JOURNAL OF BACTERIOLOGY, Mar. 1988, p. 1220-1226 Vol. 170, No. 30021-9193/88/031220-07$02.00/0Copyright C 1988, American Society for Microbiology

Partial Characterization of an Electrophoretically LabileHydrogenase Activity of Escherichia coli K-12KAREL STOKER,* L. FRED OLTMANN, AND ADRIAAN H. STOUTHAMER

Department of Microbiology, Vrije Universiteit, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands

Received 18 June 1987/Accepted 19 November 1987

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenaseisoenzyme. By means of TnS-mediated insertional mutagenesis, a class of mutants was isolated (class I) that hadretained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis,the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenaseactivity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such anactivity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identicalor part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated fromhydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically withcell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity.However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in theassay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzymecomplex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about itsphysiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogenlyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementationwith F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized themutation (hydL) close to metC at approximately 64.8 min.

Hydrogenase catalyzes the reversible reaction H2= 2H++ 2e- (Eo'=-420 mV) (2, 20). In Escherichia coli, H2uptake and, under conditions of low redox potential, also H2production can be observed. By the formate-hydrogen lyasecomplex (FHL) formate, formed during fermentation, isconverted into CO2 and H2. Hydrogenase and formatedehydrogenase form part of this system (9, 22-24). Also bymeans of a hydrogenase, E. coli is able to consume H2,passing the electrons through an anaerobic respiratory chainto, e.g., fumarate (14), thus generating ATP (9, 10, 17, 31).When the hydrogenase content of E. coli is analyzed on

neutral, activity stained polyacrylamide gel, a diversity ofactive bands is observed (1, 31), which can be ascribed to atleast two immunologically distinct isoenzymes, hydroge-nases 1 and 2 (4, 5, 26). Recently, a third hydrogenase, whichcould not be visualized on gel, was suggested (hydrogenase3) (25). Interestingly, also in Salmonella typhimurium such a(electrophoretically) labile hydrogenase is probably present(27). In E. coli the presence and physiological role of thelatter activity were based on indirect evidence. It mightfunction in the FHL reaction. Mutants specifically lackingone of the hydrogenase activities mentioned above would beof great value in linking biochemical data to physiologicalfunction. Therefore, we isolated a class of hydrogenasemutants (class I), that, although it had lost 80% of the totalactivity, showed a normal activity banding pattern on neutralpolyacrylamide gels. It therefore lacked a (electrophoreti-cally) labile hydrogenase activity. Here we provide biochem-ical, functional, and genetic data on this labile isoenzyme(hydrogenase L). In contrast to hydrogenase 3, it is, how-ever, involved in H2 uptake activities rather than in the FHLpathway.

* Corresponding author.

MATERIALS AND METHODS

Bacterial strains. For mutant isolation E. coli RR1 (pro leuthi lacZ gal xyl ara mtl hsdS phx supE rpsL) or its recA13derivative HB101 was used. Growth of X421 was on strainJMsu6 [A(lac pro) ara argE(Am) nalA rif thi supB(Am)] (19).For coconjugation experiments we used as a donor strainPK191 [Hfr thi A(lac pro) phx sup (Am) colV], a noncoli-cinogenic derivative of PK19 (11). PK191-I (Hfr, hydL::TnS)was constructed by P1 vir-mediated transduction from aclass I mutant strain. Recipient trains were CSH57B (F- leupurE trp his argG ilvA met thi lac gal xyl ara mtl tonA tsxphx sup rpsL) (18), KMBL1418 (thyA his phx rpsL), andLBE1930 (thyA serA his metG galE phx drm rpsL). Thesestrains were obtained from the Phabagen Collection, Univer-sity of Utrecht, Utrecht, The Netherlands. For cotransduc-tion experiments we used AT2699 (hisG thyA metC argGlacY gal-6 malT tsx-l supE rpsL) and JM2071 (his leu ilvAAlac mglP galP: :TnlO), both supplied by the E. coli GeneticStock Center, Yale University. For Tn5 insertion mutagen-esis we used X421 (cI857 b221 Oam Pam rex::TnS), a giftfrom G. E. De Vries.Media and growth conditions. For aerobic batch growth, a

freshly grown overnight culture was subcultured (1:100) in100 ml of prewarmed brain heart infusion broth (38 g/liter,pH 7.4; GIBCO Laboratories) and vigorously shaken in500-ml bottles for several hours at 37°C. Anaerobic batchgrowth was as above, but subculturing was in completelyfilled and tightly stoppered bottles left at 37°C withoutshaking. Plate-grown cells were transferred from a freshlygrown monolayer to a slightly moist brain heart infusionplate by using a 6 x 8 grid matrix and grown overnight at37°C. This growth condition appeared to be essentiallyanaerobic. Pseudocolonies tested for hydrogenase activitywith the MV filter assay (see below) turned blue within 1 to

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HYDROGENASE ISOENZYMES IN E. COLI 1221

2 min. When indicated, 40 mM KNO3 was included. Lambdamedium was 1% (wt/vol) tryptone, 0.7% (wt/vol) NaCl, and0.2% (wt/vol) maltose. SV medium contained (per liter) 7.5 gof K2HPO4, 4.5 g of KH2PO4, 2 g of NH4Cl, 5 mg of FeSO4,and 50 mg ofMgSO4 supplemented with 0.1 mM CaCl2, traceminerals, and amino acids (6). Glucose was applied in aconcentration of 0.5% (wt/vol); streptomycin, kanamycin,and rifampin were applied at 25 ,ug/ml. The pH was 7.0.TnS insertion mutagenesis. X421 was plaque purified and

grown on JMSu6 at 42°C to a titer of 109 PFU/ml. Cells to beinfected were cultured overnight in lambda medium andgrown to a density of 5 x 108 CFU/ml. The cells were spundown and suspended in 1/10 volume of 10 mM MgSO4. Forinfection, 0.1 ml of phage was mixed with 0.5 ml of cellsuspension. After an absorption period of 20 min at 35°C, 2ml of brain heart infusion broth supplemented with 5 mMNa4P2O7 was added, and the incubation was prolonged foranother hour. Transposants were scored by plating 0.1 ml onbrain heart infusion agar plates containing kanamycin (6, 12).MV filter assay. Colonies were screened for hydrogenase

activity by transferring them to a filter paper drenched in 20mM methylviologen (MV) (E'0 = -440 mV)10 mM Trischloride (pH 7.4) and placing this in an atmosphere of 5%H2-95% N2 as described by Glick et al. (7). Positive coloniesturned blue within a few minutes.Sample preparations. Whole cell samples were prepared

by scraping off, washing, and suspending the plate-growncells in 50 mM phosphate buffer (pH 7) at 4°C. Densitieswere measured at 600 nm in cuvettes of 1 cm path length andranged from 25 to 150. Solubilized membranes were made bysonicating the whole cell preparations described above onice by five pulses of 20 s (Branson sonifier, amplitude 16 atmedium power). Triton X-100 was added at a final concen-tration of 0.5% (vol/vol). An S100 extract was the superna-tant obtained after centrifugation of the solubilized mem-branes at 100,000 x g at 4°C during 1 h. Solubilizedmembranes and S100 extracts were prepared aerobically.

Assays. Hydrogenase activity was measured manometri-cally in Warburg vessels at 35°C. The 3-ml reaction mixturescontained 150 mM phosphate buffer (pH 7), 5 mM MV, and0.2 to 10 mg of protein. After 15 min of flushing with 100%H2 gas and equilibration for another 15 min, the reaction wasstarted by adding the electron acceptor; the H2 consumptionwas followed in time. Alternatively, hydrogenase activitywas determined colorimetrically, measuring the rate of MVreduction, by titration of serial dilutions in 0.3-ml microdi-lution wells by mixing 100 RI of diluted sample with 100 ,ul of20 mM MV-200 mM phosphate buffer (pH 7). The plateswere placed in an anaerobic environment filled with 5%H2-95% N2 and left for 24 to 48 h at room temperature.Activity was expressed in arbitrary units, being the inverseof the highest dilution still showing coloration. This methodappeared to be very reliable for the determination of relativehydrogenase activities. Formate dehydrogenase activity wasassayed manometrically on sonicated cells as for hydrogen-ase, except that benzyl viologen (E'0 = -350 mV) and 100%N2 were applied. The substrate was 50 mM sodium formate,and the CO2 production was followed. No production wasfound in the presence of KOH. The formate-to-NO3 routewas tested on intact cells manometrically as for hydrogen-ase, except that 50 mM sodium formate was the electrondonor and 40 mM KNO3 was the acceptor. CO2 productionwas measured under an N2 atmosphere. FHL activity wasdetermined on intact cells as for formate dehydrogenase,except that benzyl viologen was omitted. The total gasproduction was measured (CO2 and H2). The H2-to-fumarate

(Hup) activity was assayed manometrically on intact cells asfor hydrogenase, except that 50 mM sodium fumarate wasused as the electron acceptor. H2 consumption was moni-tored in the presence of KOH in the center well. Proteinconcentration was determined by the method of Lowry et al.(16). Cell suspensions having an optical density of 1 at 600nm typically contained 2.5 x 108 cells and 0.2 mg of proteinper ml.

Polyacrylamide gel electrophoresis. S100 extracts wereelectrophoresed on neutral, nondenaturating 7% (wt/vol)polyacrylamide gels (0.2 by 12 by 15 cm) as describedpreviously (8) at 50 mA for S h at 15 to 20°C. Activity stainingwas overnight as described previously (4). The intensity ofthe stained bands was proven to be directly proportional tothe amount of active enzyme applied. Therefore, relativehydrogenase activities could be determined by this method.DEAE ion-exchange chromatography. S100 extracts (10 mg

of protein before centrifugation) were chromatographed in0.5-ml samples on a TSK DEAE-5-PW (Bio-Gel; Bio-RadLaboratories) high-pressure liquid chromatography anion-exchange column. The mobil phase was 0 to 0.9 M KCI-50mM phosphate buffer (pH 7.0), applied in a continuouslyincreasing gradient from 0 to 20 min. Elution time was 20 minat a flow of 1 ml/min. Fractions of 1 ml were collected at 4°C.Active fractions appeared to be well separated from un-bound Triton X-100.G3000 gel filtration. Active DEAE fractions (150 ,u) were

mixed with 25 to 250 ,ug of marker proteins (Combitek;Boehringer Mannheim Biochemicals), and 0.2-ml sampleswere chromatographed on a G3000 SW (Chrompack) high-pressure liquid chromatography gel filtration column, sepa-rating in the molecular weight range between 1 x 104 and 5x 105. The mobil phase was 50 mM phosphate buffer (pH7.0), the flow rate was 0.7 ml/min, and the elution time was20 min. Protein was monitored at 280 nm, and hydrogenaseactivity was determined by microdilution (see above). Frac-tionation was in 40 fractions of 0.35 ml, collected at 4°C.

Genetic experiments. Conjugation and P1 vir-mediatedtransduction were carried out as previously described (18).

RESULTS

Isolation of partially defective hydrogenase mutants. Theisolation of mutants of E. coli specifically impaired in one ofthe hydrogenase isoenzymes was achieved by Tn5-mediatedinsertional mutagenesis with X: :TnS as the vector. Thefrequency of insertion, 10' per infected cell, was such thatwe could be sure that in every mutant only one gene was hit.Furthermore, the mutations were highly stable and select-able by the capacity of TnS to confer kanamycin resistanceupon its host.

E. coli RR1 or HB101 was infected with X421, selected forkanamycin resistance, and tested for loss of hydrogenaseactivity by means of the MV filter assay. Among the Kmrtransposants, white colonies occurred at a frequency ofabout 10'-. After purification, transfer on a 6 x 8 gridmatrix, and retesting 32 mutant strains were isolated. Sincethe hydrogenase phenotypes might be dependent on thegrowth conditions applied, they were cultured as they wereselected, i.e., plate grown, and the total hydrogenase activ-ity was determined quantatively. In addition, the hydrogen-ase isoenzyme content was analyzed on neutral polyacryl-amide gels. Since we were looking for mutants which werepartially defective in hydrogenase activity, i.e., defective inonly one hydrogenase isoenzyme, one group of six mutantstrains got our special attention. These strains showed a

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1222 STOKER ET AL.

HB 101 II...........,....... ,,.,... ..

a b c d e f g

FIG. 1. Electrophoretically stable hydrogenase isoenzymes of E.coli HB101 and a representative class I mutant (B02). A neutral,nondenaturing polyacrylamide gel is shown, stained for hydrogen-ase activity, on which S100 extracts from plate-grown cells indifferent concentrations were run: 300 ,ug (lanes a and e), 150 ,ug(lanes b and f), 30 ,ug (lanes c and g), and 3 ,ug (lane d). Isoenzymenumbers 1, 2, and 3 correspond with those described by Ballantineand Boxer (4). See Materials and Methods for procedural details.

reduced overall hydrogenase activity of about 20% of theparent strain. Unexpectedly, however, they displayed acompletely normal banding pattern, although they had lostover 80% of the overall activity. In Fig. 1, the bandingpattern of one representative strain is shown. (The otherstrains behaved physiologically identically. The bandingpattern differed in our experiments somewhat from thatdescribed by Ballantine and Boxer [4]. The intensities of thebands appeared to be dependent on the culture conditionsbut differed also from experiment to experiment. The aber-rant Rf values might be explanable by the absence of TritonX-100 in the system and/or slight differences in the pH atwhich the separation took place. To avoid confusion: band 1corresponds with hydrogenase 1, and bands 2 and 3 corre-spond with hydrogenase 2; the presumed third isoenzyme isnot visible.) This reduced level was also observed in brokencells and Triton X-100-dispersed membranes. This ruled outthe possibility that these mutants might be deficient in a

separate transmembranous transport protein, coupling hy-drogenase to MV, as was suggested for some other hydro-genase mutants (7, 15). Thus, the decreased activity re-flected the presence of less, intact enzyme. The mutationcould not be restored by growth in the presence of 0.5 mMNiCl2 (28, 29). Therefore, these mutants were apparentlylacking a labile hydrogenase activity, which is for somereason inactivated upon neutral or alkaline polyacrylamidegelelectrophoresis and therefore provisionally called herehydrogenase L. Whether this activity is brought about byone or more hydrogenase isoenzymes could not be stated atthis point. Also, the relation between hydrogenase L andhydrogenase 3 (25, 27) is discussed in the Discussion.

Physiological characterization. To determine the functionalimplications of the absence (100% or near 100%) of thishydrogenase L, the six mutants were examined for a rele-vant set of physiological parameters (Table 1). Of the twopathways in H2 metabolism, we observed no effect on theFHL activity, but a complete inhibition of fumarate depen-dent H2 uptake. Within the limits of the examination, noother functional defects could be demonstrated. Since theroute from glycerol to fumarate is unimpaired, the latterinhibition might be caused by the absence of hydrogenase L.This finding suggests that this activity functions in H2uptake. Physiologically the mutants did not differ signifi-cantly and were therefore supposed to belong to one geneticclass (class I).

Kinetic measurements. Since E. coli may contain at leastthree different hydrogenase isoenzymes, the kinetics ofunpurified preparations must be complex, composed ofpartially overlapping curves of the individual isoenzymes.Kinetic measurements were performed on strain HB101 andclass I mutants. From the data in Fig. 2A and B thecontribution of hydrogenase L to the overall kinetics couldbe derived. In Fig. 2A a preparation of sonicated and TritonX-100 dispersed membranes of parent strain HB101 is as-sayed at different protein concentrations. In such experi-ments, the initial rate varied in a linear fashion with theprotein concentration, but always the initial rate was also themaximal rate, notwithstanding the fact that traces Of 02could be detected in the reaction vessel. When the same wasdone with class I mutants (Fig. 2B), these strains displayeda characteristic lag phase, the length of which appeared to beinversely proportional to the enzyme concentration appliedand directly proportional to the time during which thepreparation had been exposed to air after sonication (25 to 60min). With high concentrations of enzyme and/or very freshpreparations, the lag phase seemed to be absent. When S100preparations were analyzed (Fig. 2C and D), in which casethe HB101 activity was somewhat reduced (Fig. 2C), theclass I activity seemed to have disappeared completely,since no H2 consumption occurred during the assay period (1to 1.5 h) (Fig. 2D). This was remarkable, since thesepreparations, analyzed on gels, showed normal bandingpatterns. When these experiments were performed in thepresence of the reducing agent sodium dithionite, we indeedfound H2 consumption (Fig. 2D), although the lag phase didnot disappear completely. The shape of the HB101 curve didnot change at these concentrations of dithionite (Fig. 2C).The time-dependent inactivation mentioned above is there-fore likely due to the necessarily prolonged period of expo-sure to air, which process is probably enhanced by theremoval of the membranes. So, we might conclude that theisoenzymes present in class I mutants not only are reversiblyinactivated by 02 but also need a very low redox potential

TABLE 1. Physiological properties of parent strain E. coliHB101 or RR1 and class I mutants lacking hydrogenase L'

Activity (nm mg-'Enzyme or reaction min-') Relative

activitybMutant Parent

HydrogenasecWhole cells 0.9 5.6 16Solubilized membranes 2.3 12.5 18S100 extractd 15-20

Formate dehydrogenase 3.1 3.2 98Formate -* NO3` 87 94 92Formate -*CO2 + H2 (FHL) 6.6 6.6 100H2 -* fumarate (Hup) 0-0.1 13 0-1

aCells were plate grown unless otherwise stated. Procedures were asdescribed in Materials and Methods. The data are averages. The mutantstrains tested were RH2, RH4, RH5, B02, B022, and B027. Parents andmutants all had wild-type polyacrylamide gel electrophoresis patterns andgrew normally in batch culture with or without 02 and on SV supplementedwith 40 mM glycerol and 40 mM fumarate, flushed with N2, and left at 37°Cwith gentle shaking.

b (Activity of mutant/activity of parent) x 100.' H2: MV oxidoreductase activity was determined manometrically and by

microtitration.d Because of the strongly aberrant behavior of these preparations in

Warburg experiments, no quantitative data are given. Relative activities weredetermined by microtitration.

' Growth in the presence of 0.35% KNO3.

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HYDROGENASE ISOENZYMES IN E. COLI 1223

1.75

.2 1.252

c 0.75g

B 0.25

-Lt 1.75

_ 1.25E2C 0.75

0.25

0

.0t. 1;-. 25 315 4:5 55 0 0 30*0 6t(min.) t(min.)

FIG. 2. Kinetics of hydrogenase activities in E. coli HB101 and arepresentative class I mutant (BO2). H2:MV oxidoreductase activitywas determined manometrically. Shown are HB101 (A) and mutant(B) solubilized membranes at different protein concentrations andHB101 (C) and mutant (D) S100 extracts in the presence or absenceof 0.1 to 1 mM dithionite (added dropwise from a freshly prepared 15mM stock solution). Samples in C and D are the same as samples inA and B, respectively (10 mg of protein, before centrifugation).Symbols: 0, 10 mg of protein; 0, 5 mg of protein; A, 2 mg ofprotein. -, without dithionite; ------, with dithionite. Cellswere plate grown. See Materials and Methods for procedural details.

for their action, and even that might not be sufficient (Fig.2D). We therefore hypothesize that this rest activity, com-prising hydrogenase 1 and 2, needs to reach an activated or

reduced state. The presence of hydrogenase L in extracts ofHB101 brings about a decrease in redox potential during theassay, and it seemes likely that this decrease is involved inthe activation of the other hydrogenase isoenzymes.Whether in HB101 extracts this role is performed by hydro-genase L remains a question. Finally, upon close examina-tion of the data, the curves of S100 extracts of HB101 wererepeatedly found to be biphasic, showing an activity increaseafter 10 to 15 min, when all 02 traces had been consumedand the MV was already partially reduced. (Fig. 2C). Thisobservation fitted very well with the hypothesis outlinedabove.DEAE ion-exchange chromatography. In an attempt to

separate hydrogenase L physically from hydrogenase 1 and2, S100 extracts ofHB101 and a representative mutant (BO2)were eluted on a DEAE high-pressure liquid chromatogra-phy column, and the fractions were analyzed on neutralpolyacrylamide gels and tested for total hydrogenase activity(Fig. 3). For both strains, hydrogenase 1 (under these growthconditions the faintest band) and the two forms of hydrogen-ase 2 appeared in fractions 7 and 8 (Fig. 3C). The lanescontaining fractions 9 and 10 revealed no hydrogenaseactivity at all. Fraction 11 was the peak fraction of acomplexed form of hydrogenase, either still bound to mem-brane fragments or artificial aggregates, of which hydrogen-ase forms a part, formed during isolation. Although beingnegatively charged it did not migrate into the gel, in contrastto, e.g., ferritin (450 kilodaltons [kDa]); therefore its weightexceeds 450 kDa.As expected, for HB101 the distribution of the total

activity over the different fractions (Fig. 3A) did not corre-late with the distribution of the stable activities, visualizedon gel (Fig. 3C); e.g., the total activities were equal infractions 8 and fraction 7, two fractions greatly differing inhydrogenase 1 and 2 content. Furthermore, the fraction

exhibiting the most intensive activity on gel, fraction 11, hadthe lowest relative overall activity, whereas empty lanes,containing fraction 9 and 10, correlated with high activity.Therefore the latter fractions contained almost exclusivelyelectrophoretically unstable hydrogenase activity. Thus, al-though the peak fractions for both types of activity may beapproximately the same (fraction 7), we practically suc-ceeded in their physical separation. When these data werecompared with those obtained with class I extracts (Fig. 3A)yielding the same electrophoretic pattern (not separatelyshown), we had to conclude that under these growth condi-tions the contribution of the electrophoretically stable activ-ities is small, about 20%, if we assume that all the activity inclass I mutants is brought about by this type of hydrogenase(hydrogenases 1 and 2). It is not impossible, however, that inthe mutant a fraction of the rest activity is still of the labilekind. This is not unlikely, since also for class I mutants thecorrelation between the total activity and the activity thatcould be visualized on gel (Fig. 3A and C) is not perfect (seeDiscussion). In that case, the contribution of hydrogenase 1and 2 activities is even smaller. This is in agreement with theresults of Sawers and Boxer (25), obtained after fermentativegrowth on glucose and formate.

Preliminary estimation of the molecular weight and pHoptimum. DEAE fraction 9 or 10 (Fig. 3A), i.e., predomi-nantly hydrogenase L, in absence of free Triton X-100 waseluted, together with marker proteins, from a high-pressureliquid chromatography G3000 SW (Chrompack) gel filtrationcolumn (Fig. 4). From this experiment a molecular size of

A

B 7 819 101111

4-F

FIG. 3. Hydrogenase activities of different fractions of S100extracts of E. coli HB101 and a representative class I mutant (B02)after elution from a DEAE ion-exchange column, analyzed by twomethods. (A) Total relative activity of each fraction, determined bymicrotitration and schematically represented. (B) DEAE fractions.(C) Activity-stained neutral, nondenaturing polyacrylamide gel, onwhich the DEAE fractions were run (shown is those of HB101, butan identical pattern was obtained with mutant extracts). Abbrevia-tions and symbols: F, 250 ~tg ferritin; 1, 2, and 3, isoenzymes (4); 0,HB101; U, mutant. Experimental details were as described inMaterials and Methods.

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1224 STOKER ET AL.

IActivity

"n

a b/c d e f 9

1I 11IAi

15 17 19 21 23 25 27 29 31 33 35Fraction number

FIG. 4. Estimation of the molecular size of hydrogenase L.DEAE fraction 9 (Fig. 3) was eluted from a G3000 SW (Chrompack)high-pressure liquid chromatography column in the presence of thefollowing molecular size markers; ferritin, 450 kDa (a); catalase, 240kDa (b); aldolase, 158 kDa (c); bovine serum albumin, 68 kDa (d);hen egg albumin, 45 kDa (e); chymotrypsinogenA, 25 kDa (f);cytochrome c, 12.5 kDa (g). For experimental details, see Materialsand Methods. The activity profile was determined by microtitrationand schematically represented in relative units; the molecular sizemarkers were monitored by their absorbance at 280 nm. (Theamount of hydrogenase was too small to be visible in the absorptionpattern.)

approximately 300 kDa was determined. However, since wehave neglected the influences of molecular shape, interac-tions with carrier material, possible Triton X-100 complexa-tion, and the poor resolution power of the column in the 500-to 250-kDa range, this figure can only be a rough estimate.Also, whether this value represents the molecular weight ofthe pure enzyme or of an active complex cannot be answeredat this moment. When the activity of the DEAE fractions 9and 10 was determined by the microtiter method as de-scribed above but at different pHs (pH 4.5 to 9), mostactivity was found between pH 7 and 8 (data not shown).

Genetic analysis of the hydL locus. To map the locusaffected in class I mutants (hydL), complementation exper-iments with a series of strains harboring different F-prime

factors (3) were carried out. Complementation was foundwith F'116 but not with F'143 or any other genomic fragmenttested, thus delimitating the position of the locus between61.5 and 66 min. Further localization was achieved by theP1-mediated transduction of the TnS-interrupted hydL locusfrom prototype strain B02 to Hfr strain PK191 (origin at 43min, clockwise transfer). This strain, PK191-I (HfrhydL::TnS, Kmr), was used as donor in conjugation experi-ments to suitable F- host strains. Selection was for kanamy-cin resistance and was followed by a MV filter test forhydrogenase activity. With PK191-I(hydL::TnS) as the do-nor strain, all Kmr colonies were also defective in hydrogen-ase activity. The coconjugation frequencies of hydL::TnSwith several markers on the E. coli linkage map are summa-rized in Table 2 and schematically represented in Fig. 5. A90-min cross with recipient CSH57B indicated that argG (69min) was a near, although distal, marker. A similar crosswith KMBL1418 (thyA) and LBE1930 (thyA serA) suggesteda location in the proximity of thyA (61 min) and serA (62.8min). To confirm that, a second conjugation was performedwith recipient LBE1930, this time for 35 min, and selectionwas made for Kmr (hydL::TnS), Ser+, and Thy'. From thatcross one could conclude that hydL was located distally fromserA and thyA, at a distance comparable with the geneticlength between thyA and serA, i.e., about 2 min from serA,close to 65 min. These results were confirmed by cotrans-duction experiments. hydL::TnS was transduced to JM2071(gaiP::TnJO), and P1 was grown on the resulting strain,JM2071-I (gaiP::TnJO hydL::TnS). The lysate obtained wasused to transduce strain AT2699 (metC) to phenotype Tetr orMetC+. A cotransduction frequency of 50% was foundbetween metC and hydL::Tn5, whereas a frequency of 9%was found between gaiP::TnJO (63.8 min) and hydL::TnS.gaiP: :TnJO and metC were found to be linked for about 1%.Taking into account that 50% linkage might be a slightunderestimation (see Discussion) and using Wu's map func-tion (30), we deduced a map position for hydL betweengaiP::TnJO and metC at about 1 min from galP and 0.2 minfrom metC, i.e., around 64.8 min (Fig. 5). This map positionfitted well with the F' complementation data.

DISCUSSIONUntil recently, the study of H2 metabolism in E. coli

covered three rather independent fields of interest, physio-logical, biochemical, and genetic. From the first it was

TABLE 2. Conjugation experiments establishing the frequencies of coconjugation of the hydL::TnS locuswith several markers on the E. coli chromosomea

Recipient Selected Unselected markerstrain Donor strain phenotype No. testedstrain phenotype ~~~~~~~~~~~~~~~~~~~~~Marker(min)

CSH57B PK191-I (Hfr hydL::Tn5) Kmr (hydL) 200 his (44) 43argG (69) 35metB (88.3) 2

KMBL1418 PK191-I (Hfr hydL::Tn5) Kmr (hydL) 150 thyA (61) 95LBE1930 PK191-I (Hfr hydL::TnS) Kmr (hydL) 150 serA (62.8) 90LBE1930 PK191-I (Hfr hydL::TnS) Kmr (hydL) 200 thyA 29

serA 38Ser+ 200 thyA 83

hydL 35Thy+ 200 serA 54

hydL 20Kmr Thy+ 200 serA 98

a See Materials and Methods for detailed genotypes. Conjugation was for 90 min, except the second cross with LBE1930, which lasted 35 min. Origin of PK191was at 43 min with clockwise transfer.

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HYDROGENASE ISOENZYMES IN E. COLI 1225

350

38

29

54*~~~~~s

-A1

9I 5

9 so

his thyA lysA serA galP::TnlO hyd L metC argG

44' 59' 60' 61' 62' 63' 64' 65' 66' 69'

PK191 +

F'143 *

F'116 l

FIG. 5. Position of the hydL locus on the E. coli linkage map (3). Numbers indicate percentages coconjugation or contransduction (seeTable 2). Symbols: 4 , origin of Hfr PK191; H *, origin and length of F-prime factors; A @-n-4 B, selection was made for markerA, n% coconjugation with marker B; A 4 n-* B, n% cotransduction between markers A and B.

reported that hydrogenase was involved not only in H2production (FHL) (22) but also in H2 uptake activities (Hup)(17). The second approach revealed the existence of twodistinct, stable forms of hydrogenase (hydrogenases 1 and 2)(4). A third activity, comprising one or more isoenzymesinactivated upon electrophoresis and therefore probablylabile (hydrogenase 3), was suggested (25). The last wasthought to play a role in the FHL reaction. Finally, at leastsix genetic loci were described (15, 21, 28, 29), regulating theoverall hydrogenase activity, albeit always in a coordinatedfashion in relation to the individual isoenzymes. The situa-tion in S. typhimurium seems to be very similar (27). At thismoment, therefore, it is necessary to deepen our knowledgeof the different aspects of H2 metabolism; of equal impor-tance is to find out the complex interrelationships whichexist between them. To solve this problem we need hydro-genase mutants, particularly those defective in only oneisoenzyme activity. Here we described such a mutant, whichlacks only the labile isoenzyme but has the two unaffectedstable forms. Experiments were carried out to establish thegene-product-function relation.

Locus. By means of coconjugation experiments the locusaffected (hydL) was mapped close to 65 min. Of all E. colihydrogenase mutants characterized thus far, only one classis complemented by F-prime factor F'116 as well; the hupmutation mentioned by Lee et al. (15), also mapped close to65 min on the E. coli chromosome. Although the mutationwas only partially mapped and characterized, the authorsfound a cotransduction frequency of 76% of hup with metC.Since it is not unlikely that in our case the 5-kilobase TnSinterruption causes a slight decrease in the chance of recom-bination (50%), the hydL mutation might be identical to thehup mutation. However, the hydrogenase activity of thismutant class appeared to be quite normal when this wasdetermined by tritium exchange, a method that excludesmembrane effects. In addition, in contrast to class I mutantsthis group was also found to be partially impaired in formatedehydrogenase activity (23 to 53%). Therefore, a goodalternative explanation might be that at this map position

more than one gene is present that regulates hydrogenaseactivities in E. coli. At this moment, we are not able to verifythis without additional studies.The mutation might be structural, only inactivating hydro-

genase L or may have pleiotropic effects, inactivating hy-drogenase L and other genes essential for H2 uptake activity.In that case it might be a positive regulator or, alternatively,constitute part of an operon, in which case an insertion mayhave a polar effect on genes in downstream position.

Physiological function. Suggestions about the physiologicalrole of the individual isoenzyrmes have been made, based oninduction experiments in which E. coli and S. typhimuriumgrown under different conditions and their distinct hydro-genase activities were measured (25, 27). Although some-times the results of these experiments can be explained indifferent ways, one observation was striking. Sawers et al.reported that when E. coli was cultured on glycerol (H2) andfumarate, in comparison with when it was grown on glucose,a 10-fold increase of the hydrogenase 2 content was found(25), accompanied by an almost inversely proportional de-crease of the labile activity, so no significant change inoverall activity could be observed. The FHL activitydropped, whereas the Hup activity increased, thus suggest-ing a production role for the labile activity and an uptakefunction for hydrogenase 2.Our results presented in this paper were obtained by the

alternative approach. Two different strains of E. coli, parentand mutant, were assayed under the same conditions. Amutant class was described that lacked the major part of thelabile activity, whereas a minor portion of this type ofactivity still could be observed (Fig. 3). The FHL pathwaywas unimpaired, in contrast with H2 uptake pathway, whichwas completely abolished under these conditions. The hy-drogenase isoenzymes 1 and 2 were present at normal levels.

If we assume that the labile activity is involved in H2production (25), we must conclude that one locus (hydL)regulates H2 production as well as H2 uptake simulta-neously, which is rather unlikely. Therefore, we propose amodel in which the major portion of the labile hydrogenase

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activity (hydrogenase L) has an uptake function. (If we wantto maintain that hydrogenase 2 is an uptake enzyme, wemust explain the absence of any uptake activity in class Imutants-while hydrogenase 2 is present at normal levels-by assuming that the hydL locus regulates the expression ofother genes involved in Hup activity as well.)

Indirect support for the idea that the electrophoreticallylabile (or nonimmunoprecipitable, in the case of S. typhimu-rium) part of the hydrogenase activity is involved in Hupactivities, rather than hydrogenase 2, is found in reference27. Two spontaneous mutants of S. typhimurium weredescribed in which the hydrogenase 2 activities amounted toless than 0.1% of that of the parent strain, whereas the Hupactivities were 6 and 20%, respectively, of the parental level.In contrast to that, however, the correlation of the latteractivity with the levels of the nonimmunoprecipitable (labile)hydrogenase activities (15 and 45%, respectively) was quitestrong.FHL-linked H2 production might be catalyzed by the

minor portion of the labile hydrogenase activity that still canbe observed in class I mutants, possibly hydrogenase 3. Inthat case hydrogenase 3 is not identical to hydrogenase L,and there are two types of labile activity. But since we havenot been able to discriminate between different types oflabile activity, the latter suggestion is speculative.

It is obvious that the data available at this moment are notsufficient to enable us to discriminate between the possiblemodels. Perhaps that the physiological and genetic charac-terization of other hydrogenase mutants and the molecularcloning of genes involved may do so.

ACKNOWLEDGMENTS

This work was supported by a grant from the NetherlandsOrganisation for the Advancement of Pure Research under theauspices of the Netherlands Foundation for Chemical Research.We thank Eveline Kampert and Richard Giltay for their assistance

in the genetic experiments and Fred Wansell for his careful exami-nation of the manuscript.

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