before inoculation of
fed-batch medium. When stationary phase was
reached, the labelled bacteria were washed in 40
mM phosphate buffer p H 7.0 and inoculated into
the growth vessel, to which was added 0.2 mg
unlabelled e-histidine • ml-1. Release of label was
followed by removing cells by filtration (0.22/~M),
and counting the filtrate.
A similar procedure was used with (DL +
meso)-2,6-diarrfino [G-3H]pimelic acid, to measure
cell wall turnover, except that 5 /LCi (0.4 Ci.
mmo1-1) was used to label cells, and 2 mM lysine
and 1 mM diaminopimelic acid were used to
dilute released radioactive products. Low-phosphate medium was used with 10 ~tCi (200 mCimmo1-1) of KH2132p]PO4 to label cells with 32p.
Washed 32p-labelled cells were inoculated into
normal growth medium to determine the release of
32p.
3.4. A utoradiography
Cells growing on glucose in fed-batch culture at
0.067 h -1 were pulsed with 2 mCi (4.2 Ci.
mmo1-1) [1-3H]glucose. After 1 h, samples were
fixed by heat on glass microscope slides and
stained with carbol fuchsin. The slides were dipped in photographic emulsion (Ilford type L4),
dried and developed after 6 weeks. Grains resulting from incorporated radioactivity were then visible by light microscopy.
4. RESULTS A N D DISCUSSION
Fed-batch culture differs from chemostat culture in that a steady state is never achieved. The
specific growth rate (#) of the bacteria falls continuously. However, the procedure used in the
present experiments resulted in a very slow rate of
change of ~t (e.g., 0.039-0,015 h -1 in 24 h), and
there was no indication that an approximation to
steady-state conditions was not valid for the determination of maintenance [12].
The method of calculation of m and Yc; differed from that used with chemostats [3,5], although the assumptions made were identical to
those made by Pirt [3]. Instantaneous values of/z
were calculated, not from dilution rate, but from
the rate of increase of cell mass divided by the
total cell mass. The rate of increase of cell mass
could be determined by C balance, by subtraction
319
Table 1
Influence of growth temperature on maintenance coefficients
and molar growth yields of E. coil limited by different carbon
sources in fed-batch culture
Carbon source
Growth
temperature
m
(mmol. g x
.h ~1)
Yc
(g. m o l - 1)
Glucose
40
37
30
25
0.260
0.038
0.026
0.026
115.2
95.0
103.9
111.9
Glycerol
42
40
38.3
37
33.5
30
25
0.393
0.232
0.111
0.103
0.041
0.038
0.040
52.6
54.7
53.2
50.7
57.2
57.3
56.8
Malate
40
37
30
0.473
0.127
0.068
38.9
43.3
44.1
Lactose
40
37
30
0.086
0.037
0.013
210.0
218.3
217.2
of the rate of CO 2 evolution from the rate of
supply of substrate, since C recovery as cells and
CO: was complete throughout these experiments
[12]. Maintenance coefficients were then calculated from plots similar to those of Stouthamer
and Bettenhaussen [5]. There was no evidence of
non-linearity of any sort, in contrast to the multiple growth phases observed in a recycling fermenter [13,14]. Furthermore, microautoradiography indicated that all cells in the culture incorporated similar amounts of radioactivity and no
dead bacteria were present.
Maintenance coefficients increased rapidly as
the growth temperature increased above 37 ° C for
the 4 substrates tested (Table 1), a pattern similar
to those observed previously with E. coil growing
on glucose in chemostats [8]. YG was not affected
to a significant extent. Thus it can be inferred that
the increased m at higher temperatures reflected
an increased demand for ATP for maintenance
reactions, rather than a poorer ATP yield from the
glucose metabolised. The latter would have caused
a corresponding fall in Yr.
Addition of 1.0 M NaC1 to yeast increased m
[15], indicating an energy requirement for ionic
homeostasis. With E. coli in fed-batch culture at
37°C, addition of 0.50 M NaC1 also increased m,
from 0.038 to 0.054 mmol glucose (g dry wt) -1.
h -1, with Y~ almost unchanged at 93.2 gmo1-1.
However, the same addition at 40°C actually
caused a slight fall in the much higher m, the
0.242 mmol (g dry w t ) - l h -1, with a Yo of 102.2
gmol--1. Thus, whether the extra maintenance requirement at 37°C was for the exclusion of NaC1
or an altered K ÷ gradient [2], this was evidently a
minor component of maintenance at 40°C and
not responsible for the marked increase in m.
The absolute measurement of bacterial protein
turnover cannot be achieved by the method used
here, i.e. the labelling of bacteria with a radioactive amino acid and diluting label released as the
result of protein breakdown by adding excess unlabelled amino acid. Problems such as the equilibration of pools, the effects of excess cold acid
on observed turnover rate and many others militate against this [12,16]. However, there is no
reason to suppose that the present method does
not give results useful for comparing relative rates
of protein turnover under different conditions.
The same limitations doubtless apply as well to
cell wall and phosphate turnover. Indeed, phosphate turnover measured by 32p-release must be
Table 2
Influence of growth temperature on turnover in E, coli growing in fed-batch culture
Protein turnover was determined from the rate of loss of
[3H]histidine from a pre-labelled inoculum growing in medium
containing 0.2 mg cold histidine ml-1. Turnover of phosphate
and cell walls were measured in a similar way (see METHODS)
Carbon
Growth
Rate of turnover (% h - 1)
source
temperature
Protein
Cell walls
Phosphate
40
37
30
3.97
2.28
1.26
-
-
40
37
30
25
3.74
3.01
1.15
0.41
3.73
3.90
2.80
1.62
2.44
2.96
1.39
0.94
Glucose
Glycerol
(°C)
320
Table 3
Activation energies (E~) of maintenance and turnover in E.
coli
The Arrhenius activation energy (kJ-mol 1) of each of the
processes described in Tables 1 and 2 was calculated from the
Arrhenius equation
E~
log k = constant- - RT
where k is the reaction rate at absolute temperature T. R is
the gas constant. Since m appeared to be constant at 30°C
and below, the values of m for these temperatures were not
included in the calculation.
Reaction(s)
Substrate
Ea
Maintenance coefficient
Glucose
Glycerol
Malate
Lactose
515
212
352
226
Protein turnover
Glucose
Glycerol
86
115
Cell wall turnover
Glycerol
44
Phosphate turnover
Glycerol
57
due to the b r e a k d o w n of a variety of m a c r o m o l e cules, i n c l u d i n g nucleic acids a n d phospholipids,
a n d is therefore poorly defined. Nevertheless, it
was clear from the results (Table 2) that p r o t e i n
t u r n o v e r increased with temperature, as did cell
wall a n d p h o s p h a t e turnover, except above 37 ° C,
b u t i n a different m a n n e r to m a i n t e n a n c e .
A r r h e n i u s activation energy coefficients, E a,
were calculated from the results i n Tables 1 a n d 2,
to c o m p a r e the effects of t e m p e r a t u r e o n different
processes. It should be n o t e d that these values of
Ea are based o n o n l y a few points (Tables 1 a n d 2)
a n d over a small t e m p e r a t u r e range. Nonetheless,
c o m p a r i s o n of their a p p r o x i m a t e m a g n i t u d e s provides useful i n f o r m a t i o n a b o u t the n a t u r e of the
process. M a i n t e n a n c e a n d t u r n o v e r h a d quite different values of E a (Table 3). E~ values for
t u r n o v e r were 4 4 - 1 1 5 k J . m o 1 - 1 , a n d therefore
w i t h i n the range n o r m a l l y e n c o u n t e r e d with enzyme reactions [17]. M a i n t e n a n c e , o n the other
h a n d , had E~ values of 2 1 2 - 5 1 5 kJ- mo1-1, more
typical of n o n - e n z y m e reactions such as d e n a t u r a tion [17]. T h u s d i s r u p t i o n of cell structure, result-
ing perhaps i n energetic inefficiency b u t without
c a u s i n g b r e a k d o w n to m o n o m e r s like a m i n o acids,
seems to be largely responsible for the observed
m a i n t e n a n c e energy r e q u i r e m e n t of E. coli at
growth temperatures above 3 0 ° C . The precise nature of this d i s r u p t i o n r e m a i n s to be determined.
F u r t h e r m o r e , because the t e m p e r a t u r e sensitivities
of m a i n t e n a n c e in E. coli ML308, used here, a n d
E. coli B [8], were higher t h a n that of E. coli W
[7], there m a y be differences b e t w e e n strains in the
extent to which the process uses m a i n t e n a n c e energy.
ACKNOWLEDGEMENT
R.J. Wallace was in receipt of a n M.R.C. Research Studentship.
REFERENCES
[1] Monod, J. (1942) Recherches sur la Croissance des Cultures Bacteriennes. Hermann, Paris.
[2] Tempest, D.W. and Neijssel, O.M. (1984) Annu. Rev.
Microbiol. 38, 459-486.
[3] Pirt, S.J. (1965) Proc. R. Soc. London, B, 163, 224-231.
[4] Marr, A.G., Nilson, E.H. and Clark, D.J. (1963) Ann. NY
Acad. Sci. 102, 536-548.
[5] Stouthamer, A.H. and Bettenhaussen, C.W. (1973) Bioclaim. Biophys. Acta 301, 53-70.
[6] Hempfllng, W.P. and Mainzer, S.E. (1975) J. Bacteriol.
123, 1076-1087.
[7] Farmer, I.S. and Jones, C.W. (1976) FEBS Lett. 67,
359-363.
[8] Mainzer, S.E. and Hempfling, W.P. (1976) J. Bacteriol.
126, 251-256.
[9] Holms, W.H. and Bennett, P.M. (1971) J. Gen. Microbiol.
65, 57-68.
[10] Harvey, N.L., Fewson, C.A. and Holms, W.H. (1968) Lab.
Pract. 17, 1134-1136.
[11] Hamilton, I.D. and Holms, W.H. (1970) Lab. Pract. 19,
795-807.
[12] Wallace, R.J. (1975) Thesis, University of Glasgow.
[13] Chesbro, W., Evans, T. and Eifert, R. (1979) J. Bacteriol.
139, 625-638.
[14] Arbige, M. and Chesbro, W. (1982) J. Gen. Microbiol.
128, 693-703.
[15] Watson, T.G. (1970) J. Gen. Microbiol. 64, 91-99.
[16] Pine, M.J. (1973) J. Bacteriol. 116, 1253-1257.
[17] Dixon, M. and Webb, E.C. (1964) Enzymes, 2nd ed.
Longmans, Green, London.