Biochemical Society Transactions
Integration of catabolism and anabolism in microbial systems
David W. Tempest
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield SI 0 2TN, U.K.
972
After being taken into the cell, substrate metabolism generates three flows - those of intermediary metabolites, reductant and ATP (via
substrate-level phosphorylation reactions). Aerobically, a substantial portion of the reductant is oxidized by way of the respiratory chain to generate
the bulk of the biologically useable energy which,
together with the remaining reductant, serves to
convert the intermediary metabolites into polymers
that contribute to net cell synthesis and growth. Any
shortfall of reductant or energy can, of course, be
made up by further oxidation of intermediary
metabolites. However, it is important to recognize
that any surfeit of reductant or energy cannot be
stored at the levels of the pyridine and adenine
nucleotide pools. These compounds serve essentially as flux mediators; their concentrations within
the living cell are relatively low (about 10 pmol g - '
for the adenine nucleotide pool; see [2]) and therefore must be rapidly turned over for catabolism to
proceed. In the actively growing cell this turnover is,
of course, effected primarily by the reactions of
anabolism. For example, in a rapidly growing
Escherichiacolicell (p=2.0 h -'; Y,v,:,,=10 g mol-' i.e., utilizing 100 mmol of ATP for the synthesis of
each g of cell biomass) the whole adenine-nucleotide pool will turn over more than five times per
second. Thus if the formation of ATP were to cease
suddenly, then net cell synthesis would stop within
200 ms! The same holds true for the pyridinenucleotide pool, and if at some moment in time the
It was clearly established by Monod [l] that for
growth to proceed at any particular rate, all essential
nutrients must be consumed at correspondingly
proportional rates. Hence, if p is the specific rate of
growth (l/x-dx/dt) and q the specific rate of consumption of some substrate (i.e., - l/x.ds/dt) then:
p 0:q, or p = Y-q
where Y is the proportionality factor or yield value.
This yield value assumes particular significance
when applied to the utilization of carbon substrate
by heterotrophic micro-organisms in that here the
substrate serves an amphibolic function - that is,
both as a source of cell carbon and, through its catabolism, as a source of utilizable energy. So what,
one might ask, is the significance of a yield value of,
say, 90 or 100 g of cells mol-' for growth on
glucose? What biosynthetic and bioenergetic considerations does such a value embody? What precise relationships exist between catabolism and
anabolism, and how are the two processes regulated
and integrated? These are the core questions that
need to be addressed in the teaching of microbial
growth energetics.
The problem can be brought into sharper
focus by looking at the overall reactions involved in
converting substrate carbon to biomass, such as is
depicted in Fig. 1.
Abbreviations used: p, specific growth rate; Y, yield
value: DNP. dinitrophenol.
Fig. I
A schematic representation of the pathways of carbon and energy flow in
aerobic bacteria, showing potential control points
From [8].
Oxidative
phosphorylation)
D>
2
Reducing
equivalents
HI0
p;A
Polymers
phosphorylation)
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Growth
unassociated
functions
-
Growth
Bioenergetics
oxidation of reduced pyridine nucleotides was to
cease, then further substrate catabolism would
cease almost instantly. It follows, therefore, that
there are ample control points that ought to allow a
tight coupling between catabolism and anabolism,
thereby ensuring that growth proceeded with a
maximum efficiency with respect to carbon-substrate utilization and energy generation. So what are
the realities of this situation?
This is most effectively studied by resort to
chemostat culture in which the anabolic rate can be
limited to any value less than a well-defined maximum (pmax,,
the maximum specific growth rate).
One does this by limiting the rate of supply of one
essential anabolite (e.g. N-, S- or P-source) while
maintaining a surfeit of the carbon and energy
supply.
When such a culture is established and analysed, what is often found is typified by the results
obtained with Klebsiella pneumoniue (Table 1). As is
evident in this Table, under all glucose-sufficient
conditions, carbon substrate was taken up at a
greatly elevated rate relative to that of a glucoselimited culture growing at the same rate. So how
can one reconcile this observation with the scheme
depicted in Fig. l ? If catabolism becomes partially
dissociated from anabolism, what happens to the
excess intermediary-metabolite flow, and what happens to the excess reductant? The situation is even
more starkly evident with washed cell suspensions
that will often oxidize carbon substrate at high rates
Table 1
Glucose and oxygen consumption rates, and corresponding yield values, of Klebsiella pneumoniae cells
growing aerobically on glucose in a simple-salts
medium
Cells were grown in chemostat culture at a fixed rate (Dilution
rate, D = 0. I7 h - ' ; 35°C; pH 6.8)
Specific
consumption rate
(pmol min-' g - '
dry wt of cells)
Yield values
(g mol- ')
Limitation
Glucose
Oxygen
Glucose
Oxygen
Glucose
SuIphate
Ammonia
Phosphate
Magnesium
Potassium
35
83
98
I05
I I2
I75
69
I23
I40
I63
I86
272
81
34
29
27
25
16
41
23
20
17
15
10
while being totally incapable of effecting net cell
synthesis. With chemostat cultures of glucose-sufficient K. pneumoniue the excess intermediarymetabolite flow is disposed of by excreting selected
metabolites (e.g. acetate, pyruvate, 2-oxoglutarate)
into the culture, and the excess reductant clearly is
disposed of by way of a greatly enhanced rate of
respiration (Table 1). However, if respiration is
tightly coupled to ATP generation, then in solving
the problem of redox imbalance in this particular
way, one must create a problem of energy overplus.
So how is the excess energy dissipated? Presumably
as heat; but by what molecular mechanism? This is
yet another crucial question, and one that can be
answered properly only by examining more closely
precisely how energy is transduced in the living cell.
When this is done, one becomes aware of a number
of possibilities, all of which are of relevance to the
teaching of bioenergetics.
In the above context, it is necessary to bear in
mind the fact that energy is transduced at two levels
within the microbial cell; (i) in the cytoplasm by the
scalar reactions of substrate-level phosphorylation,
and (ii) across the plasma membrane as an electrochemical gradient of protons and other cations. So
in seeking to understand the mechanism of dissociation of catabolism from anabolism one must look
primarily at mechanisms that can either dissipate
the membrane potential or turn over the ATP pool
at a high rate in the absence of net biosynthesis.
There would appear to be several possibilities,
some of which have received direct experimental
support, but all of which require an understanding
of membrane physiology.
The least easy to test experimentally is the
proposition that there can be slippage in the pumps
- that is, particularly in the membrane ATPase.
Here it is postulated that when the proton motive
force exceeds a value of about 300 mV, protons
enter the cell through the membrane ATPase either
without effecting ADP phosphorylation, or else
effecting phosphorylation with a greatly decreased
efficiency. On thermodynamic grounds, it can be
readily argued of course, that the -,H'/P quotient
must be a function of the phosphorylation potential
extant within the cell, the higher the latter, the
greater the number of protons needed to effect
ADP phosphorylation. One variant of this hypothesis is the suggestion that a specific membrane protein is present that functions as a gated proton
channel: when the proton motive force is below
about 250-300 mV the gate is closed, but above
300 mV it opens to allow a leakage of protons into
the cell.
1991
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Biochemical Society Transactions
974
Whether or not a leakage current of protons
occurs naturally, it is easy to effect one artificially
by adding a protonophorous uncoupler such as
dinitrophenol (DNP) to the culture. With chemostat
cultures of K. pneumonzize the net effect was found
to be a greatly enhanced rate of respiration [3], no
doubt needed to pump excess protons from the
cytoplasm and to maintain pH homoeostasis. An
effect similar to that of DNP was found to be
invoked in chemostat cultures of the yeast Cundida
utilis by high concentrations of weak organic acids
at, or close to, their p K , values [4].
Not only may a proton leakage current be
invoked under some conditions, but also a leakage
current of other ions - most particularly potassium
ions [5, 61. Potassium is present intracellularly in
relatively high concentration and in a largely
unbound form. Moreover it seemingly can flux
from the cell down its concentration gradient when
the extracellular K + concentration is low [ S ] . Under
K+-limiting growth conditions the transmembrane
K + gradient can be extremely high (that is, more
than 10000-fold) and such cultures tend to express
a correspondingly high respiration rate as K + flux
from the cell and are pulled back across the cytoplasmic membrane by the ATP-driven high-affinity
uptake system (kdp). In this connection, it is worth
mentioning that K+-limited cultures of K. pneumoniae excrete gluconic acid and 2-ketogluconic
acid when growing on glucose, and these products
are also found with glucose-sufficient (otherwise
limited) cultures growing in the presence of 1 mMDNP [3]. These products arise by direct oxidation
of glucose (and gluconate) at the level of the membrane by the action of specific dehydrogenases - a
quinoprotein glucose dehydrogenase and a flavoprotein gluconate dehydrogenase (see [7]). The
former enzyme also is present in K+-limited cells of
E. coli, as well as in cells grown in the presence of
DNP, but no gluconic acid is produced because
these organisms seemingly lack the prosthetic
group, PQQ [ 7 ] . The importance of this enzyme
resides in the fact that it is coupled to the respiratory chain at the level of cytochrome b, and thus can
utilize the excess capacity of the terminal oxidases
and act as a low-impedance high-flux auxiliary
energy-generating system (a kind of super-charger).
Whereas glucose-limited K. pneumonzize cells
exhibit only a very low glucose dehydrogenase
activity, they can rapidly oxidize any excess glucose
transiently added to the culture, and can do so without there being a concomitant increase in growth
rate. Hence at least over short periods of time these
cultures extensively dissociate catabolism from anabolism and, in so doing, excrete much acetate and
pyruvate. The accumulation of pyruvate is of particular interest in that K. pneumonzize contains constitutively two enzymes that implicate this molecule a pyruvate reductase that is NADH-linked, and a
iAactate dehydrogenase that is a flavoprotein,
generating pyruvate and donating its electrons to
the quinone pool (Q). Hence, when acting in concert, these two enzymes serve to oxidize NADH by
a route that circumvents the proton-translocatingsite-1 segment of the respiratory chain and, significantly, pyruvate reductase is homotropic with
Fig. 2
Organization of the respiratory chain of Klebsiello pneurnonioe showing
possible mechanism for circumventing the proton-translocating segment of
the NADH dehydrogenase (site I )
From [ 101 Abbreviations used cyt, cytochrome. fp, flavoprotein. Q, quinone. PQQ,
pyrroloquinoline quinone
Succinate
-
fp
\
(CN insensitive
oxidase)
-2
Pyrivate
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D-Lactate
dehydrogenase
Bioenergetics
respect to pyruvate. Hence, any transient rise in the
intracellular pyruvate level will activate this enzyme
and invoke the bypass reaction which circumvents
one of the proton-translocating segments of the
respiratory chain (Fig. 2).
These, then, are some of the potential
mechanisms that may function at the level of the
membrane and may serve to allow catabolism to be
extensively dissociated from anabolism. But if ATP
is still formed at an elevated rate, what mechanisms
conceivably might be utilized to accelerate its rate of
turnover? Microbial cells certainly possess the
potential to invoke a so-called ‘futile cycling’ of
metabolites leading to net ATP hydrolysis. However, here the problem once again is in determining
whether, and to what extent, these futile cycles
operate. Indeed, if they were to function as a primary mechanism for dissociating catabolism from
anabolism then one might expect them to be most
obviously manifest with cultures or washed suspensions of organisms incubated anaerobically and fermenting glucose. Indeed, such anaerobic cultures
are able to extensively dissociate catabolism from
anabolism when growing on (or incubated with)
glucose [8]. Here it has been found that the potential problem of excess energy flux through the
adenine-nucleotide pool is circumvented by the
organisms invoking a metabolic reaction sequence
(the methylglyoxal bypass; [91) that circumvents the
energy-conserving steps of glycolysis. However,
when incubated with substrates such as pyruvate,
K. pneumonke is unable to effect a dissociation of
catabolism from anabolism [8]. This strongly suggests that futile cycles offer no realistic mechanism
of energy dissipation, and probably do not participate in dissociating catabolism from anabolism.
The fact that there seemingly is no tight coupling between catabolism and anabolism in microbial
systems poses yet other, more philosophical, questions such as ‘why do organisms behave in this
seemingly profligate manner?’ and ‘what possible
ecophysiological advantages might accrue from
substrate catabolism not being tightly controlled
such as to meet the minimum biosynthetic and
energetic requirements of cell synthesis?’. However,
though these questions are well worth pondering,
they go well beyond my brief to discuss (in the context of teaching bioenergetics) the integration of
catabolism and anabolism in microbial systems.
1. Monod, J. (1942) Recherches sur la Croissance
Bacterienne, Masson, Paris
2. Harrison, D. E. F. (1976) Adv. Microb. Physiol. 14,
243-309
3. Neijssel, 0.M. (1977) FEMS Lett. 1, 47-50
4. Hueting, S. & Tempest, D. W. (1977) Arch. Microbiol. 115,73-78
5. Heuting, S., de Lange, T. & Tempest, D. W. (1979)
Arch. Microbiol. 123, 183- 188
6. Mulder, M. M., Teixeira de Mattos, M. J., Postma, P.
W. & van Dam, K. (1986) Biochim. Biophys. Acta
851,223-228
7. Neijssel, 0. M., Hommes, K. W. J., Postma, P. W. &
Tempest, D. W. (1989) Antonie van Leeuwenhoek
56,Sl-61
8. Streekstra, H., Buurman, E. T., Hoitink, C. W. G.,
Teixeira de Mattos, M. J., Neijssel, 0. M. & Tempest,
D. W. (1987) Arch. Microbiol. 148, 137-143
9. Cooper, R. A. & Anderson, A. (1970) FEBS Lett. 11,
273-276
10. Tempest, D. W. & Neijssel, 0. M. (1987) in Escha’chia coli and Salmonella typhimurium: Cellular and
Molecular Biology (Neidhardt, F. C., Ingraham, J. I,.?
Brooks Low, K., Magasanik, B. & Schaechter, M.,
eds.), vol. 1, pp. 797-806. Am. SOC. Microbiol.,
Washington D.C.
Received 24 July 1991
1991
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