Energetics of Bacterial Growth: Balance of Anabolic and Catabolic

15 Pages·1995·0.32 MB·English

MICROBIOLOGICALREVIEWS,Mar.1995,p.48–62 Vol.59,No.1
0146-0749/95/$04.00(cid:49)0
Copyright(cid:113)1995,AmericanSocietyforMicrobiology
Energetics of Bacterial Growth: Balance of Anabolic
and Catabolic Reactions
JAMESB.RUSSELL1,2*ANDGREGORYM.COOK2
USDAAgriculturalResearchService1andSectionofMicrobiology,
CornellUniversity,2Ithaca,NewYork14853
INTRODUCTION.........................................................................................................................................................48
Y VALUES................................................................................................................................................................49
ATP
FactorsAffectingY Determinations..................................................................................................................49
ATP
EstimationofATPproduction............................................................................................................................49
Energysourceutilizationforcarbon..................................................................................................................50
Changesincellcomposition................................................................................................................................50
Maintenanceenergy..............................................................................................................................................50
Y .....................................................................................................................................................................52
ATP/MAX
ISMAINTENANCEENERGYACONSTANT?.......................................................................................................52
OTHERMECHANISMSOFENERGYLOSS.........................................................................................................53
OverflowMetabolism................................................................................................................................................53
MetabolicShifts........................................................................................................................................................53
Uncoupling.................................................................................................................................................................54
ENERGY-SPILLINGREACTIONS...........................................................................................................................54
FutileCycles..............................................................................................................................................................54
Futileenzymecycles.............................................................................................................................................54
Futilecyclesofpotassiumandammonium.......................................................................................................56
Futilecycleofprotons..........................................................................................................................................56
Comparisonoffutileioncycles...........................................................................................................................57
AreEnergy-SpillingReactionsConstitutiveorInducible?.................................................................................57
HowAreEnergy-SpillingReactionsRegulated?...................................................................................................58
IsEnergySpillingAdvantageous?..........................................................................................................................59
Bacterialcompetition...........................................................................................................................................59
Dielectriceffects....................................................................................................................................................59
Methylglyoxaltoxicity...........................................................................................................................................59
MAINTENANCEVERSUSENDOGENOUSMETABOLISM...............................................................................59
APPLICATIONS...........................................................................................................................................................60
CONCLUSIONS...........................................................................................................................................................60
ACKNOWLEDGMENTS.............................................................................................................................................60
REFERENCES..............................................................................................................................................................60
INTRODUCTION clearthat‘‘inthelifeofabacterium,anynumberofessential
nutrientscananddooftenbecomelimiting’’(72).
Since Leeuwenhoek first observed bacteria more than 250
Whenbacteriaaregrownonagarplates,quantitativeaspects
years ago, microbiologists have made extraordinary progress
ofbiomassformationarecompletelydisregarded,andinmany
in studying this extremely diverse group of living organisms.
casesthegrowthassessmentofbrothcultureshasbeenlimited
The processes of energy source degradation, ATP formation, to semiquantitative scorings (e.g., (cid:49)(cid:49)(cid:49) to (cid:49)/(cid:50)) (118). With
monomer synthesis, macromolecular polymerization, DNA rep-
the advent of continuous-culture techniques in the 1950s, mi-
lication, and cell duplication are surprisingly well understood.
crobiologistswereabletogrowbacteriaunderdefinedgrowth
However,despitetheabundanceofinformationonthedetails
ratesandsteady-stateconditions,butmanycontinuous-culture
of bacterial metabolism, there has been little quantitative in-
experiments were simply an exercise of feeding and weighing
formation regarding the thermodynamics and kinetics of bac-
bacteria. The distinction between energy and carbon source
terialgrowth.
utilization,preciseestimationsofATPgeneration,andpoten-
Thegrowthstrategiesofbacteriaaresometimesmanifested
tialvariationsinbacterialcompositionwereoftenoverlooked.
by rapid cell division, but the mathematics of exponential
Standardtextbooksofbiochemistryhaveoftenpromotedthe
growth readily illustrate the point that bacteria cannot grow
ideathat‘‘cellsarecapableofregulatingtheirmetabolicreac-
rapidly for long periods. If a bacterium had an intracellular
volumeof1(cid:109)m3andadoublingtimeof20min,thissinglecell tions and the biosynthesis of their enzymes to achieve maxi-
wouldgenerateavolumeofprotoplasm2.2(cid:51)1025m3in48h. mum efficiency and economy’’ (55), and microbiologists have
Because the volume of the Earth is only 1.1 (cid:51) 1021 m3, it is generally assumed that the ‘‘yield of cells is directly propor-
tionaltotheamountofATPproduced’’(8).Thisassumption
ofastrictcouplingbetweenanabolismandcatabolismiscon-
*Correspondingauthor.Mailingaddress:WingHall,CornellUni- tradictedbytheobservationthat‘‘resting-cellsuspensions’’can
versity,Ithaca,NY14853.Phone:[phone].Fax:[phone]. utilize energy sources in the complete absence of growth and
Electronicmailaddress:[email protected]. by the fact that the correlation between ATP and biomass
48
VOL.59,1995 BALANCE OF ANABOLIC AND CATABOLIC REACTIONS 49
TABLE 1. ATPrequirementfortheformationof TABLE 2. Freeenergychangeofvariousphosphate
bacterialcellsfromglucosea transferreactionsa,b
ATPrequirement Phosphoryldonor (cid:68)G(cid:57)
(mmol/gofmacromolecule) (kcal/mol)b
%Dry
Macromolecule wt Phosphoenolpyruvate................................................................. (cid:50)12.8
Withamino Withoutamino
acids acids 1,3-Diphosphoglycerate............................................................. (cid:50)11.8
(cid:98)-L-Aspartylphosphate............................................................. (cid:50)11.5
Protein 52.2 Acetylphosphate........................................................................ (cid:50)10.1
Aminoacidformation 0.0 1.4 ATP.............................................................................................. (cid:50)7.6
Polymerization 19.0 19.0 PP ................................................................................................ (cid:50)6.6
i
Polysaccharide 16.6 ADP............................................................................................. (cid:50)6.4
G6Pbformation 1.0 1.0 Galactose1-phosphate.............................................................. (cid:50)5.0
Polymerization 1.0 1.0 Glucose1-phosphate.................................................................. (cid:50)5.0
RNA 15.7 2-Phosphoglycerate.................................................................... (cid:50)4.2
Nucleosideformation 1.5 1.5 Fructose6-phosphate................................................................. (cid:50)3.8
Polymerization 0.9 0.9 Glucose6-phosphate.................................................................. (cid:50)3.3
DNA 3.2 3-Phosphoglycerate.................................................................... (cid:50)3.1
Nucleosideformation 0.4 0.4 Fructose1-phosphate................................................................. (cid:50)3.1
Polymerization 0.2 0.4 Glycerol1-phosphate................................................................. (cid:50)2.3
Lipid 9.4 0.1 0.1
Otherfunctions aModifiedfromreference128.
mRNAturnover 1.4 1.4 b1kcal/mol(cid:53)4.184kJ/mol.
Transportofammoniumions 0.0 4.2
Transportofaminoacids 4.8 0.0
Transportofpotassium 0.2 0.2 that‘‘yieldvaluespersearenotreadilyinterpretableinprecise
Transportofphosphate 0.8 0.8 bioenergetic and/or physiological terms, and, unless treated
Total 31.3 32.3 withconsiderablecircumspection,theymayleadtotheforma-
tionofconceptsthatareatbestdubious.’’
Y (gofcells/molofATP) 32 31
ATP
aModifiedfromreference107. FactorsAffectingY Determinations
bG6P,glucose6-phosphate. ATP
EstimationofATPproduction.Theroleofphosphateesters
in energy transduction of living cells was first recognized by
formationisoftenverypoor.Someofthevariationingrowth HardenandYoungin1906(41),butitwasnotuntilthe1940s
efficiency can be explained by maintenance energy expendi- thatthesignificanceofphosphateesterswasmorefullyappre-
tures, but bacteria have other mechanisms of nongrowth en- ciated.Lipmann(56)usedtheterm‘‘energyrich’’todescribe
ergydissipation. ATP and other phosphorylated intermediates, and with time,
phosphate bond formation and breakage were recognized as
means of energy exchange. ATP is often assigned a standard
YATPVALUES freeenergy((cid:68)G)valueof(cid:50)7.6kcal/mol((cid:50)31.8kJ/mol)(Table
When Monod (68) studied the growth of Bacillus subtilis, 2),butasNicholls(77)noted,itisthedisplacementofthemass
Escherichia coli, and Salmonella typhimurium batch cultures, action ratio ‘‘from equilibrium which defines the capacity of
the dry weight of the organisms was directly proportional to thereactantstodowork,ratherthantheattributesofasingle
theamountofenergysourceadded,buttherewasnoestima- component.’’ Since it is often difficult to determine the mass
tionofATPproductionfromcarbohydratefermentation.En- actionratiounderphysiologicalconditions,the(cid:68)Gvaluesare
terococcus (Streptococcus) faecalis produced more biomass usuallylittlemorethanconjecture.
from glucose fermentation than did Lactobacillus mesen- Thestudyofbioenergeticshasalsobeenconfoundedbythe
teroides(25),andsubsequentworkshowedthatthesetwobac- factthatcellscanusetwodistinctlydifferentmethodsofATP
teria used different pathways of fermentation and produced generation. Soluble phosphate transferases (kinases) have a
differentamountsofATP/glucose(43).Bythelate1950s,bac- well-defined ATP stoichiometry (Table 2), but ATP produc-
teriologistshadgenerallyacceptedtheideathatcellyieldwas tionfromchemiosmoticmechanisms(56,66)hasbeen‘‘alively
roughlyequivalenttoenergyyield(102,104). topic of debate’’ that has yielded little lasting consensus (40).
In 1960, Bauchop and Elsden (5) studied the growth of Sometextbooksofmicrobiologystillindicatethatglucoseox-
severalanaerobicbacteriaandcorrelatedbiomassproduction idationinvolvesthreecouplingsites,withthecompleteoxida-
withATPavailability(Y ).Theyobtainedanaveragevalue tionofglucoseproducing38ATPmolpermol(8),butbacteria
ATP
of10.5gofcellspermolofATP,buttherangewasactually8.3 donotusuallyhavethreephosphorylationsites(37,39).InE.
to 12.6 g/mol. Despite the more than 50% variation, the 10.5 colithereareseveralpathwaysofrespiration,andthephysio-
valueforY wastreatedasabiologicalconstant(8,37,47, logicalmechanismsregulatingtheflowofelectronsarestillnot
ATP
105). Cellular dry weight and Y have even been used as a wellunderstood(39).
ATP
methodofestimatingtheATPproductionofasuspectedcat- Mitchell and Moyle (67), by relating the standard free en-
abolicscheme(47,105). ergyofATPhydrolysis((cid:68)G(cid:57)p)totheprotonmotiveforce((cid:68)p),
By the 1970s, the notion of a constant Y , however, was indicated that the proton stoichiometry of the mitochondrial
ATP
being questioned. A review of the literature indicated that membrane-bound ATPase was approximately 2, but similar
there was at least a fivefold range in Y values (107), and measurements with bacteria indicated that the stoichiometry
ATP
Stouthamer’scalculations(Table1)indicatedthatY should couldbe3,orevengreater(59,60,82).Eachoftheseestimates
ATP
be threefold higher than the value derived by Bauchop and assumedanintracellularmagnesiumconcentrationofapprox-
Elsden (32 versus 10.5 g of cells per mol of ATP). These imately10mM,anequilibriumbetweenthebulkphase(cid:68)pand
inconsistencies led Tempest and Neijssel (118) to conclude localized charge movement through the membrane-bound
50 RUSSELL AND COOK MICROBIOL.REV.
FIG. 1. EffectofATPontheamountofcarbonthatwouldbeusedasan FIG. 2. Growthofabacteriuminanenergy-limitedchemostatundersteady-
energysourceversustheamountthatwouldbeincorporatedintocellcarbon. state conditions. As the dilution rate increases, the concentration of energy
TherelationshipsarebasedonaY of32gofcellspermolofATPforthe sourceremaininginthechemostatvesselincreasesuntilitisequaltothecon-
ATP
growthofbacteriainminimalmedia.Carbonandenergywerecalculatedfrom centration of energy source in the medium reservoir. As the energy source
thesimultaneousequations:carbon(cid:49)energy(cid:53)1and1/32gofcellspermolof accumulatesinthechemostatvessel,thecelldensitydeclinesandtheculture
ATP(cid:49)ATP/energysource.Energyrequirementswerenotcorrectedformain- washes out when the concentration of energy source in the chemostat vessel
tenanceenergy. equalsthereservoir((cid:109) ).Atlowdilutionrates,thecelldensityalsodeclines,
max
becausealargerfractionoftheenergysourceutilizationmustbedevotedto
maintenancefunctions.Thedottedlineshowsthecelldensitywhichwouldbe
obtainediftherewerenomaintenanceenergy.
ATPase,andanegligiblecontributionofpassiveprotonleaks
(46, 53, 133). More direct estimates that were based on (cid:68)pH
relaxation(84)andheatdissipation(6)indicatedthatthepro- than protein but the Y was only 25% higher than for car-
ATP
ton/ATP ratio was only 1.9. Given these considerations, it bon-limitedcells(94).
not surprising that there is large variations in the Y of Maintenanceenergy.TheconceptofY assumesthatall
ATP ATP
aerobes. of the energy from catabolism can be used for growth; how-
Energy source utilization for carbon. Bacterial-cell yields ever, bacteria also expend energy on functions that are not
are often calculated from energy source depletion, but such directlygrowthrelated.Directestimatesofmaintenancewere
simple estimates of energy source utilization do not account confoundedbythefactthatuntilrecently,microbiologistsdid
fortheincorporationofenergysourceintocellmaterial.The not have sensitive equipment for measuring very low rates of
overestimationofATPproductionisdependentontheamount catabolism(92).Additionalconfusionarosefromtheobserva-
ofATPthattheenergysourcegenerates.IftheATPproduc- tion that the maintenance rate of growing cells is not always
tion from the catabolic scheme is low, most of the energy the same as the endogenous metabolic rate of cells that are
sourcewillbeusedforenergy,evenifothercarbonsourcesare starving (see the section on maintenance versus endogenous
notavailable.AstheATPproductionincreases,however,the metabolismbelow).
fraction of energy source that is used for carbon can be very In the 1920s, Buchanan and Fulmer (10) noted that low
significant.Onthebasisofgrowthinaminimalmediumanda levels of energy sources were not effective in subculturing
Y value of 32 g of cells per mol of ATP, as much as 90% bacteria,evenifthetransferintervalwasshortandsuggested
ATP
of the energy source could be used for cell carbon (Fig. 1). that bacteria needed some energy to ‘‘maintain’’ the cells.
Bauchop and Elsden (5) indicated that Enterobacter faecalis Monod(68)consideredthepossibilityofmaintenanceenergy
divertedonly4%oftheenergysourcetocellcarbon,butother in his classic treatise on bacterial growth, but the approach
researchers have not determined the source of cell carbon ofestimatingmaintenancefromthenegativeinterceptofglu-
(38). cose concentration versus optical density indicated that the
Changesincellcomposition.Inthe1970s,Stouthamer(107) maintenanceenergiesofE.coliandB.subtiliswereessentially
calculated the amount of ATP which would be needed to zero. In the early 1960s, McGrew and Mallette (65) tried to
produce bacterial biomass and based these calculations on a estimate bacterial maintenance energy by determining the
cellcompositionthatwastypicalofE.coli(seeTable1).These amount of glucose which would be needed to prevent a de-
calculationsillustratedatleastthreemajorpoints.First,poly- creaseinopticaldensity,butonceagainthisapproachdidnot
merization reactions, and in particular protein synthesis, are provide a clear-cut distinction between the maintenance en-
clearly the most demanding steps of biomass formation. Sec- ergy of growing cells and endogenous metabolism of starving
ond, monomer biosynthesis per se (amino acids, nucleotides, cultures.
etc.) utilizes only a small fraction of the total ATP. Third, Duclaux (28) provided a mathematical derivation of main-
transport of carbon sources and osmolytes accounts for less tenancein1898,buttherewerefewdatawithwhichtotesthis
thanone-quarterofthetotalATPrequirement. model.Withtheadventofcontinuous-culturetechniquesand
RNA and polysaccharide are the components of the bacte- thegrowthofbacteriaatdefinedandsubmaximalgrowthrates
rialcellthataremostlikelytochange.WhenE.coliincreases (69,79),theestimationofmaintenanceenergybecameamore
its growth rate, there is a commensurate decrease in protein straightforwardexercise.Sincemaintenanceisafunctionthat
levels(57),butevena2.5-foldchangeinRNAwouldcauseless detractsfromgrowth,thecontributionofmaintenanceismore
thana7%variationinY .Theenergeticdifferencebetween pronouncedwhenthegrowthrateislow(Fig.2).Herbertetal.
ATP
polysaccharideandproteinisgreater,butonceagainitwould (45)conceptualizedmaintenanceenergyas‘‘negativegrowth,’’
take a fairly large increase in polysaccharide to affect Y . andthisthemewascontinuedinthemaintenancederivationof
ATP
Whentheruminalbacterium Prevotellaruminicola wasgrown Marr et al. (63). According to Marr et al. (63), maintenance
undernitrogenlimitation,polysaccharidewas1.5timesgreater energycanbedescribedbyanegativegrowthrateconstant(a),
VOL.59,1995 BALANCE OF ANABOLIC AND CATABOLIC REACTIONS 51
Ifonedefinesx asthetheoreticalmaximumcellmass(the
max
cell mass produced if there were no maintenance), one can
derivetheequationofastraightline(Fig.3a):
1 a 1 1
(cid:53) (cid:122) (cid:49)
x x (cid:109) x
max max
A few years later, Pirt (85) indicated that the ‘‘negative
growth’’conceptofMarretal.(63)was‘‘artificialandindirect’’
and proposed a less hypothetical approach. The negative
growth rate concept was circumvented by describing mainte-
nance by a ‘‘coefficient’’ (m) that described the amount of
energy needed to maintain cells for a given period (energy/
cells/time).InPirt’sderivation,maintenancehasnodirectef-
fectongrowthrate,buttheyieldisdecreased.Onthebasisof
theassumptionthatYistheactualyieldofbacteria(gramsof
bacteriapergramofenergysource)andY isthetheoretical
G
maximumyield(Yiftherewerenomaintenance),totalenergy
utilization ((cid:109)x/Y) can be partitioned into maintenance (mx)
andtruegrowth((cid:109)x/Y ):
G
(cid:109)x/Y(cid:53)mx(cid:49)(cid:109)x/Y
G
UsingthesametypeofalgebraictransformationsasMarretal.
(63),Pirtsucceededinderivinganotherstraight-lineequation
(Fig.3b):
1 m 1
(cid:53) (cid:49) (1)
Y (cid:109) Y
G
Thetwomaintenanceparameters,manda,arerelatedby
m(cid:53)a/Y
G
More recently, Tempest and colleagues (75, 117) algebra-
ically modified the derivation of Pirt (equation 1) to get an-
other linear relationship (Fig. 3c). Assuming that the specific
rateofenergysourceconsumptionq(cid:53)(1/Y)(cid:109)and1/Y(cid:53)q/(cid:109),
q m 1
(cid:53) (cid:49)
(cid:109) (cid:109) Y
G
Multiplyingby(cid:109),
q(cid:53)m(cid:49)(cid:109)(cid:122)1/Y
G
TheplotsusedbyPirtandTempestbothdefinemasaspecific
coefficient, but the experimental error of the plots is parti-
tioned differently. In the Pirt plot (Fig. 3b), the error is pri-
marilyintheslope,whereastheerroroftheTempestplot(Fig.
3c) is primarily in the intercept. Since the intercept of the
Tempestplotisusuallysmall,thedataalwayslookbetter.
FIG. 3. Effectofmaintenancerate(a)orthemaintenancecoefficient(m)on Thelinearityofmaintenanceplotsisbasedontheassump-
theyieldofabacteriumgrowninanenergy-limitedchemostat.(a)Derivationof tionsthattheATPproductionperunitofenergysourcedoes
Marr et al. (63); (b) derivation of Pirt (85); (c) derivation of Tempest and not change, cell composition remains the same, and mainte-
Neijssel(118).Y isdefinedasthetheoreticalmaximumgrowthyield.
G nanceisastrictlymass-andtime-dependentfunction.Pirt(85)
notedthatthemaintenanceplotofSelenomonasruminantium
wasnotlinearandindicatedthatthechangeinyieldcouldnot
and the total rate of substrate utilization for growth [((cid:50)dS/ readilybeattributedtomaintenance.Laterworkshowedthat
dt)(cid:122)Y]canthenbepartitionedintogrowth((cid:109)x)andnegative S. ruminantium switches fermentation end products and gets
growth(ax): more molecules of ATP per molecule of glucose when the
growthrateislow(97).Pirtplotsoftheaminoacid-fermenting
(cid:126)(cid:50)dS/dt)(cid:122)Y(cid:53)(cid:109)x(cid:49)ax
bacteriumClostridiumsticklandii(124)indicatedthatarginine-
Fromtheseassumptions,acausesadecreaseinthetheoretical limitedcellshadtwiceashighamaintenancecoefficient(based
maximumgrowthrateoftheorganism,and(cid:109) canbeenvi- on ATP) as did lysine-limited cells, but this difference was
sionedasthegrowthratethatwouldbeobtainmeadxiftherewere caused by transport rather than maintenance per se. C. stick-
nomaintenance: landii always used facilitated diffusion to take up lysine, but
argininewastakenupbyeithersodiumsymport(activetrans-
(cid:109) (cid:53) (cid:109) (cid:49) a port)orfacilitateddiffusion.Whenthedilutionrateandargi-
max
52 RUSSELL AND COOK MICROBIOL.REV.
TABLE 3. Y ofvariousmicroorganismsgrownin
ATP/MAX
glucose-limitedcontinuousculturea
Microorganism Y b
ATP/MAX
Lactobacilluscasei....................................................................... 24.3
Aerobacteraerogenes.................................................................... 14.0
Escherichiacoli............................................................................. 10.3
Saccharomycescerevisiae............................................................. 13.0
Candidaparapsilosis.................................................................... 12.5
aModifiedfromreference107.
bGramsofcellspermoleofATP.
nineconcentrationdecreased,C.sticklandiigraduallyswitched
from facilitated diffusion to sodium symport. The increased
cost of arginine transport at low dilution rates in continuous
culture caused a decrease in cell yield and an increase in the
slopeofthePirtplot.
Inthemathematicalderivations,maintenanceislooselyde- FIG. 4. Relationshipbetweenthespecificgrowthrateandthespecificrateof
finedasanydiversionofenergyfrom‘‘growth’’to‘‘nongrowth’’ O consumptioninchemostatculturesofKlebsiellaaerogenesgrowinginglucose-
2
reactions,butthisdefinitiongiveslittlemechanisticinsightinto containingmediathatwerelimitedbycarbon,phosphorus,orammonia.Re-
printedwithpermissionfromreference117.
the nature of maintenance functions. In many cases, the dif-
ferencebetweenmaintenanceandgrowthisdictatedbynoth-
ing more than the difference between net and gross. For ex-
ample, protein synthesis is a growth-related function, but Lactobacillus casei grown in glucose-limited continuous cul-
protein turnover (degradation and resynthesis) is a mainte- tureshadaYATP/MAXofonly24.3gofcellspermolofATP.
nanceexpenditure. Because other values of YATP/MAX were even lower, it ap-
Radiolabelingexperimentsindicatedthattherateofprotein peared that maintenance energy alone could not explain the
turnoverinexponentiallygrowingE.colicellsrangedfrom0.5 variations in growth efficiency (Table 3). As noted by Harold
to2.5%/handthatRNAturnoverparalleledthebreakdownof (42), ‘‘there is something misleading about the fundamental
protein (61). During stationary phase, protein turnover was assumption that the free energy of catabolism is fully con-
higher(5%forE.coliand8%forBacilluscereus[61]),buteven servedasATPandexpendednecessarilyeitherforbiosynthesis
theseratescannotaccountforallofthemaintenanceinE.coli. orforusefulwork.Anydeparturefromperfectcoupling,either
Onthebasisofacellcompositionof0.5gofproteinpergof inthegenerationofATPorinitsutilization,willshowupasa
cells,4ATPequivalentsperaminoacidpolymerized,anaver- shortfall of the yield and exaggerate the apparent cost of cel-
age molecular mass for an amino acid of 100 Da, and a max- lularupkeep.’’
imum ATP production of 24 ATP per glucose, the glucose
consumptionrateneededtosustainaproteinturnoverrateof
ISMAINTENANCEENERGYACONSTANT?
5%/hwouldbe0.04mmolofglucosepergofcellsperh.The
maintenance rate of E. coli is 0.31 mmol of glucose per g of Hempfling and Mainzer (44) grew E. coli in continuous
cellsperh(85). cultureandnotedthatthespecificrateofoxygenconsumption
Ingrahametal.(47)listedthe‘‘accumulationofsubstratesto wasdependentonthecarbonsourcelimitinggrowthaswellas
a higher concentration’’ as a maintenance function, but onthegrowthrateofthecultures.Evenwhencorrectionswere
Stouthamerindicatedthatmosttransportfunctionsaregrowth madefordifferencesinATPproduction,themaintenanceco-
related (Table 1). Once again, the difference between growth efficients varied by as much as 2.5-fold. Anderson and von
and maintenance is related to turnover. It was originally as- Meyenburg(1)likewiseobservedthatthespecificrateofres-
sumedthatbacterialmembraneswereperfectinsulators(66), pirationbyE.coliwasnotwellcorrelatedwiththegrowthrate
but bacterial membranes have an inherent or passive perme- when the carbon source was changed. Since the variation in
abilitytomostions(59,120).Ionfluxesacrossthemembranes respiration was far greater than the amount that could be
of growing bacteria have not been measured directly, but ion ascribed to ATP production, it appeared that the cells had a
turnover is likely to be a very significant component of main- variable maintenance coefficient or were wasting (spilling)
tenance.Becausetheflagellaofbacteriaaredrivenbyproton ATP.
or sodium motive force, motility can be viewed simply as a When Neijssel and Tempest (74, 75) grew Klebsiella aero-
specialcaseofionturnover.MacNabandKoshland(58)indi- genes in continuous cultures which were limited by carbon,
catedthatasmuchas1%ofthetotalenergyinE.colicouldbe ammonia, sulfate, or phosphate, the rate of carbon source
devotedtomotility.BecausethemaintenancerateofE.coliis utilization was always higher when carbon was in excess. On
only 1.25% of the glucose consumption rate of exponentially the basis of Tempest plots (Fig. 4), ‘‘these carbon-sufficient
growingcells(85),thisvalueisprobablynotapreciseestimate. cultures had a greatly increased maintenance energy require-
ment,butneverthelessusedtheremainingenergywithamuch
Y increased efficiency compared with carbon-limited cultures.’’
ATP/MAX
Maintenance(theinterceptoftheTempestplot)increasedbut
In an effort to account for the impact of maintenance on theslope(1/Y )decreasedwhencarbonwasinexcess.
G
Y ,StouthamerandBettenhaussen(108)introducedanew Becausemannitol-andglucose-limitedculturesofKlebsiella
ATP
term,Y ,whichwascorrectedformaintenanceenergy. aerogenes had significantly lower growth yields than did glu-
ATP/MAX
However,DeVriesetal.(26)notedthatnoteventhiscorrec- conate-limited cultures, Neijssel and Tempest (76) indicated
tion could give Y values as great as 32 g of cells per mol; that‘‘themannitolandglucose-limitationsmustbeessentially
ATP
VOL.59,1995 BALANCE OF ANABOLIC AND CATABOLIC REACTIONS 53
carbon (and not energy) limitations.’’ In an effort to explain
thedifferencebetween‘‘energy-andcarbon-limited’’cultures,
Neijssel and Tempest concluded that ‘‘maintenance energy is
composed of at least two factors: (i) maintenance of cell in-
tegrity, and (ii) maintenance of growth potential (involving
slips reactions).’’ On the basis of these latter assumptions, an
additionalvariable,c,wasaddedtothestandardPirtequation
(75):
1 m 1
(cid:53) (cid:49) (cid:49) c (cid:122) m
Y (cid:109) Y
G
Sinceq(cid:53)(1/Y)(cid:109)and1/Y(cid:53)q/(cid:109),
q m 1
(cid:53) (cid:49) (cid:49) c (cid:122) m
(cid:109) (cid:109) Y
G
Multiplyingby(cid:109),
q(cid:53)m(cid:49)(cid:109)[(1/Y )(cid:49)c(cid:122)m]
G
In this model of energy excess cultures, the c term is given a
negative value. The negative c term allows for a decrease in
1/Y , the slope, but this adjustment alone would not account
G
foranincreaseinm,theintercept(Fig.4).
Pirt(86)addressedtheideaofvariablemaintenancerateby
redefining maintenance with both growth rate-independent
(m) and growth rate-dependent (m(cid:57)) components in which
equation 1 is modified. k(cid:109) is defined as the specific growth
rate-dependentmaintenancerate:
1 m 1 m(cid:57)(cid:126)1-k(cid:109)(cid:33)
(cid:53) (cid:49) (cid:49)
Y (cid:109) Y (cid:109) FIG. 5. Effectofdilutionrateontheglucoseyield(a)andATPyield(b)of
G
Streptococcusboviswhenitwasgrownincontinuousculture.Redrawnfromthe
Sinceq(cid:53)(1/Y)(cid:109)and1/Y(cid:53)q/(cid:109), dataofRussellandBaldwin(97).
q m 1 m(cid:57)(cid:126)1(cid:50)k(cid:109)(cid:33)
(cid:53) (cid:49) (cid:49)
(cid:109) (cid:109) Y (cid:109) concluded that microbial growth yields were ‘‘50% less than
G
they theoretically could be’’ and that ‘‘anabolism is incom-
Multiplyingby(cid:109),
pletelycoupledtocatabolism.’’AccordingtoWesterhoffetal.
q(cid:53)m(cid:49)(cid:109)(1/Y ) (cid:49) m(cid:57)(1 (cid:50) k(cid:109)) (129), ‘‘some thermodynamic efficiency may be sacrificed to
G maketheprocessrunfaster,’’butthecarbon-sufficientcultures
k is assigned a value of 1 when the culture is growing at its of Neijssel and Tempest (75) had their greatest unexplained
maximum rate or is carbon limited, but k increases to values energydissipationwhenthegrowthratewaslow(Fig.4).
greater than 1 when carbon is in excess. This unified model
providedarealisticmethodofdescribingthevariationinmbut OverflowMetabolism
did not address the biological mechanisms affecting the vari-
ablemaintenance.AsPirtnoted(86),‘‘Whyshouldthespecific Bacteriasometimesexcreteorleakpartiallyoxidizedmeta-
maintenancerate(a)varyupto30fold(fromabout0.01to0.3 bolicintermediates,capsularmaterial,andproteinintoculture
h(cid:50)1)dependingonthenatureofthecarbonandenergysource media (21, 38, 101, 107, 119). Tempest and Neijssel (74, 117)
which limits growth?’’ In his review of factors affecting the noted that Klebsiella aerogenes produced pyruvate, 2-oxoglut-
growth rate of E. coli, Marr (62) indicated that there is a arate, gluconate, 2-ketogluconate, and succinate when energy
‘‘considerablebodyofevidence’’indicatingthatATPproduc- wasinexcess,buttheseintermediatesaccountedforlessthan
tiondoesnotnecessarilydeterminethegrowthrateofE.coli, 50% of the unexplained energy source utilization (75). The
evenifallnecessarynutrientsareinexcess.Recentworkwith cellsalsoexcretedsomeextracellularpolysaccharideandpro-
Zymomonas mobilis indicated that overexpression of fermen- tein,buteventheseproductscouldnotaccountfortheabnor-
tativegenesinsomecasescauseda50%reductioninglycolytic mallyhighratesofglucoseconsumption(74).Sincethecarbon
rate,buttherateofgrowthdidnotdecrease(2). balances were nearly 100%, it appeared that the cells were,
indeed,respiringtheglucoseandnotjustsecretingorstoring
carbon.
OTHERMECHANISMSOFENERGYLOSS
MetabolicShifts
The terms ‘‘uncoupling,’’ ‘‘energy spilling,’’ ‘‘overflow me-
tabolism,’’‘‘futilecycles,’’‘‘slipreactions,’’and‘‘wastage’’(11, Fermentative bacteria can change their end products and
62,76,102,107,116)haveallbeenusedascorrectivemeasures alterATPproduction.Manystreptococciandlactobacillithat
to justify variations in yield, but the mechanism(s) of the ad- were originally classified as homolactic produce acetate, for-
ditional energy expenditure was not defined. In the 1980s, mate,andethanolwhentherateofglucosefermentationislow
Westerhoffetal.(129–131)appliedtheprinciplesofnonequi- (26, 97). The change from a homo- to heterofermentative
librium thermodynamics to the study of bacterial growth and scheme increases the ATP/glucose ratio from 2 to 3. These
54 RUSSELL AND COOK MICROBIOL.REV.
changes are regulated by fructose 1,6-diphosphate (FDP), an thesis, conceptualized any anomaly as ‘‘uncoupling.’’ This all-
allosteric effector of lactate dehydrogenase (LDH) (132). inclusive definition did not differentiate between the produc-
Whentherateofglucosefermentationdecreases,thelevelof tionofATPandtheutilizationofATPinnongrowthreactions.
intracellularFDPdeclinesandtheLDHisnolongeractivated. Because the latter process would be more aptly termed ATP
In Streptococcus bovis, the FDP activation is also regulated spilling(seethesectiononATPspilling,below),wewilldefine
by changes in intracellular pH. As intracellular pH declines, uncoupling as the inability of chemiosmotic mechanisms to
theLDHrequireslessFDPandthefermentationishomolactic generatethetheoreticalamountofmetabolicenergy.
eventhoughtherateofglucosefermentationislow(98).Sel- Oxygen consumption is often used as an indicator of respi-
enomonas ruminantium also regulates lactate production as a ration, but as Haddock (39) noted, ‘‘it is important to appre-
function of fermentation rate (97), but this regulation is not ciatethatnotallmembrane-boundredoxenzymessynthesized
mediated by the effect of FDP on the LDH (126). Wallace inE.coliarenecessarilyinvolvedinenergyconservation.Many
(126)indicatedthatlactateregulationbyS.ruminantiummight serve simply for the reoxidation of reduced coenzymes, the
bemediatedbyahomotropicactivationoftheLDHbypyru- removalofpotentiallytoxicmetabolicproducts,orthereduc-
vate, but later work indicated that the intracellular pyruvate tionofintermediatesrequiredforbiosyntheticreactions.’’Azo-
concentration did not increase (112). In S. ruminantium, the tobactervinelandiiusesaportionofitsrespiratorychainsolely
decreaseinlactateproductionatlowratesofglucosefermen- toscavengeoxygenandprotectnitrogenase(51),andwhenE.
tation is associated with an increase in levels of acetate, pro- coli was grown under sulfate limitation, the NADH dehydro-
pionate,andsometimessuccinate(97,112). genasebecameanon-proton-translocatingenzyme(87).E.coli
WhenS.boviswasgrowninaglucose-limitedchemostat,the has two different respiratory pathways involving different
glucoseyielddeclinedathighdilutionrates,andthisdecrease NADH dehydrogenases (NDH-1 and NDH-2) and terminal
wascausedbyachangefromhetero-tohomolacticfermenta- cytochromes (o and d) (40). An E. coli mutant defective in
tion(97).Theplotof(1/dilutionrate)versus(1/glucoseyield) cytochrome o grew less efficiently than the wild type, but a
waslinearatlowdilutionrateswhenvirtuallyalloftheglucose mutant defective in NDH-2 grew with greater efficiently than
was converted to acetate, formate, and ethanol, but 1/yield the wild type (15). From these comparisons, it appears that
increased at high dilution rates (Fig. 5a). When corrections bacteriacanhavemultiplestrategiesofelectronflowandcou-
were made for differences in ATP production, the plot was pling.
linear (Fig. 5b). On the basis of the intercept of the plot, the Because ATPase-negative mutants can use electron trans-
Y was 22 g of cells per mol of ATP. Even when cor- port systems to create a (cid:68)p but not ATP, ATPase-negative
ATP/MAX
rections were made for the incorporation of glucose into cell mutants have been used as a method of estimating coupling.
carbon, the Y was only 25 g of cells per mol of ATP Jensen and Michelsen (49) recently reported that ATPase-
ATP/MAX
(93). negative mutants of E. coli had much higher rates of oxygen
In the 1930s, methylglyoxal was believed to be a normal consumption than could be explained by a simple shift from
intermediateinthecatabolismofglucose,butbythe1940s,the oxidative phosphorylation to glycolysis and concluded that
methylglyoxalshuntwaslargelydismissedasanartifact(19).In wild-typeE.coliwasnotcouplingrespirationandATPsynthe-
1970, Cooper and Anderson (20) showed that E. coli used a sisinahighlyefficientmanner(49).Onthebasisoftheinher-
pathway involving methylglyoxal to convert dihydroxyacetone entconstraintsofestimatingthedegreeofcouplingthatexists
phosphatetoD-lactate.Sincethispathwaydoesnothavephos- inoxidativephosphorylation,onemustviewtheYATPvaluesof
phate transferases, the free energy change of glucose catabo- aerobes with a high degree of skepticism. Only in anaerobic
lism does not generate ATP. The methylglyoxal pathway has systems which depend solely on substrate level phosphoryla-
been demonstrated in Pseudomonas saccharophilia, Clostrid- tion can the rate of ATP production be estimated with any
iumsphenoides,andEnterobacter(Klebsiella)aerogenes(19,34, certainty.
35). StouthamerandBettenhaussen(109)notedthatanATPase
When anaerobic glucose-limited continuous cultures of K. mutantgrownaerobicallyhadaY thatwasmorethan
ATP/MAX
aerogeneswerepulsedwithglucose,thespecificrateofglucose twice as high as that of a wild type grown anaerobically and
consumption increased markedly, and much of the additional concludedthatwild-typeE.coliwasusingmorethanhalfofits
glucose was converted to D-lactate (114, 115). From this and energytosustainamembranepotential.Sincetheturnoverof
subsequent work (110, 111), it appeared that anaerobic cul- ionsthroughthecellmembraneisclearlyamaintenancefunc-
turesofK.aerogenescouldshifttheirmetabolismandproduce tion,onewouldhaveexpectedadifferenceinthemcoefficient,
less ATP when an energy source was in excess. However, not but there was little difference in either m or m . 2,4-
glucose ATP
even the methylglyoxal shunt could explain all of the non- Dinitrophenol, an uncoupler which acts as a protonophore,
growth, nonmaintenance energy dissipation of K. aerogenes. causedadecreaseintheoreticalmaximumgrowthaswellasan
When K. aerogenes was grown aerobically with an excess of increaseinmaintenanceenergy(73).
glucose under steady-state conditions, the specific rate of ox-
ygenconsumptionwasgreaterthanunderglucose-limitedcon-
ENERGY-SPILLINGREACTIONS
ditionsandD-lactatecouldnotbedetectedasanendproduct
(74,75).
FutileCycles
In some cases, metabolic shifts account for some of the
variations in yield, but such a phenomenon alone cannot ex- Futile enzyme cycles. Certain sequences of metabolism can
plainwhytheY ofmostbacteriaissignificantlylower serve as catabolic and anabolic pathways (e.g., glycolysis and
ATP/MAX
thanthetheoreticalvalueof32gofcellspermolofATPthat gluconeogenesis)andactinanantagonisticfashion(e.g.,phos-
wasderivedbyStouthamer(106). phofructokinaseandfructose-1,6-diphosphatase,glycogensyn-
thetaseandglycogenglycogenolysis,glucokinaseortheglucose
Uncoupling phosphotransferasesystem(PTS)andglucose-6-phosphatase)
(Fig. 6a). These antagonistic enzymes must be regulated to
Senez (102), in describing the link between energy-yielding preventafutilecycleofATPutilization.Bacteriahaveevolved
reactions and the energy-consuming reactions of cell biosyn- avarietyofallostericmechanismstocounteracttheseopposing
VOL.59,1995 BALANCE OF ANABOLIC AND CATABOLIC REACTIONS 55
FIG. 6. Potentialmethodsofenergyspillinginbacteria.(a)Futilecyclesofenzymesinvolvingphosphorylationanddephosphorylation.(b)AfutilecycleofK(cid:49)via
K(cid:49)influxbyhigh-affinityK(cid:49)transportandK(cid:49)effluxvialow-affinityK(cid:49)transport.(c)UptakeofNH(cid:49)viaK(cid:49)/NH(cid:49)antiporter,dissociationofNH(cid:49),passiveefflux
ofNH,andeffluxofH(cid:49)viatheFF-ATPase.(d)UptakeofNH(cid:49)byhigh-affinityK(cid:49)transport,di4ssociationofN4H(cid:49),thepassiveeffluxofNH,4andexpulsionof
H(cid:49)via3theFF-ATPase.(e)Expu1lsi0onofH(cid:49)viatheFF-ATPa4seandtheinfluxofH(cid:49)viaaresistance(R)change4inthecellmembrane. 3
1 0 1 0
processes,andthesemechanismscanrespondtokeyintracel- phosphoenolpyruvate carboxylase in E. coli, the cell yield in-
lularmetabolitesaswellastotheenergystateofthecell. creased,butmostofthiseffectwasexplainedbyanincreasein
Otto(81)reportedthatLactococcus(Streptococcus)cremoris ATPproduction(lessfermentation)ratherthanlessATPturn-
hadbothphosphofructokinaseandfructose-1,6-diphosphatase over per se. From these results, it did not appear that phos-
activityandsuggestedthatthesetwoenzymeswereresponsible phoenolpyruvate carboxylase and phosphoenolpyruvate car-
fortheincreasedrateoflactosecatabolismbyleucine-limited boxykinasewereoperatingasasignificantfutilecycle.
cells. This conclusion was based on the observation that the
2-Deoxyglucose(2-DG)isoftenusedtodeenergizebacteria.
leucine-limitedcellshadfivefold-lowerintracellularAMPlev-
2-DG is taken up by the glucose PTS, 2-DG-6-phosphate is
elsandlessphosphoenolpyruvatethandidthelactose-limited
accumulatedtohighconcentrations(approximately100mM),
cells. AMP is an inhibitor of fructose-1,6-diphosphatase, and
2-DG-6-phosphate is dephosphorylated by hexose-6-phos-
phosphoenolpyruvate is an inhibitor of phosphofructokinase.
phatase,and2-DGleaksoutofthecell(121).Sincethephos-
Directfluxthroughthiscycle,however,wasnotdemonstrated.
phoenolpyruvate must be replenished, this cycle represents a
Using32P labeling,DaldalandFraenkel(22)notedthatthere
i net loss of ATP. The potential involvement of sugar uptake
was little, if any, gluconeogenic futile cycling in exponentially
andeffluxinbacterialgrowthkineticshasneverbeenassessed,
growingE.colicells.
Patnaik et al. (83) recently examined the potential cycle of but it is unlikely that glucose PTS and hexose-6-phosphatase
pyruvate kinase and phosphoenolpyruvate synthase in E. coli wouldoperateasafutilecycleunderphysiologicalconditions.
byusingoverexpressionmutants.Themutantsconsumedmore Theintracellularconcentrationofglucose6-phosphateingly-
oxygenthandidthewildtype,buta30-foldoverexpressionof colyzing(mostprobablyenergy-spilling)cellswasonly1.6mM
phosphoenolpyruvatesynthaseincreasedoxygenconsumption (121), the affinity of the phosphatase for hexose 6-phosphate
only twofold. When Chao and Liao (16, 17) overexpressed was very low (K of approximately 20 mM) (122), and the
m
56 RUSSELL AND COOK MICROBIOL.REV.
TABLE 4. EffectoffutileenzymecyclesontheATPturnoverratebybacteriaunderdifferentgrowthconditions
Nongrowthenergy AdditionalATPconsumption
Bacterium Growthlimitation dissipation ((cid:109)molofATP/mgofprotein/h) Reference
E.coli Glucose Maintenance 5.1–8.7 109
E.coli Potassium Energyspilling 19.2 70
E.coli Potassium(cid:49)excessammonium Energyspilling 25.4 13
S.bovis Glucose Maintenance 5.0 97
S.bovis Chloramphenicol Energyspilling 50.8 18
additionofglucosebrokethecycleofdeenergizationby2-DG mulationof[14C]methylammoniumandammoniumcompetes
(121). with methylammonium uptake, it appears that E. coli has a
E.colicarefullyregulatesitsratesofglycogensynthesisand K(cid:49)/NH (cid:49) antiporter (4). Active uptake of ammonium ion,
4
glycogenolysis (88), but such control may not be a ubiquitous however, is counteracted by a more alkaline interior and the
feature of all bacteria. When the glycogen reserves of the passiveeffluxofammonia.Kleiner(52)estimatedthatupto6
cellulolytic bacterium Fibrobacter succinogenes were labeled mol of ammonium may be transported before 1 mol can be
with [13C]glucose and the cells were then incubated with fixed by the glutamine synthetase/glutamate synthase cycle
[1-13C]glucose and [2-14C]glucose, there was a rapid loss of (Fig.6c).Thisfutilecycleofammoniumandammoniawould
both 1-13C and 2-13C (36). From these results, the authors decrease (cid:68)p, dissipate the potassium gradient, and increase
concludedthat‘‘glycogenwasdegradedatthesametimeasit F F -ATPaseactivity.
1 0
was being stored, suggesting futile cycling of glycogen.’’ Sub- When potassium is limiting, some bacteria can use ammo-
sequentwork,however,indicatedthatglycogenrecyclinginF. niumasareplacementionforintracellularpotassium(13),and
succinogeneswasnotaprimarycomponentofitsmaintenance undertheseconditionsE.coliappearstotransportammonium
energy.ThespecificrateofglycogencatabolisminF.succino- ions via the Kdp potassium uptake system (12). When E. coli
geneswasalwaysatleastthreefoldlowerthanthemaintenance was grown under potassium-limiting conditions, addition of
rateofgrowingcells(127). ammonium chloride caused a significant increase in the spe-
While it is impossible to rule out futile enzyme cycles as a cific rate of oxygen consumption (12). On the basis of the
potentialmechanismofATPturnoverinbacteria,thereislittle observation that a mutant which lacked the Kdp potassium
evidence to suggest that such cycles are highly significant or transport system grew more efficiently under potassium limi-
likelytoplayamajorroleinATPspilling. tationwhenammoniumwasinexcess,itappearedthatammo-
Futilecyclesofpotassiumandammonium.InE.coli,intra- niumwasbeingtakenupbyKdpandwasthendiffusingoutof
cellular potassium is a prime factor in regulating turgor pres- the cell as ammonia (Fig. 6d). In this case, the futile cycle
sure,andthisbacteriumhasmultipletransportsystemswhich wouldresultinadirectconsumptionofATPbyKdpaswellas
are involved in potassium uptake and efflux (3). Similar sys- increasedATPconsumptionbytheF F -ATPase.
1 0
temsappeartooperateinavarietyofgram-negativebacteria Futilecycleofprotons.ThefermentativebacteriumStrepto-
andStaphylococcusaureus(27).Mulderetal.(70)studiedthe coccusbovisderivesallofitenergyfromsubstrate-levelphos-
impactofpotassiumtransportsystemsonthegrowthefficiency phorylation,andcontinuous-culturestudiesindicatedthatthis
of E. coli when potassium was limiting. Because a mutant bacteriumhadahighY (30gofcellspermolofATP)
ATP/MAX
which was defective in high-affinity potassium transport uti- ifitwasglucoselimited(93).WhenS.boviswasgrowninbatch
lized glucose more efficiently, it appeared that the wild type culturewithanexcessofglucose,theY declinedmore
ATP/MAX
was taking up potassium by the high-affinity, ATP-driven than 15%, and chloramphenicol-treated batch cultures fer-
(Kdp) system and losing potassium through the low-affinity mentedglucoseatahighrateeventhoughgrowthandprotein
protonsymport(Trk)system(Fig.6b).Onthebasisofdiffer- synthesis were completely inhibited (18, 93). This nongrowth
encesinATPproductionratesandthesteady-stateconcentra- glucoseconsumptionratewas10timesthemaintenancerateof
tions of biomass in continuous culture, it appeared that the glucose-limitedcellsandnearlyone-thirdtherateofexponen-
futilecycleofpotassiumwasdecreasingATPavailability(Ta- tiallygrowingcells(approximately28(cid:109)molofglucosepermg
ble4). ofproteinperh).
TheuptakeandeffluxofpotassiumproposedbyMulderet Because S. bovis spilled energy even when potassium and
al.(70),however,wouldnotnecessarilyleadtoafutileenergy ammoniumwereinexcess(18),itappearedthatthecellshad
cycle (Fig. 6b). The uptake of potassium via the ATP-driven amechanismofenergyspillingthatdidnotinvolvehigh-affinity
Kdp system would consume ATP, but electrogenic efflux of potassiumorammoniumcarriers(Fig.6candd).Thishypoth-
potassium via the Trk potassium proton symport would gen- esis was supported by the observation that N,N(cid:57)-dicyclohexyl-
eratea(cid:68)(cid:67)anddecreaseF F -ATPase-dependentATPhydro- carbodiimide (DCCD), an inhibitor of the membrane-bound
1 0
lysis. A significant energy loss would only occur if there was F F -ATPase, completely inhibited nongrowth glucose fer-
1 0
anotherpathwayofpotassiumefflux(e.g.,apotassiumchannel mentation and ATP turnover. Thus, the ATP spilling by S.
operating as a uniporter). Zoratti and Ghazi (134) recently bovisseemedtobecausedbyadirectcycleofprotonsthrough
summarizedevidencefortheexistenceofturgor-activatedpo- the cell membrane (Fig. 6e). This model was consistent with
tassium channels in E. coli. These potassium uniporters seem the effect of 3,3(cid:57),4(cid:57),5-tetrachlorosalicylanilide (TCS), an un-
to provide a more plausible explanation for the results of couplerthatdecreasesmembraneresistancetoprotons.When
Mulderetal.(70)thandoestheTrkperse. the chloramphenicol-treated S. bovis cells were treated with
Whenammoniaconcentrationsarehigh,facilitateddiffusion TCS, the rate of energy spilling increased more than twofold
appears to be the dominant mechanism of ammonia uptake, (100). The idea that bacteria can decrease their membrane
but bacteria also have active uptake mechanisms for ammo- resistance is supported by the work of Taylor and Jackson
nium ions (4, 52). Since internal K(cid:49) is required for the accu- (113), who showed that the phototrophic bacterium,
VOL.59,1995 BALANCE OF ANABOLIC AND CATABOLIC REACTIONS 57
Rhodobactercapsulatus,hadacurrent-carryingpathwayinthe
cell membrane that was capable of dissipating a light-driven
membranepotentialwhenenergywasinexcess.Becauseonly
net charge transfer was measured, the current could not be
precisely defined, but proton flux was ‘‘the most likely candi-
date.’’
The best-documented example of a proton cycle occurs in
the mitochondria of brown adipose tissue, and this cycle is
mediatedbyaspecificprotonchannel.Formanyyears,itwas
assumed that energy spilling was a unique characteristic of
newborn and hibernating animals, but recent work indicated
that mitochondrial proton leak is a general characteristic of
most mammalian tissues (7). Brand et al. (7) estimated that
proton leaks could account for 26% of the total oxygen con-
sumption of animals. The mechanism of the proton leak in
tissueslikemuscleandliverhasbeenlinkedonlytolongterm
effects(hormoneactions,increasedsurfaceareaofmitochon-
drialmembranes,andchangesinthefattyacidcompositionof
mitochondrialmembranes).
Comparison of futile ion cycles. The experiments with E. FIG. 7. Relationshipbetweentherateofglucoseconsumptionandintracel-
coli, S. bovis, and R. capsulatus indicate that bacteria can dis- lularATPconcentrationinnongrowing,energy-spillingStreptococcusboviscells.
RedrawnfromthedataofCookandRussell(18).
sipateenergyinfutilecyclesofionsthroughthecellmembrane
and that these cycles can be regulated by short-term mecha-
nisms that do not involve additional protein synthesis. Some
ion cycles will occur only under specific nutrient limitations acids caused large increases in the yield and growth rate of
(e.g.,potassiumandammonia),butprotoncyclescanoperate batch cultures. Because the amino acid-dependent change in
wheneverthereisanimbalanceofcatabolicandanabolicrates. growth rate and yield of the batch cultures was at least five
Thisadditionalnongrowthenergydissipationwilldetractfrom times greater than the maintenance rate of glucose-limited
cell production in the same manner as maintenance, but the cells, maintenance alone could not explain the difference in
magnitudeisoftenmuchgreater.InS.bovis,therateofenergy growthefficiency.
spillingis10-foldgreaterthanthemaintenancecoefficientand The effect of amino acids on the growth ofS. bovis is most
approximately one-third of the glucose consumption rate of easilyexplainedbyenergyspillingandthebalanceofanabolic
exponentially growing cells. Given such observations, energy and catabolic rates (95). In continuous cultures, the rate of
spilling could have a very significant effect on the overall effi- anabolismwasregulatedbytherateofglucoseentry(dilution
ciency of bacterial cell production and explain much of the rate),thecellswereenergylimited,andtheefficiencyofgrowth
discrepancybetweenactualandtheoreticalgrowthyields(Ta- washigh.Inthebatchcultures,thesituationwasquitediffer-
ble4). ent. Glucose was always in excess, and the rate of anabolism
was controlled by amino acid availability. When S. bovis was
transferredfromarichmedium(containingaminoacids)toa
AreEnergy-SpillingReactionsConstitutiveorInducible?
minimal medium (only ammonia as a nitrogen source), the
Because ATP-spilling reactions were traditionally demon- specificgrowthratedecreasedby50%butthespecificrateof
strated in continuous cultures that were limited by nutrients glucoseconsumptionremainedthesame(95).Sincethecata-
other than energy, it appeared that ATP spilling might be an bolic and anabolic rates were no longer in balance, the addi-
induciblephenomenon.Thisassumptionwassupportedbythe tionalATPwashydrolyzedbyenergy-spillingreactions.
observationthathigh-affinitytransportsystemsforammonium
andpotassiumareclearlyinducible(Fig.6btod).Pulsedose
experiments,however,indicatedthatbacteriainevenrichme-
diacouldspillexcessenergy.Whenenergy-limitedcontinuous
culturesofPseudomonassp.(9),K.aerogenes(76),Selenomo-
nasruminantium,Prevotellaruminicola(92),andStreptococcus
bovis(18)weregivenapulsedoseofenergysource,therewas
animmediateincreaseintherateofenergysourceutilization
thatdidnotcorrespondtoanincreaseincellproduction(en-
ergyspilling).BecauseS.bovisshowedanimmediateincrease
in energy spilling when it was treated with chloramphenicol
(18, 100) and energy-limited Rhodobacter capsulatus had the
sameinherentcapacitytospillenergyasenergy-excesscultures
(113), it does not appear that energy spilling is an adaptive
phenomenonthatrequiresadditionalproteinsynthesis.
It has long been recognized that many bacteria grow faster
and more efficiently when amino acids are present in the
growthmedium(24,30,31,48),butStouthamer’scalculations
indicatedthataminoacidsshouldhaveverylittleimpactonthe
efficiencyofbiomassproduction(Table1).WhenS.boviswas
FIG. 8. Relationshipbetween(cid:68)pandamperageinnongrowing,energy-spill-
growninglucose-limitedcontinuouscultures,aminoacidshad
ingStreptococcusboviscells.Reprintedwithpermissionfromreference18.The
no effect on either Y or the m coefficient; however, amino brokenlinerepresentsahypotheticalOhmicrelationship.
G