JOURNAL OF BACTERIOLOGY, Aug. 1982, p. 591-599 Vol. 151, No. 20021-9193/82/080591-09$02.O0/0

dha System Mediating Aerobic and Anaerobic Dissimilation ofGlycerol in Klebsiella pneumoniae NCIB 418

R. G. FORAGE AND E. C. C. LIN*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 30 December 1981/Accepted 22 April 1982

In Klebsiella pneumoniae NCIB 418, the pathways normally responsible foraerobic growth on glycerol and sn-glycerol 3-phosphate (the glp system) aresuperrepressed. However, aerobic growth on glycerol can take place by theintervention of the NAD-linked glycerol dehydrogenase and the ATP-dependentdihydroxyacetone kinase of the dha system normally inducible only anaerobicallyby glycerol or dihydroxyacetone. Conclusive evidence that the dha system isresponsible for both aerobic and anaerobic dissimilation of glycerol was providedby a TnS insertion mutant lacking dihydroxyacetone kinase. An enzymaticallycoupled assay specific for this enzyme was devised. Spontaneous reactivation ofthe glp system was achieved by selection for aerobic growth on sn-glycerol 3-phosphate or on limiting glycerol as the sole carbon and energy source. However,the expression of this system became constitutive. Aerobic operation of the glpsystem highly represses synthesis of the dha system enzymes by cataboliterepression.

Klebsiella pneumoniae 1033 (previously clas-sified as K. aerogenes [37; D. P. Nierlich, per-sonal communication]) uses two different sets ofinducible pathways for growth on glycerol, asdiagrammed in Fig. 1. In the presence of anexogenous electron acceptor, glycerol is primar-ily phosphorylated to sn-glycerol 3-phosphate(G3P) by an ATP-dependent kinase which issubject to noncompetitive feedback inhibitionby fructose-1,6-diphosphate; the G3P is thenconverted to dihydroxyacetone (DHA) phos-phate by an aerobic dehydrogenase if molecularoxygen is available or by an anaerobic dehy-drogenase if fumarate or nitrate is availableinstead.Exogenous G3P can also be utilized directly

by a concentrative permease. Thus, this meta-bolic system closely resembles the one specifiedby the repressor-controlled glp regulon of Esch-erichia coli K-12. In the absence of an exoge-nous electron acceptor, glycerol is primarilyconverted to DHA by an NAD-linked dehydrog-enase, and the DHA is then phosphorylated toDHA phosphate by an ATP-dependent kinase(21). The NAD consumed is in part regeneratedin a two-step parallel pathway that involvesconversion of glycerol to 3-hydroxypropionalde-hyde by a glycerol dehydratase dependent uponcoenzyme B12 and subsequent reduction of 3-hydroxypropionaldehyde to trimethylene glycol(TMG) by an NADH-dependent enzyme termedTMG dehydrogenase (10). (An early fermenta-tion study indicated that about one-half of theglycerol consumed is converted to TMG [29].)

Glycerol dehydrogenase, DHA kinase, glyceroldehydratase, and TMG dehydrogenase are alsohighly induced when DHA is provided anaerobi-cally as sole carbon and energy source. Theseenzymes are members of a separate regulon (thedha system) which is apparently under repressorcontrol (10, 43). Evidently, E. coli cannot growanaerobically on glycerol alone because of theabsence of this system (44).

In addition to respiratory control of the glpand dha regulons, the existence of another mod-ulating factor was proposed on the basis ofchemostat studies on K. pneumoniae NCIB 418.It appeared that, whereas under anaerobic con-ditions the dha system was induced irrespectiveof the glycerol concentration in the growth medi-um, under aerobic conditions the dha systemwas induced when glycerol was abundant andthe glp system was induced when glycerol waslimiting. Since the Km for glycerol is 1 to 2 ,uMfor the kinase (34) and 20 to 40 mM for thedehydrogenase (23, 28), a rationalization wasmade that the preferential synthesis of the ki-nase under substrate-limiting conditions is aphysiologicall; adaptive response to enhancesubstrate scavenging power and that the gradualswitch to the synthesis of the dehydrogenasewith rising external substrate concentration is toavert the accumulation of traumatic metabolites(34, 35, 47). However, it should be cited thatharmful accumulation of metabolites from glyc-erol had been observed only in specially con-trived situations (5, 11, 18, 39, 45, 53). Normal-ly, noncompetitive kinetic feedback inhibition of

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Trirnetrylene Glycol

i1I

Foci/iator DehydrolGse Oxidoreduc/ose iGlycerol Glycerol ----*---- - - 3-Hydroxypropionaldehyde -4 eTrimethyene Glycol

ATPS NAD+ +v~~~i~~~11i %~~~~NADH NAD

G3P

FIG. 1. Pathways for glycerol and G3P dissimilation in K. pneumoniae 1033. Solid lines represent metabolicsteps in the glp system; broken lines represent metabolic steps in the dha system.

glycerol kinase by fructose-1,6-diphosphateserves as an adequate protective mechanism.Furthermore, previous studies on K. pneumo-niae 1033 gave no clue to aerobic modulation ofglp and dha gene expression by substrate con-centrations (42, 43), During the course of afurther study on strain NCIB 418, we foundunexpectedly that the glp system actually can-not be expressed unless a regulatory mutationtakes place. The results reported here show thatthe apparent modulation of the aerobic expres-sion of the dha and glp systems by substrateconcentration reflects a genetic shift of the pop-ulation rather than a physiological adaptation.

MATERIALS AND METHODSStock strains. K. pneumoniae ATCC 15380 was

obtained from the American Type Culture Collection.According to R. L. Gherna of the American TypeCulture Collection, the strain was received from theNational Collection of Type Cultures as K. aerogenesNCTC 418 in 1964. A seed stock of K. pneumoniaeNCIB 418 lyophilized in 1975 and another one pre-pared in 1979 were obtained from the National Collec-tion of Industrial Bacteria. According to I. J. Bousfieldof the National Collection of Industrial Bacteria, thestrain is equivalent to NCTC 418 and is believed to bedescended from Pfeiffer's original "capsule bacillus"(Bacillus aerogenes) (38). The organism was depositedat the National Collection of Industrial Bacteria in1950 by the National Collection ofType Cultures, whoin turn obtained it from the American Museum of

Natural History in 1920. They received it from theJohns Hopkins Hospital in 1911.

Cells from the 1975 sample ofNCIB 418 retrieved byovernight growth in Luria broth (LB) medium wereplated on LB agar. A nonmucoid colony was used in aprevious study by Forage and Foster (9). In the workdescribed here, that cell line was designated as strainECL2 and used as the parental strain for mutantisolation.

Isolation of mutants. Strain ECL4 is a methionine-requiring mutant isolated from strain ECL2 by thestreptozotocin enrichment method (10). Strain ECL8is a spontaneous streptomycin-resistant mutant ofstrain ECL4.For mutagenesis of the dha system by Tn5 insertion,

E. coli 1830(pJB4JI), kindly provided by J. Mekalanos,was mated with strain ECL8. The promiscuous plas-mid vector pJB4JI codes for gentamicin resistance andbears a defective Mu prophage and a TnS elementconferring kanamycin resistance. The plasmid is de-graded after transfer into the recipient, so cells thatbecome kanamycin resistant without becoming genta-micin resistant represent those that have gained onlyTnS (3, 4). Strain ECL8 was fully grown in LB mediumcontaining streptomycin, and E. coli 1830(pJB4JI) wasgrown in LB medium containing gentamicin and kana-mycin. A portion of each culture was diluted 50-fold infresh medium and grown at 37°C for 2 h. Then 0.1 ml ofthe donor and 1.0 ml of the recipient cell culture weremixed in 10 ml of sterile saline and filtered onto asterile membrane filter (Millipore Corp.). After beingwashed with another 10 ml of saline, the membranewas laid face up for 4 h on an LB agar plate prewarmedto 30°C. The cells were then freed from the membrane

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GLYCEROL DISSIMILATION IN K. PNEUMONIAE 593

by blending in a Vortex mixer in 5 ml of LB, andappropriate dilutions of the suspension were plated foranaerobic growth for 40 h on MacConkey agar base(Difco Laboratories) containing glycerol, cyanocobal-amin, streptomycin, kanamycin, and methionine.Kanamycin-resistant derivatives of strain ECL8 werefound at frequencies between 5 x 10-5 and 2 x 10-4per recipient cell, and more than 90%o of this popula-tion remained sensitive to gentamicin. Mutants defec-tive in the dha system were detected as colorlesscolonies at frequencies between 10-4 and 5 x 10-4 perkanamycin-resistant colony.Ten independent derivatives of strain ECL8 that

were defective in the dha system were found andpurified. All were sensitive to gentamicin. All of theirglycerol revertants became kanamycin sensitive, indi-cating that the mutant phenotype arose from singleTnS insertion. One dha-defective mutant, strain ECL14, was used in the present study.The bacterial strains used in this study are listed in

Table 1.Culture media. A mineral medium buffered by 0.1 M

phosphate was used for growth experiments (46),except when DHA was used as the carbon and energysource. To minimize phosphate-catalyzed decomposi-tion of DHA, a medium at pH 7.2 with the followingcomposition was used: 100 mM N-morpholinopropane-sulfonic acid (MOPS), 1 mM KH2PO4, 40 mM KCl, 34mM NaCl, 20 mM (NH4)2SO4, and the trace elementsused in the high-phosphate medium described above.As carbon and energy source, glycerol was added at100 mM and acid casein hydrolysate was added at0.75%. For anaerobic growth, the glycerol mediumwas supplemented with casein hydrolysate (300 mg/liter), and the casein hydrolysate medium was supple-mented with sodium pyruvate (10 mM). When appro-

priate, supplements were added at the following con-centrations: cyanocobalamin, 50 nM; methionine, 40,/ml; streptomycin sulfate, 200 ,ug/ml; kanamycinsulfate, 40 pLg/ml; and gentamicin sulfate, 5 g/ml. LBmedium (rich) was used for growing cells to startgrowth experiments.Growth conditions. Cells were grown at 37°C, and

growth was monitored by using a Klett-Summersoncolorimeter with a no. 42 filter. (A reading of 100 Klettunits corresponds to an optical density at 600 nm of0.89, or 5 x 108 cells per ml.)For aerobic growth, conical flasks containing a

volume of medium that was 12 ± 2% of the nominalcapacity were agitated at 300 cycles per min on arotary platform. For enzyme assays, the cultures wereharvested when their density reached 100 Klett units.For anaerobic growth, conical flasks were filled tocapacity with medium and left undisturbed.

Preparation of celi extracts. All procedures were

carried out at 0 to 40C. For estimation of enzymes ofthe glp system, cells from 500 ml of culture were

washed and suspended in 50mM potassium phosphate(pH 7.0)-i mM glycerol-1 mM 2-mercaptoethanol-1mM EDTA. For estimation of enzymes of the dhasystem, cells from 500 ml of culture were washed with20 mM Trizma (free base) and suspended in 10 mMpotassium phosphate (pH 8.2)-S0 mM (NH4)2S04-10mM DL-1,2-propanediol. The resuspended cells weresonically disrupted, and the debris was removed bycentrifugation.Enzyme and permease assays. Glycerol kinase was

estimated at 30°C by a modification of the method ofRichey and Lin (41). The reaction mixture (final vol-ume, 100 ,ul) contained 1 mM [U-14C]glycerol (1 mCi/mmol), 1 mM ATP, 2 mM MgCl2, 0.5 mg of bovineserum albumin per ml, 20 mM triethanolamine-HClbuffer (pH 7.0), and appropriately diluted cell extract.At the end of a 10-min incubation period, a 50-1±lsample was transferred to a disk of DEAE-cellulosewhich was dropped into a beaker containing 80%oethanol. The filters were washed in 10 changes ofdistilled water, dried, and suspended for radioactivecounting in 4 ml of Aquasol (Packard Instrument Co.,Inc.). Aerobic G3P dehydrogenase activity was esti-mated at 30°C by the rate of reduction of a tetrazoliumdye (12). G3P permease activity was estimated by therate of cellular uptake of 14C-labeled substrate (12).Glycerol facilitator was tested by an optical osmoticmethod (16).Glycerol dehydrogenase activity was assayed at

30°C (42). Glycerol dehydratase activity was assayedat 37°C. (Strain NCIB 418 produces only the glyceroldehydratase which is associated with the dha system,unlike strain 1033 and a number of other subspecieswhich also produce a diol dehydratase both inducibleby and active on TMG and glycerol [10, 49, 50].) TMGdehydrogenase activity was assayed at 30°C (9, 10).DHA kinase activity was estimated at 30°C by thedisappearance ofNADH in a coupled system in whichthe reaction product, DHA phosphate, was convertedto G3P by an NAD+-linked G3P dehydrogenase. Be-fore assay, a 2-ml portion of cell extract was subjectedto centrifugation (4°C) for 3 h at 145,000 x g to removemembrane particles, which apparently catalyze theoxidation of NADH. The reaction mixture contained 1mM DHA, 1 mM ATP, 1 mM MgCl2, 0.1 mM NADH,2 p.g of G3P dehydrogenase per ml from rabbit muscle(Boehringer Mannheim Biochemicals), 5 mM glycerol,10 mM at,ca-dipyridyl, 50 mM triethanolamine-HCl

TABLE 1. Origin and phenotypes of strains used

Strain Parent Descriptiona

ATCC 15380 (See text) Mixture of mucoid andnonmucoid colonies

NCIB 418 (See text) Mixture of mucoid andnonmucoid colonies

ECL2 Wild-type Prototrophic nonmucoidNCIB 418 colony

ECL4 ECL2 Met- auxotroph select-ed by streptozotocinenrichment

ECL8 ECL4 Spontaneous Smr mu-tant

ECL14 ECL8 dhaK(D,B,T)c mutantresulting from Tn5 in-sertion

ECL33 ECL8 Spontaneous glp consti-tutive mutant

ECL50 ECL2 Spontaneous glp consti-tutive mutant

a For further details, see the text. Symbols: dha,dihydroxyacetone system; glp, sn-glycerol 3-phos-phate system; K, dihydroxyacetone kinase; D, glycer-ol dehydrogenase; B, B12-dependent glycerol dehydra-tase; T, TMG dehydrogenase; (D, B, T )C, constitutivein D, B, and T.

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buffer (pH 7.0), and 10 to 50 ,ul of extract in a finalvolume of 1.0 ml. Glycerol kinase has a much higherapparent affinity for glycerol (Km = 105 M [48]) thanfor DHA (Km = 0.5 mM [14]). Fortunately, DHAkinase does not act on glycerol, nor is the enzymeinhibited by the compound even at 100 mM (S. Burkeand R. Forage, unpublished data). Therefore, thepresence of 1 mM glycerol almost totally prevents theaction of any glycerol kinase on DHA without affect-ing the activity of DHA kinase. Glycerol dehydroge-nase also interferes with the assay by catalyzing thereduction of DHA at the expense of NADH. Thisreaction can be strongly inhibited by a,a-dipyridyl (23)at 10 mM, under which condition there was no effecton DHA kinase. DHA kinase activity is defined as theATP-dependent increase in the rate of NADH oxida-tion. The protein concentration in the cell extractbefore the ultracentrifugation step was used for calcu-lation of specific activity.

Protein concentrations were estimated by the Folin-Ciocalteu reagent method (24).

RESULTS

Inability of stock strains to grow on G3P. StrainECL2 subcloned from NCIB 418 (stock of 1975)and its derivative, strain ECL8, both grew aero-bically or anaerobically on agar with glycerol ascarbon and energy source. No growth occurredaerobically on G3P or anaerobically on G3P plusfumarate. Three mucoid and three nonmucoidclones from each of two other stocks, NCIB 418(batch of 1979) and ATCC 15380, showed thesame growth pattern. Even though the mucoidstate is likely to be a trait of the original isolate(38), the nonmucoid clones were adopted forfurther study because of their more desirablesurface properties.Revertants that grow on G3P. When cells of

strain ECL2, strain ECL8, and a nonmucoidclone from the 1979 stock of NCIB 418 wereseparately plated on agar containing 25 mM G3P

as the sole aerobic source of carbon and energy,phenotypic revertants appeared at frequenciesbetween 1 x 10-6 and 5 x 10-6. A revertantfrom strain ECL2 (designated as strain ECL50)and a revertant from strain ECL8 (designated asstrain ECL33) were adopted for further charac-terization.

Aerobic and anaerobic enzymatic profiles ofstrains ECL8 and ECL33. Strain ECL8 and itsG3P revertant strain ECL33, grown aerobicallyon glycerol or casein hydrolysate, were com-pared for enzyme activities. Strain ECL8 lackedboth glycerol kinase and aerobic G3P dehydrog-enase activities when grown on either carbonsource (Table 2). G3P permease and glycerolfacilitator were similarly missing (data notshown). Significant levels of the four dha en-zymes, however, were induced in glycerol-grown cells. By contrast, strain ECL33 constitu-tively produced glycerol kinase, aerobic G3Pdehydrogenase (Table 2), G3P permease, andglycerol facilitator (data not shown). The en-zymes of the dha system, however, were notinduced during aerobic growth on glycerol.The question arises as to whether the failure

of strain ECL33 to be aerobically induced in thedha system reflects its high sensitivity to catabo-lite repression. To test this, cells of strainsECL8 and ECL33 were grown on glycerol in thepresence of 5 mM cyclic AMP (cAMP). As mayalso be seen from the data presented in Table 2,exogenous cAMP allowed pronounced inductionof the dha enzymes in strain ECL33, althoughthe induced levels of these enzymes were alsoraised in strain ECL8.When glycerol served as the anaerobic carbon

and energy source, the dha enzymes were in-duced in both strains ECL8 and ECL33 to aboutequally high levels, significantly exceeding those

TABLE 2. Enzyme specific activities in cells grown aerobicallySp act (nmol/min per mg of protein)

Strain ECL8 Strain ECL33EnzymeEnzyme Grown on Grown on Grown on Grown on

casein glycerol casein Grolhydrolysate glycerol hydrolysate glycerol

Glycerol kinase 7 9 310 360Aerobic G3P dehydrogenase 8 6 550 310Glycerol dehydrogenase 3 470 (860)a 3 3 (470)DHA kinase 1 54 (62) 2 7 (41)Glycerol dehydratase 6 410b (780) 4 0 (390)TMG dehydrogenase 3 240 (470) 3 4 (190)

a Values in parentheses were obtained from cells grown in media containing 5 mM cAMP in addition.b The activity was measured with 1,2-propanediol as substrate, because, unlike glycerol, the analog did not

cause reaction inactivation under the assay conditions. The in vivo activity might be much lower. K. pneumoniaeATCC 8724 grown anaerobically in a glycerol medium contains 35 ,ug of vitamin B12 per g of dry cells; the valuewas only 14% of this amount in cells grown aerobically. Even anaerobically, the fermentation of 1,2-diols isbelieved to be rate limited by the B12-dependent dehydratase activity (49).

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TABLE 3. Enzyme specific activities in cells grown anaerobicallySp act (nmol/niin per mg of protein)

Strain ECL8 Strain ECL33Enzyme Grown on Grown on

casein Grown on casein Grown on

hydrolysate glycerol hydrolysate glycerol

Glycerol kinase 10 10 140 21 (39)aAerobic G3P dehydrogenase 4 3 190 44 (83)Glycerol dehydrogenase 310 6,100 730 5,600DHA kinase 1 110 1 120Glycerol dehydratase 1 1,100 1 850TMG dehydrogenase 9 1,300 8 1,000

a Values in parentheses were obtained from cells grown in media containing 5 mM cAMP in addition.

attained by strain ECL8 grown aerobically onglycerol, even with cAMP (Table 3). More strik-ingly, in strain ECL33, dominance of the twoglycerol systems was reversed by anaerobiosis;the functioning of the dha system strongly re-duced the constitutive expression of the gIpsystem. This effect was only counteracted to aslight extent by the addition of cAMP to theglycerol medium. The strong anaerobic effect onthe glp system, however, is not limited togrowth on glycerol. A similar effect was foundwith sorbitol (data not shown). Only a moderateanaerobic reduction of the expression of the glpsystem was observed with casein hydrolysateand pyruvate as the carbon and energy source.Thus, it seems that aerobiosis and cataboliterepression impede the expression of the dhasystem, whereas anaerobiosis and a yet to beidentified metabolic factor impede the expres-sion of the glp system.

A _1000.

Ni! mM~~~~~~~~~~801X 1

4100

14 10.

Advantage of the glp system for aerobic growthon glycerol. To compare the substrate scaveng-ing power of strain ECL8 relying on the dhasystem with that of strain ECL33 relying on theglp system, cells grown in LB medium wereinoculated at low culture density into mineralmedia containing 1 to 50 mM glycerol. In allcases, growth of strain ECL33 occurred with ageneration time of 44 min (Fig. 2A). By contrast,the growth rate of strain ECL8 diminished as theconcentration of glycerol was reduced from 50(generation time, 44 min) to 3 mM; the lag periodprogressively lengthened. Below 3 mM, nogrowth occurred even after 8 h (Fig. 2B). Prein-duction of strain ECL8 reduced the lag periodwithout affecting the growth rate between 10 and50 mM glycerol. Below 3 mM glycerol, onlytransient growth occurred after 5 h of lag (Fig.2C). Thus, the threshold concentrations for in-duction and maintenance of the dha system in

4 6 8HOURS

FIG. 2. Effect of glycerol concentration on aerobic growth of strains ECL8 and ECL33. Strains ECL33 andECL8 grown overnight in LB medium (A and B) or standard medium containing 50 mM glycerol (C) wereinoculated at low culture density into standard medium containing different concentrations of glycerol. Growthwas followed turbidimetrically.

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FIG. 3. Selection of strains constitutive for the glp system by growth on limiting glycerol. Cells of strainsECL2, ECL8, ECL33, and ECL50 were spread in counterclockwise order on quadrants of MOPS agar platescontaining either 50mM (A and B) or 1 mM (C and D) glycerol. Plates A and C were inoculated with 4 x 106 cellson each quadrant, and plates B and D were inoculated with 4 x 102 cells on each quadrant. Growth was aerobicfor 40 h at 37°C.

strain ECL8 are close, between 3 and 10 mM.This is in contrast to the clear-cut difference inthe behavior of the lac operon in E. coli towardsa gratuitous inducer, thiomethyl-a-D-galactoside(30, 36).

Revertants in the glp system selected by limitingglycerol. The superior substrate-extracting pow-er of the glp system over the dha system alsoallowed the selection of Glp+ revertants withlimiting glycerol. Cells of strains ECL2 andECL8 and those of their respective G3P+ rever-tants (glp constructive), strains ECL50 andECL33, were spread on separate quadrants ofMOPS agar plates containing either 50 or 1 mMglycerol (Fig. 3). On 50 mM glycerol, luxuriantgrowth of all four strains occurred as expected.

On 1 mM glycerol, strains ECL2 and ECL8 gaverise to few discrete colonies when 4 x 106 cellswere spread, whereas strains ECL50 and ECL33formed thin confluent lawns; strains ECL2 andECL8 yielded minute colonies when 400 cellswere spread, whereas strains ECL50 and ECL33yielded medium-sized colonies. Three discretecolonies arising from the film of strain ECL2plated on 1 mM glycerol were picked and puri-fied. All had become glp constitutive. The sameresults were obtained with strain ECL8.dha system in strain ECL8 serving for both

aerobic and anaerobic growth on glycerol. StrainECL14, a mutant of strain ECL8, failed to growboth aerobically and anaerobically on eitherglycerol or DHA. DHA kinase activity disap-

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GLYCEROL DISSIMILATION IN K. PNEUMONIAE 597

peared, but glycerol dehydrogenase, glyceroldehydratase, and TMG dehydrogenase wereproduced at high levels even in cells grown oncasein hydrolysate alone. The apparent constitu-tivity of the three remaining enzymes might beattributable to the endogenous accumulation ofthe inducer, DHA (10). Lack of galactokinase isknown to cause endogenous induction of thegalactose operon of E. coli (51).When strain ECL14 was plated on MOPS agar

containing 50 mM DHA and incubated anaerobi-cally, revertants were found at a frequency ofabout 2 x 10-7. Of 10 revertants examined, allwere found to be sensitive to kanamycin, indi-cating that reversion was accompanied by lossof TnS, as is usual in E. coli (2).When strain ECL14 was plated on agar con-

taining G3P and incubated aerobically, rever-tants were found at a frequency of about 2 x10-6. Of 10 revertants examined, all had ac-quired the ability to grow aerobically on glycer-ol, but not anaerobically on glycerol or DHA.All were found to retain resistance to kanamy-cin. Thus, it appeared that the TnS remained insitu.When strain ECL14 was plated on agar con-

taining 50 mM glycerol and incubated aerobical-ly, two kinds of revertants were found as expect-ed. The majority were glp constitutive andremained resistant to kanamycin, and the minor-ity were restored in the dna system and lostkanamycin resistance.

DISCUSSIONBy analogy with the GlpRn (noninducible in

the glp system) state of E. coli (7), the revertibil-ity of the glp system in strain NCIB 418 only toconstitutivity suggests that this regulon is undersuperrepression. This cryptic state of the glpsystem and the expansion of the role of the dhasystem to include aerobic glycerol dissimilationprovide a basis for explaining why cells harvest-ed from aerobic growth in a chemostat underglycerol-abundant conditions contained glyceroldehydrogenase activity but no detectable glycer-ol kinase activity, whereas the opposite wasfound when growth occurred under glycerol-limiting conditions (34, 35, 47). It is likely that,by the time a steady state was reached withglycerol-limited growth, the parental cells werereplaced by Glp+ revertants because of theirsuperior substrate scavenging power. Indeed, itwas shown that cells containing glycerol kinaseexhibited a maximal initial rate of substrate-stimulated oxygen consumption even when theconcentration of glycerol was reduced to <1mM, whereas cells containing glycerol dehy-drogenase decreased their rate of oxygen con-sumption progressively as the glycerol concen-tration in the incubation medium was reduced

from 50 to 0.4 mM. (The unexpectedly lowglycerol dehydrogenase specific activity of only9 nmollmin per mg of protein at 35°C wasattributed to measurement of the enzyme undersuboptimal conditions [17, 34], but intense self-catabolite repression during growth limited byammonium must also be considered.) Parentalcells relying on the dha pathway may actually behandicapped in three other ways: inadequatepermeability to glycerol at low concentrations,partial escape of the intermediate DHA into themedium, and respiratory repression.

It is reasonable to suppose that the glp regulonwas at one time inducible in the organism, as inall the other enteric bacteria so far studied (21).With both inducible glp and dha systems avail-able, glycerol could be channeled through theappropriate pathway by a set of controls. Aero-bically, the primacy of the glp system seems tobe assured by respiratory control together withhierarchic catabolite repressibility. This catabo-lite effect can be sustained over a range oflimiting glycerol concentrations because diminu-tions of metabolic flow through the glp systemwould be largely compensated by the lifting offeedback inhibition of glycerol kinase (54). Onthe other hand, the fall of glycerol concentrationwould render it increasingly difficult to maintainthe induction of the low-affinity dha system (seereference 17). Anaerobically, the primacy of thedha system appears to be assured by respiratorycontrol acting in concert with a physiologicalmechanism yet to be defined. Restraint of anaer-obic induction of the glp system is important foravoiding growth-inhibiting levels of G3P (8)when there is an inadequacy of exogenous hy-drogen acceptors.Permanent repression of the glp regulon in K.

pneumoniae 418 might be a first step towardsregressive evolution. If the records are correct,the original isolate was first described in 1889(38). It is quite possible that replacement of thewild-type population by a mutant with the dys-function in the glp system occurred during culti-vation under laboratory conditions. The moreinteresting point is why a rare mutant of thisparticular kind should be the one to supersedethe wild-type population. It is understandablethat constitutive expression of the regulon (themost likely consequence of random mutations inglpR) would be unsuccessful because of theresulting metabolic burden. It is also under-standable that mutants lacking aerobic or anaer-obic G3P dehydrogenase would not emergesince they would be at a serious disadvantage ifexposed to glycerol or G3P. On the other hand,the loss of glycerol facilitator, glycerol kinase,or G3P permease activity should be almost se-lectively neutral in environments deficient inglycerol and G3P or rich in a preferred carbon

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and energy source, such as glucose. The ab-sence of a demonstrable glycerol facilitator inShigellaflexneri may be a case in point (41). Theabsence of P-galactoside permease in Shigelladysenteriae is a more clear-cut example of re-gression by loss of a transport system (15, 25).The establishment of an unusual regulatory mu-tant instead of one of the more common mutantsblocked in the initial step of metabolism there-fore points to positive selection, and a plausibleexplanation can be offered. The true inducer ofthe regulon is G3P (13). Because of its essentialrole as a structural unit in phospholipids andother macromolecules, G3P must also be madeavailable through biosynthesis. Consequently,some degree of endogenous induction is difficultto circumvent. Without opportunities to utilizeexternal glycerol or G3P, an altered repressorthat is indifferent to the inducer can contributefurther economy to the cell by lowering the basalexpression of the four operons coding for thestructural genes (7). More than that, noninduci-ble repressors, by virtue of their enhanced bind-ing to the operator, can be more effective thanthe normal repressor in blocking basal geneexpression (1).

Evolvants with revertible genetic defects areprobably not very rare in nature. Revertiblelactose-negative strains of E. coli have beenisolated on several occasions (20, 27, 31). Mu-tants that can grow on succinate, fumarate, andmalate (possibly as the result of the restorationor acquisition of a dicarboxylic acid permease)were observed in Moraxella lwoffi to occur at afrequency of about 10-8 to 10- (26). Strongerevidence for the accumulation of only minorgenetic defects in natural isolates are providedby the discovery that a number of amino acidrequirements by lactobacilli can be abolished bymutations (32).So far we have focused on regressive evolu-

tion and considered the mutations cited as exam-ples of reversion, because the metabolic path-ways thereby appearing are widespread innature. However, mutational gains in metabolicability often do not represent function rescue.Cases of acquisitive evolution have been report-ed in which functional gains are achieved byrecruitment of existing genetic systems throughmutations in regulatory and structural genes (6,15, 22, 33, 52). There exist functional gains thatmust be viewed from yet another perspective. Ahighly interesting example is the activation of anE. coli pathway for utilizing aromatic P-gluco-sides by an insertion element. It was suggestedthat this semipermanent control evolved to pro-tect the population from substrates that wouldyield toxic products upon hydrolysis. In thepresence of such hazardous substrates, variantswith the operon rendered inactive by deletion of

the element would be immune. When only utiliz-able p-glucosides are present, a new progenywith an activated operon can again emerge (40).Although the various kinds of metabolic gainscited have different biological significance, theyall underscore the dynamic state of the genomesof living organisms.

ACKNOWLEDGMENTS

We thank R.-Z. Jin for verifying the growth properties ofstrain ATCC 15380 and Sarah Monosson for editorial assist-ance.

This work was supported by grant PCM7924046 from theNational Science Foundation and Public Health Service grant5 RO1 GM11983 from the National Institute of GeneralMedical Sciences.

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