Energy Excess: Nutrient Limitations

Cessation of growth in a bacterial culture in the presence of energy source, with some other factor limiting, raises the question of the constancy or tight-ness of coupling between energy-liberating reactions and cell growth.

T h e behavior of the cells and the fate of carbon during glycolysis or respira-tion b y cell suspensions become the limiting case. T h e intermediate condi-tions, nutrients present b u t growth limited without substrate exhaustion, result from an unknown factor limiting the growth, i.e., unfavorable physical or chemical environment (pH, toxic chemicals accumulated) or the end point reached when a single limiting chemical, not energy substrate, is exhausted. T h e latter case is used in growth factor assay. T h e behavior of the culture and its cells has been studied to a limited extent (Mcllwain^{2 0 8}).

I n the case of limiting factor other t h a n energy source, the substrate m a y continue to be degraded either at full or reduced rate. I n these cases, the available energy cannot be used for growth and must be dissipated in some other fashion. T h e condition of energy release without coupled growth m u s t be understood if variables in growth yield and substrate carbon use for non-energy functions are to be understood. As part of the larger problem of un-coupling in the presence of limiting factors, three mechanisms of energy dissimilation other t h a n the formation of new protoplasm will be briefly discussed. T h e y a r e : (1) accumulation of polymeric products, either in storage form or as unusable waste; (2) dissipation as heat by " A T P a s e mechanisms"; and (3) activation of shunt mechanisms bypassing energy-yielding reactions or requiring a greater expenditure of energy for priming.

A. ASSIMILATION: POLYMER FORMATION

T h e relationship between energy metabolism and carbon assimilation in washed cell suspensions (resting cells) has long been associated with the incorporation of a fraction of the substrate carbon into cells. During res-piration, the "oxidative assimilation" can use a large fraction of the

sub-strate; during glycolysis, "fermentative assimilation,'' the a m o u n t is smaller. B a r k e r^{2 0 9} established the oxidative assimilation of more t h a n half the carbon from organic acids during oxidation b y resting cells of the color-less alga, Prototheca zopfii. Since then the occurrence of massive accumula-tion has been observed in m a n y organisms with a wide variety of sub-strates.^{6 8} Anaerobic "fermentative assimilation^{, ,} was established by v a n Niel and Anderson^{2 1 0} with suspensions of both resting and growing yeast.

T h e products of anaerobic glucose fermentation accounted for only 70 % of the carbohydrate utilized, with the remainder presumably incorporated into "cellular material.^{, ,} Under nutrient-limited conditions, the assimilated carbon is not incorporated into new protoplasm b u t is found to be in ac-cessory polymeric (storage or waste) products, e.g., capsular slime (3), polypeptides,^{2 1 1} glycogen^{2 1 2} or lipids of t h e poly-jS-hydroxybutyrate t y p e .^{2 1 2 a} N o t all compounds degraded result in polymer accumulation; formate oxi-dation b y suspensions of E. coli proceeds to completion without measurable accumulation of new cellular or polymeric material.^{2 1 3} Only those com-pounds yielding "useful" energy to the cell promote assimilation.

Carbohydrate polymers, internal or external, are among the principal products of substrate "assimilation" during nutrient-limited substrate turn-over. Cellulose synthesis provides the most striking example of polysac-charide synthesis. Both A. xylinum^2U and A. acetigenum^21b accumulate u p to one-quarter of the glucose metabolized as extracellular cellulose. E a c h hexose unit converted to cellulose requires one A T P for activation; the poly-merization occurs via uridine-diphosphoglucose catalyzed by particle-bound enzymes associated with the cell m e m b r a n e .^{2 1 6} T h e other principal form of extracellular polysaccharides is capsular material.^{2 1 7}

Internal storage, usually analyzed as glycogen, is common in bacteria.

Dagley and Dawes,^{2 1 2} for example, observed glycogen accumulation after growth had ceased in cultures due to limitations other t h a n depletion of energy source. Palmstierna and co-workers2 1 7 - 2 1 9 found glycogen accumula-tion to be maximum in continuous cultures in nitrogen-limited medium.

Glycogen synthesis was much greater after nitrogen exhaustion with either glucose or lactate as energy source. T h e maximal amounts of glycogen stored internally accounted for 20 % of the dry weight.

Among the compounds, in addition t o carbohydrate, which are found frequently in the cytoplasm of bacterial cells are polyphosphates, usually as granules. Such granules accumulate in b o t h aerobic and anaerobic bac-teria and appear to react directly with A T P b y transphosphorylation, reaction (32).

ATP + (P0⁸)n - ADP + (P0⁸)„⁺i (32)

An enzyme catalyzing this reaction has been purified from extracts of E. coli,

1. ENERGY-YIELDING METABOLISM I N BACTERIA 49 b u t the cultural conditions leading to polyphosphate formation have not been established.

B . A T P A S E : D I R E C T AND INDIRECT

T h e role of limiting metabolite levels in the control of metabolism has been considered in m a n y cases since cell-free yeast glycolysis was found to be phosphate limited and H a r d e n and Young found fructosediphosphate to be the product of bound phosphate and missing carbon.⁵ A clue in the initial experiments was the shift in stoichiometry from 2 moles of alcohol per glu-cose to 1 mole per gluglu-cose on change from whole cell suspensions to extracts.

T h e mechanism of this apparent return of phosphate to the inorganic level in t h e intact cell, b u t not the extract, engaged Meyerhof's curiosity as late as 1949.^{2 2 7} One could envision the return of A T P phosphorus to the inor-ganic level in growing cultures through the work functions of cellular trans-port, biosynthesis of intermediates, polymerization—but the continued fermentation b y suspensions, without apparent work functions, remained obscure. Meyerhof convinced himself t h a t in the yeast cell the cause is the occurrence of a very unstable phosphatase which does not withstand drying or prolonged storage in yeast extracts. I n very careful and system-atic studies, Meyerhof examined the level of phosphatase, its disappear-ance from dry cells and extracts, and re-established t h e normal alcoholic fermentation balance by the addition of ATPases to yeast extracts.

Whether or not M e y e r h o f s " A T P a s e " of intact yeast fermentation is a direct hydrolytic enzyme or occurs b y other mechanism, it appears to be the prototype for nonproliferating glycolysis or respiration, i.e., energy-linked system without apparent work function. T h e cell confined to gly-colysis without growth must either regenerate orthophosphate and energy-rich phosphate acceptor ( A D P or A T P ) or transform metabolites b y an alternate noncoupled mechanism. This problem is the more important from the need t o understand t h e mechanisms of coupling or its lack in grow-ing and nonproliferatgrow-ing cells.

Knowledge of glycolytic control in microbial cells is in a completely unsatisfactory state. E v e n a knowledge of the phosphatases which could perform either direct or indirect A T P a s e activity is fragmentary and in most cases completely lacking. I t m a y be true t h a t ATPase activities are entirely lacking in fermentative bacteria. If this is true, combined reactions which can lead t o a net release of A T P might be sought; in fact, several re-actions have been related to glycolytic rate. I n lactic acid bacteria, glu-tamic-glutamine, and t o a lesser extent ornithine-citrulline-arginine, h a v e been implicated. Further, one can visualize organic acid mechanism such as coupled citrogenase (condensing enzyme)-citritase which would ener-getically equal an A T P hydrolysis.

T h e rate of glycolysis of washed streptococci in cell suspensions is mark-edly stimulated b y addition of glutamic acid and ammonia, histidine and ammonia, or glutamine.^{2 2 3} I n some instances, t h e rate increase is as much as 5- to 8-fold. Gale^{2 2 4} has demonstrated a glycolytic requirement for in-corporation of glutamate b y cell suspensions. M c l l w a i n^{2 2 5} has demonstrated a glucose dependency for the conversion of glutamine t o glutamic plus N H³ as well as for t h e reverse reaction. One can visualize t h e expenditure of a t least one energy-rich phosphate bond in t h e glutamate permease reaction from t h e d a t a of Gale. I t is also possible to visualize a n " A T P a s e " -type reaction b y a coupled glutamine synthetase-glutaminase reaction. A severalfold stimulation of glycolytic rate in washed cell suspensions b y chemical additions which could serve an ATPase function m a y , b u t b y no means must, state their mode of action. Mcllwain a n d co-workers^{2 2 5} have observed, in common with Meyerhof's labile A T P a s e of yeast, a complete inability t o prepare a glutaminase in extracts of streptococci, although in the same extracts they were able t o obtain active arginine dihydrolase.

T h e mechanisms of t h e glutamine hydrolysis in these organisms remains obscure.

T h e arginine dihydrolase reaction which occurs in two steps, hydrolytic-ally from arginine t o citrulline and phosphorolytichydrolytic-ally from citrulline t o ornithine plus carbamyl phosphate, requires 2 moles A T P in t h e reverse (synthetic) direction. T h u s , a repetition of t h e first step, hydrolysis of arginine t o citrulline, return of citrulline t o arginine via an ATP-dependent condensation with aspartate, would yield a net loss of high-energy phos-p h a t e .1 7 1 , 1 7 2 T h e primary question is: do these reactions constitute a mech-anism permitting nonproliferating cell glycolysis, i.e., t h e regeneration of phosphate acceptors and orthophosphate?

Several reactions of organic acids, normally energy-linked oxidations, would appear t o obviate t h e accumulation of phosphate anhydride energy.

Three examples will be given. (1) T h e acetone-butanol fermentation of Clostridia, (2) t h e acyloin fermentation of lactic acid bacteria and Aero-bacter, and (3) t h e oxidative pyruvate-acetate bypass.

T h e clostridial butanol fermentation (Wood, Chapter 2, Table V ) , as distinguished from acetate or b u t y r a t e fermentation, occurs without n e t energy gain beyond the pyruvate stage (see Table V I , this chapter). B y t h e thiolase condensation and Lynen cycle for acetoacetate generation from acetoacetyl-CoA, this product stoichiometry could account for t h e conver-sion of 2 moles of acetyl-CoA t o acetone and C 0² without net energy gain.

Acetone arises via t h e decarboxylation of acetoacetate, Scheme V. T h e mechanism of t h e microbial acetoacetate-forming system h a s n o t been clarified. Scheme V is based on Lynen's recent d a t a with mammalian

tis-sue.^{2 2 7}

1. ENERGY-YIELDING METABOLISM I N BACTERIA 5 1 2 acetyl-CoA - * acetoacetyl-CoA + CoA

acetoacetyl-CoA + Acetyl-CoA HMG-CoA + CoA HMG-CoA —> acetoacetate + acetyl-CoA

acetoacetate —> acetone + CO2

(33) (34) (35) (36) Sum: 2 acetyl-CoA —> acetone + CO2 + 2 CoA

SCHEME V

A nonenergy-generating system from p y r u v a t e yields 2 moles of C 0 ² and 1 acyloin without acyl ( ~ P generation^{7 6}). One pair of electrons formed from glucose via triosephosphate could reduce acetoin to butyleneglycol, but one residual electron pair would remain for another acceptor.

I t is not as yet clear whether glycolyzing cells form bypass systems which dissipate t h e energy of oxidation in heat without phosphorylative coupling;

aerobic E. coli cells do form a phosphate-independent p y r u v a t e oxidase.^{7 4}

Experiments of Senez et al. (see refs. 2 0 4 and 2 0 5 ) with nitrogen-limited growth ( N² or N 0³~ in place of N H³) showed a depressed growth rate without decreased substrate turnover—thus a metabolic rate independent of growth. This would be possible only if some mechanism either bypassing A T P generation or permitting its dissipation a t a uniform r a t e is present in t h e cell. T h e principle illustrated here is the apparent lack of regulation b y feedback or limiting level of essential cofactor or stoichiometric participa-tion in nonproliferating cell fermentaparticipa-tion and respiraparticipa-tion. As indicated in earlier sections of this chapter, m a n y questions are raised b y the funda-mental problems of energy metabolism and its relation t o growth, biosyn-thesis, and control of cell functions.

Cell behavior in nutrient-limited growth is a prime example of an area in which much information is needed; it has implications of control of pro-liferation and energy turnover in organized biological forms, including mammals.

Dr. S. R. Elsden has made many helpful contributions of data prior to publication and critical discussions of this manuscript for which the authors wish to express their appreciation.

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C . UNCOUPLING IN GROWTH

ACKNOWLEDGMENT

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In document Energy-Yielding Metabolism in Bacteria (Pldal 47-58)