JOURNAL OF BACTERIOLOGY, Apr. 1984, p. 43-480021-9193/84/040043-06$02.00/0Copyright X 1984, American Society for Microbiology

Vol. 158, No. 1

Alcaligenes eutrophus Hydrogenase Genes (Hox)CHRISTINE HOGREFE, DETLEF ROMERMANN, AND BARBEL FRIEDRICH*

Institut fur Mikrobiologie der Universitat Gottingen, D-3400 Gottingen, Federal Republic of Germany

Received 22 August 1983/Accepted 12 January 1984

Mutants ofAlcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew veryslowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) werenot affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizingability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconju-gants which had inherited the plasmid and the phenotype of the donor. This indicates that the structuralgenes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox-mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since theywere unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-).Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act asrecipients but could act as donors of the Hox character. Transconjugants derived from those crosses wereHox+ Nit', indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome.Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hoxgenes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multipleresistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype,we conclude that this property is not directly plasmid associated.

Hydrogen-oxidizing ability of the facultatively chemolitho-autotrophic bacterium Alcaligenes eutrophus has beenfound to be genetically linked to a large plasmid (1, 9). Theplasmid from A. eutrophus H16 is about 300 megadaltons insize and has been designated pHG1. Plasmid-cured mutantshave been described which were unable to oxidize hydrogen(Hox-). Hydrogenase activity of these mutants could berestored by conjugal tr,ansfer of plasmid pHG1. Hox+ trans-conjugants occurred at a high frequency of 10-2. From theseresults it was concluded that plasmid pHG1 is self-transmis-sible and codes for'essential Hox determinants (9).

In A. eutrophus, hydrogen is oxidized by a complexenzyme system: a soluble NAD+-reducing hydrogenase(hydrogen:NAD+ oxidoreductase, EC 1.12.1.2 [21]) and amembrane-bound hydrogenase linked to the respiratorychain (18). The formation of these enzymes is coordinatewith, but not subject to, induction by hydrogen (12). Synthe-sis of hydrogenase is derepressed during energy limitation(10). Moreover, we have shown that the formation of hy-drogenase is temperature sensitive and that this regulatoryproperty is associated with plasmid pHG1 (11).

In the present communication, we report on properties ofHox mutants and on mating experiments with donor strainsthat carry either structural or regulatory Hox gene muta-tions. The biochemical analysis of transconjugants indicatesthat the structural genes for both hydrogenases reside onplasmid pHG1. Evidence is presented which indicates thatan additional chromosomal determinant is required for theexpression of plasmid-encoded Hox genes.

MATERIALS AND METHODSBacterial strains. Strains of Alcaligenes eutrophus em-

ployed in this study and mutants derived therefrom are listedin Table 1. Auxotrophic and hydrogenase-defective mutantswere isolated as described (9) unless otherwise stated. Trans-poson mutagenesis was carried out as described (23), exceptthat TnS was transferred from Escherichia coli SM10 to A.

* Corresponding author.

eutrophus by a vector plasmid pSUP5011 containing a P-type-specific recognition site for mobilization (22). Kanamy-cin-resistant colonies were selected on fructose-containingminimal medium with 500 ,ug of kanamycin per ml.Growth conditions. The bacteria were grown either in

complex medium consisting of nutrient broth or in mineralsalts medium as described by Schlegel et al. (20). Organiccarbon sources were supplied at a concentration of 0.4%(wt/vol); the concentration of the nitrogen source was 0.2%(wt/vol). The gas atmosphere for autotrophic growth con-tained a mixture of hydrogen, oxygen, and carbon dioxide ina ratio of 8:1:1 (vol/vol). Anaerobic cultivation of cells withnitrate or nitrite as the electron acceptor was performed intubes or flasks sealed with serum stoppers. Growth wasfollowed by withdrawing samples anaerobically and measur-ing the absorbance of the cell suspension at 436 nm with aZeiss PL4 spectrophotometer. The concentration of potassi-um nitrate was 0.2% (wt/vol). Potassium nitrite was added ata concentration of 0.1% (wt/vol), and the pH of this mediumwas' adjusted to 7.5. Antibiotics were supplemented 'asindicated in the text.Enzyme assays. Cells were grown under hydrogenase-dere-

pressing conditions with a substrate combination of fructoseand glycerol as described previously (8). The activity of thesoluble hydrogenase was determined with a whole cell assay(8) by monitoring NADH formation spectrophotometricallyby the method of Schneider and Schlegel (21). Membrane-bound hydrogenase activity was assayed with the particulatefraction by spectrophotometric measurement of hydrogen-dependent methylene blue reduction (18). The particulateextract was prepared as described (8). The presence ofsoluble and particulate hydrogenase antigen in soluble ex-tracts and in the solubilized membrane fraction was deter-mined as described (19). Protein was measured by themethod of Lowry et al. (14).

Determination of nitrite. Nitrite determination was carriedout by the method of Lowe and Evans (13). One milliliter ofthe cell suspension was withdrawn from the culture at thetimes indicated. The suspension was rapidly cooled to 4°C,

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44 HOGREFE, ROMERMANN, AND FRIEDRICH

TABLE 1. Bacterial strains

Straina Relevant Reference or source

A. eutrophusH16 Hos+ Hop' Wild type: ATCC 17699,

Hox Ts DSM 428HF33 Hox- Smr Plasmid-cured mutant,

this studyHF151 Hox- Smr Nar Spontaneous Nar mutant

of HF33, this studyHF123 Hox- 11HF15 Hos- Hop' EMS-induced mutant; this

study"HF89 Hos- Hop' EMS-induced mutant; this

studyHF08 Hos+ Hop- 16HF134 Hos+ Hop- Mutant of HF08, this

Hox Tr studyHF82 Hox- Nit- Nitrous acid-induced

mutant, this studyHF149 Hox- Nit- Tn5-induced mutant, this

studyTF93 Hox+ Wild type: ATCC 17697,

DSM 531N9A Hox+ Wild type: DSM 518N9AF06 Hox- Plasmnid-cured mutant of

N9A, this studyH20 Hox+ Wild type: DSM 530A704 Hox+ Wild type: ATCC 17704

A. hydrogenophilus Hox+ 15

a Strains carrying the initials HF are derivatives of A. eutrophusH16.

b Phenotypes are designated as follows: Hox, ability to oxidizehydrogen; Hox Ts and Hox Tr, inability and ability, respectively, togrow with H2 and CO2 at 37°C; Hos, catalytically active solublehydrogenase; Hop, catalytically active particulate hydrogenase;Smr, resistance to 800 ,ug of streptomycin per ml; Nar, resistance to100 ,ug of nalidixic acid per ml.

EMS, Ethyl methane sulfonate.

and the cells were removed by centrifugation. Samples of thesupernatant were diluted if necessary so that the nitriteconcentration did not exceed 0.035 ,umol/0.5 ml. This samplewas mixed with 0.5 ml of sulfanilamide (58 mM, dissolved in3 N hydrogen chloride) and 0.5 ml of N-(1-naphthyl)-ethylene-diaminedihydrochloride (0.39 mM). The mixture was dilut-ed with 3 ml of distilled water, and the color was allowed todevelop for 20 min. Then the absorbance was measured at546 nm.

Conjugation. Matings were routinely performed on agar asdescribed previously (9).

Analysis of plasmid DNA. Crude lysates were prepared bythe method of Casse et al. (6), modified as described (9).Twenty microliters of the ethanol-concentrated DNA solu-tion was subjected to agarose gel electrophoresis and ana-lyzed as described (9).

Chemicals. NAD+ was obtained from C. F. Boehringer &Soehne GmbH, Mannheim, Federal Republic of Germany.Mitomycin C, kanamycin sulfate, nalidixic acid, and agarosetype V were purchased from Sigma Chemical Co., St. Louis,Mo. Antibiotic sensitivity disks were from Oxoid Ltd.,London, England. All of the other chemicals were purchasedfrom E. Merck AG, Darmstadt, Federal Republic of Germa-ny.

RESULTSCoincidence of plasmid curing and antibiotic resistances. In

most of the A. eutrophus wild-type strains, the pHG plasmidwas very stably maintained (9). We accidentally detectedamong spontaneous antibiotic-resistant mutants cloneswhich had lost the ability to oxidize hydrogen. Furthercharacterization revealed that these Hox- mutants were freeof plasmid DNA. Moreover, they were not only resistant tothe antibiotic they had been exposed to; they had alsocollected resistances to other drugs as indicated in Table 2.The multiresistance patterns of independently isolated Hox-mutants from a specific parent strain were identical. Howev-er, the pattern differed for each wild-type strain (Table 2).A. eutrophus TF93 was the only strain in our collection

which was'able to grow with glucose as the carbon source.The other wild-type strains were cryptic for this substrate.All of the plasmid-cured Hox- mutants of TF93 had con-comitantly lost the ability to grow with glucose (data notshown). On the other hand, plasmid-bearing Hox- mutantsexhibited neither the drug resistance markers nor the glu-cose-negative phenotype.The retransfer of plasmid pHG1 to cured Hox- mutants

resulted in trapsconjugants which had recovered hydroge-nase activity but retained the antibiotic resistance phenotypeof the recipient (Table 2). Correspondingly, Hox+ transcon-jugants derived from A. eutrophus TF93 were still unable togrow with glucose (data not shown).

Self-transmissibility of pHG plasmids. Andersen et al. (1)reported that the Hox character could only be transferred byresistance-factor-mediated mobilization. This prompted usto examine whether naturally occurring genetic transfer ofHox was restricted to A. eutrophus H16 or was commonlydist'ributed among Alcaligenes wild-type strains. Table 3 liststhe strains which were included in this study., Matings wereperformed with the plasmid-cured Hox- mutant HF151 asthe recipient. As shown, all of the wild-type strains haddonor ability of Hox. No resistance-factor mobilization wasrequired to achieve this transfer. The transfer frequency wasin the range of 1 x - to 5 x i0-. It was lower thanreported earlier (9) since Hox+ transconjugants were select-ed in the presence of antibiotics to counterselect the donorcells (Table 3). It was observed that selection on a drug-containing medium or auxotrophic markers 'in the donorstrains led to a decreased efficiency of Hox transfer.The stability of the Hox character was examined in Hox+

transconjugants derived from the plasmid-cured mutantHF151 (Table 3). The majority of Hox+ transconjugantsstably maintained the Hox marker, as did the wild-typestrain A. eutrophus H16 (data not shown). However, amongthe transconjugants derived from matings with Alcaligeneshydrogenophilus as the donor, about 7% of the testedcolonies were Hox-. Moreover, Hox+ transconjugantswhich had acquired the plasmid from A. eutrophus TF93were extremely unstable. The result suggests that the stabil-ity of the Hox character is mainly determined by theincoming plasmid.Mutants deficient in soluble and particulate hydrogenase

activity. One approach to elucidate the nature of plasmid-coded Hox gehes was through the study of appropriatemutants. This included strains devoid of soluble hydroge-nase activity (Hos-) or particulate hydrogenase activity(Hop-). The lack of soluble hydrogenase activity led to asignificantly slow lithoautotrophic growth compared withthe wild type (Table 4). Furthermore, growth of Hos-mutants commenced after an extended lag period of 30 to 60

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TABLE 2. Antibiotic resistance pattern of cured Hox- mutants and the corresponding Hox+ transconjugants

Antibiotic resistance'Strain Isolation Phenotype Plasmidb

Cm Sm Tc Na

H16 Wild type Hox+ + 22 23 30 21HF33 Cmr mutant Hox- - 0 0 19 0HF142 Transconjugant from Hox+ + 0 0 19 0

HF33N9A Wild type Hox+ + 21 14 27 15N9AF06 Mitomycin Hox- - 0 13 19 0N9AF35 Transconjugant from Hox+ + 0 14 19 0

N9AF06

a Antibiotic resistance was determined on nutrient agar by a diffusion assay. Antibiotic disks of the following concentrations (inparentheses) were employed: Cm, chloramphenicol (30 ,ug); Sm, streptomycin (25 ,ug); Tc, tetracycline (30 ,ug); Na, nalidixic acid (30 ,ug). Thenumbers represent the diameter of the inhibition zone in millimeters.

b +, Presence of plasmid pHG; -, absence of plasmid pHG.

h. Surprisingly, the Hop- mutant exhibited almost the same

growth rate as the parent strain. Biochemical studies clearlyshowed that Hos- mutants contained normal activity ofparticulate hydrogenase, and correspondingly, the Hop-mutant was not affected in soluble hydrogenase activity(Table 4). The catalytically inactive soluble hydrogenase ofHos- mutants exhibited cross-reactivity with antiserumagainst purified soluble enzyme (Table 4). The precipitationbands were only of partial identity, indicating structuralchanges in the protein (Fig. 1A, wells 2 and 4). We detectedthe catalytically inactive particulate hydrogenase protein ofthe Hop- mutant only in the cytoplasmic fraction of theextract (Fig. 1B, well 5). This indicates that it is eithersoluble or less tightly bound to the membrane than the wild-type enzyme is.

Temperature-resistant mutants which were able to growwith H2 and CO2 at 37°C were isolated from the Hop-mutant HF08. The spontaneous isolates formed hydroge-nases at the permissive temperature (Hox Tr, Table 4). Thesoluble enzyme was catalytically active (Hos+), whereas theparticulate hydrogenase was inactive (Hop-).

Transfer of hydrogenase deficiency by conjugation. Studieswere made of the properties of transconjugants which arosefrom crosses with donors devoid of soluble (Hos-) or

particulate (Hop-) hydrogenase activity (Table 4). All ofthese mutants were hosts of plasmid DNA (data not shown).Two types of Hox- mutants were employed as recipients,the plasmid-cured mutant HF151 and the plasmid-bearingmutant HF123. Both recipients had in common the lack ofcatalytically and immunologically active hydrogenase pro-teins (Table 4; Fig. 1, wells 6).

Transconjugants able to grow lithoautotrophically oc-curred with all of the donors employed in this study (Table5). It was obvious from the small size of the colonies thattransconjugants derived from Hos- donors grew remarkablyslower with hydrogen than Hox+ transconjugants whicharose from crosses with the wild type. As expected, the slowgrowth was due to the lack of soluble hydrogenase activity(Table 5). Thus, the transconjugants exhibited the samephenotype as the donor. Among the slowly growing colo-nies, larger colonies were detected at a low frequency of10-6 to 10-7 per donor. However, this type of colonyoccurred exclusively with plasmid-bearing recipients, notwith plasmid-cured Hox- mutants. Biochemical analysisrevealed that these rare clones exhibited active soluble aswell as active particulate hydrogenase (Table 5).

In a series of analogous experiments conducted with

Hop- mutants as donors, it was found that the resultingtransconjugants expressed the phenotype of the donor (Ta-ble 5). Since Hop- derivatives grew as fast as the wild type,it was not possible to discriminate between colonies ofdifferent size. Thus, in these crosses plasmid recombinantswere not identified.The use of the double mutant HF134 (Table 4) as a donor

generated transconjugants which expressed the two pheno-typic markers of the donor, namely Hop- and Hox Tr (Table5).

Evidence for chromosomally located Hox determinants. Aclass of plasmid-containing Hox- mutants which were freeof hydrogenase proteins and exhibited donor activity but norecipient activity in crosses was further characterized. It wasdiscovered not only that they were unable to grow withhydrogen, but also that they failed to grow with nitrate. Thephenotype of these pleiotropic mutants was designatedHox- Nit- (Table 4). The Nit- character was furtherexplored. The mutants scarcely utilized nitrate as the solesource of nitrogen or (anaerobically) as the electron accep-tor. Anaerobic incubation of cells with fructose as the

TABLE 3. Formation and stability of Hox+ transconjugantsStability of Hox+transconjugantsa

Donorb Transferfrequencyc No. of No. ofcolonies Hox-examined mutants

H16 2.7 x 10-4 594 0H20 1.0 x 10-3 702 1A704 1.2 x 10-4 756 0N9A 4.5 x 10-5 594 0A. hydrogenophilus 3.4 x 10-4 712 49TF93 1.5 x 10-3 1025 746

a Cells were grown in fructose-glycerol-containing mineral medi-um for 72 h as described (8). They were plated on nutrient agar, andsingle colonies were tested for the ability to utilize hydrogen as thesole energy source.

b Matings were performed on agar with the plasmid-free Hox-mutant HF151 as the recipient. This mutant was derived from thewild-type strain H16 and carried as a counterselectable marker highresistance to streptomycin and nalidixic acid.

c Transfer frequency results are expressed as the number of Hox+transconjugants obtained per donor. The titer was determined afterthe incubation on agar.

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TABLE 4. Physiological and biochemical properties of mutants impaired in hydrogenasesHydrogenase activitya

Strain Relevant phenotypeb Doubling time (h) Catalytic (U/mg of protein) Immunologicalwith H, + CO,`_

Soluble Particulate Soluble Particulate

H16 (wild type) Hos+ Hop' Hox Ts 3.6 2.8 1.4 + +

HF15 Hos- Hop' 12 0 0.7 + +

HF89 Hos- Hop' 17 0 1.6 + +

HF08 Hos+ Hop- Hox Ts 4.2 (300C) 1.4 0 + +NG (370C) 0 0 - -

HF134 Hos+ Hop- Hox Tr 4.0 (300C) 2.3 0 + +5.7 (370C) 2.4 0 + +

HF151(pHG1)- Hox- NG 0 0 - -

HF123(pHG1)+ Hox- NG 0 0 - -

HF82(pHG1)+ Hox- Nit- NG 0 0 - -

HF149(pHG1)+ Hox- Nit- Kmr NG 0 0 - -

a Cells were grown in fructose-glycerol-mineral medium as described (8). +, Presence of cross-reacting material; -, absence of cross-reacting material.

b Phenotype designation's are as described in Table 1, footnote b.c The growth temperature was 30°C unless otherwise indicated. NG, no growth.

carbon source and nitrate or nitrite as the electron acceptorrevealed that these mutants dissimilated nitrate to a certainextent since nitrite accumulated in the medium. When thecells were incubated with nitrite, the nitrite concentrationremained constant over the whole incubation period (datanot shown). Apparently, the mutants were unable to convertnitrite further, which suggests that the Nit- property is dueto a failure in nitrite reduction.To exclude the possibility that the pleiotropic effect arose

from a double mutation, reversion analysis was carried outwith independently isolated mutants. This analysis includedmutants isolated after chemical mutagenesis (nitrous acid),such as HF82, and the transposon Tn5-induced mutantHF149. Revertants specifically selected for the Hox+ pheno-type had concomitantly regained the ability to grow withnitrate. Hox+ revertants of mutant HF149 were Nit' andhad lost the kanamycin resistance marker. The resultsindicate that the Hox- Nit- phenotype is the result of asingle mutation.Hox+ transconjugants originating from crosses with Hox-

Nit- mutants as the donor did not coinherit the Nit-

A:6:'

5 2

4 3

B 6 1

.5.

K)?4 3

FIG. 1. Ouchterlony double-diffusion analysis of hydrogenasefrom wild-type and mutant extracts. Center wells contained purifiedantisoluble hydrogenase serum (A) and purified antiparticulatehydrogenase serum (B). Soluble extracts were added to the outerwells of A as follows: well 1, H16; well 2, HF15; well 3, H16; well 4,HF89; well 5, HF08; well 6, HF151. Solubilized particulate extractswere added to the outer wells of B in the same pattern, except thatwell 5 was supplied with soluble extract.

character. They expressed activity in both hydrogenases(Table 5). Finally, Hox+ transconjugants arising from cross-es with the TnS-induced mutant HF149 were also Nit' andkanamycin susceptible.

DISCUSSIONIn this study, we have demonstrated that plasmids from

other Alcaligenes wild-type strains also have transfer func-tions and restore hydrogenase activities of Hox- mutants asreported for plasmid pHG1 from A. eutrophus H16 (9).The linkage of hydrogenase genes to a 190-megadalton

plasmid was shown for Rhizobium leguminosarum (3). Ef-forts to correlate hydrogenase determinants of Rhizobiumjaponicum with a plasmid have been unsuccessful so far (5).The construction of a gene bank yielded cosmids containingDNA sequences involved in the hydrogen uptake function ofR. japonicum. It is not known yet whether the cloned DNArepresents structural or regulatory hydrogenase genes (4).Alcaligenes is the only genus among hydrogen bacteria

which has evolved two hydrogenases (2). The physiologicalsignificance of the presence of a soluble hydrogenase in A.eutrophus is evident from the mutant analysis. Mutantslacking soluble activity (Hos- Hop') grew very poorly withhydrogen. On the other hand, mutants impaired in theactivity of the particulate hydrogenase (Hos+ Hop-) werescarcely affected in lithoautotrophic growth, which agreeswell with earlier studies (17). Consequently, it appears to beextremely difficult to directly select for the Hos' Hop-phenotype.The soluble hydrogenase is a tetramer with an approxi-

mate molecular weight of 190,000 (21). According to recentstudies, it consists of four different polypeptides (K.Schneider, submitted for publication). The particulate hy-drogenase has a molecular weight of 98,000 and represents adimer of two different polypeptides (18). There is no immu-nological cross-reactivity between the native enzymes (19),which suggests that the two hydrogenase proteins are en-coded by six structural genes. In earlier studies, we haveshown that regulatory functions of Hox expression are

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HYDROGENASE GENES 47

TABLE 5. Transfer of plasmid-coded markersDonor' Transconjugants

Hydrogenase activity (U/mg ofStrain Relevant phenotype Phenotype Temp ('C) protein)

Soluble ParticulateH16 Hos+ Hop' Hox Ts Hos+ Hop' Hox Ts 30 3.8 1.7

37 0 0

HF15 Hos- Hop' Hos- Hop' 30 0 0.92Hos+ Hop' 30 2.9 1.6

HF08 Hos+ Hop- Hox Ts Hos+ Hop- Hox Ts 30 2.5 037 0 0

HF134 Hos+ Hop- Hox Tr Hos+ Hop- Hox Tr 37 2.3 0

HF82 Hox- Nit- Hox+ Nit' 30 3.1 1.8

HF149 Hox- Nit- Kmr Hox+ Nit' Kms 30 2.2 1.3a Phenotype designations are as described in Table 1, footnote b. The plasmid-free Hox- mutant HF151 and the plasmid-containing Hox-

mutant HF123 were used as recipients.

conferred by pHG plasmids in A. eutrophus and A. hydro-genophilus (7, 11). This communication presents evidencewhich supports the notion that plasmid pHG1 from A.eutrophus H16 also determines structural hydrogenasegenes. The conclusion is drawn from the observation that adeficiency in soluble hydrogenase activity in a donor strainwas inherited by transconjugants. Correspondingly, trans-conjugants which arose from crosses with mutants devoid ofparticulate hydrogenase activity exhibited the Hos+ Hop-phenotype of the donor. Since we have never observed themobilization of chromosomal genes by plasmid pHG1 (9),the transfer of markers by conjugation indicates their plas-mid localization. Because of the presence of immunological-ly active hydrogenase proteins in Hos- and Hop- mutants,it is more likely that these mutants contain structural genemutations rather than regulatory lesions. This assumption issupported by recent observations which indicate that thecatalytically inactive soluble hydrogenase of mutant HF15still contains nickel (C. Friedrich, personal communication)and catalyzes hydrogen-dependent reduction of electronacceptors other than NAD+, although at an extremely lowrate (S. Homhardt, personal communication).From preliminary results of an earlier study, we have

predicted that plasmid pHG1 does not determine the entiregenetic information for Hox expression (9). This assumptiongained further support from the newly isolated mutants,including TnS-induced derivatives. We discovered a class ofplasmid-containing Hox- mutants which not only wereimpaired in the formation of both hydrogenases but alsofailed to grow with nitrate. This pleiotropic Hox- Nit-phenotype appears to be the result of a single mutation sinceHox+ revertants had concomitantly regained the ability togrow with nitrate. Furthermore, revertants of the TnS-induced mutants were susceptible to kanamycin. The factthat all of these Hox- Nit- mutants were perfect donors ofthe Hox character but none of them acted as a recipientsuggests that the mutation which renders the cells Hox-Nit- is chromosomally located. A preliminary characteriza-tion of the Nit- marker favors the assumption that themutants are blocked in nitrite reduction rather than in thedenitrification of nitrate. The linkage between hydrogenoxidation and denitrification is still unknown. The followingpossibilities can be considered: (i) a common cofactor re-quirement, (ii) a deficiency in an electron transport compo-

nent, and (iii) a lesion in a regulatory gene whose product isnecessary for the expression of Hox and Nit genes.The observation that plasmid-cured derivatives collected

resistances to various antibiotics or lost the ability to growwith glucose led to the speculation that the plasmid codes foruptake functions. Moreover, we recently discovered thatplasmid-free mutants of A. eutrophus H16 and N9A alsofailed to grow with histidine (B. Friedrich, unpublishedresults). Although pHG+ transconjugants recovered hydrog-enase activity, they retained the other pleiotropic markers ofthe recipient. We have no evidence that during the transferplasmid pHG1 undergoes major structural changes such asdeletions or insertions since the restriction patterns of plas-mid DNA isolated from the wild type and from Hox+transconjugants appear to be identical (C. Hogrefe, unpub-lished results). At present, we are considering the possibilitythat the loss of the plasmid is associated with alterations ofmembrane constituents owing to a chromosomal mutation.

ACKNOWLEDGMENTSWe thank A. Kamienski and G. Henning for technical assistance

and V. Elles, E. Heine, and C. Rohde for the isolation of mutants.We are grateful to K. Schneider and G. Podzuweit for providinganti-hydrogenase serum. We are indepted to A. Piihler and co-workers for the use of Tn5 vector strains before publication.

This work was supported by a grant Fr 305/5-2 from the DeutscheForschungsgemeinschaft and in part by Forderungsmittel desLandes Niedersachsen.

LITERATURE CITED1. Andersen, K., R. C. Tait, and W. R. King. 1981. Plasmids

required for utilization of molecular hydrogen by Alcaligeneseutrophus. Arch. Microbiol. 129:384-390.

2. Bowien, B., and H. G. Schlegel. 1981. Physiology and biochemis-try of aerobic hydrogen-oxidizing bacteria. Annu. Rev. Micro-biol. 35:405-452.

3. Brewin, N. J., T. M. De Jong, D. A. Philips, and A. W. B.Johnston. 1980. Co-transfer of determinants for hydrogenaseactivity and nodulation ability in Rhizobium leguminosarum.Nature (London) 288:77-79.

4. CantreUl, M. A., R. A. Haugland, and H. J. Evans. 1983.Construction of a Rhizobium japonicum gene bank and use inisolation of a hydrogen uptake gene. Proc. Natl. Acad. Sci.U.S.A. 80:181-185.

5. Cantrell, M. A., R. E. Hickok, and H. J. Evans. 1982. Identifica-tion and characterization of plasmids in hydrogen uptake posi-

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7. Friedrich, B., C. G. Friedrich, M. Meyer, and H. G. Schlegel.Expression of hydrogenase in Alcaligenes spp. is altered byinterspecifc plasmid exchange. J. Bacteriol. 158:331-333.

8. Friedrich, B., E. Heine, A. Finck, and C. G. Friedrich. 1981.Nickel requirement for active hydrogenase formation in Alcali-genes eutrophus. J. Bacteriol. 145:1144-1149.

9. Friedrich, B., C. Hogrefe, and H. G. Schlegel. 1981. Naturallyoccurring genetic transfer of hydrogen-oxidizing ability betweenstrains of Alcaligenes eutrophus. J. Bacteriol. 147:198-205.

10. Friedrich, C. G. 1982. Derepression of hydrogenase duringlimitation of electron donors and derepression of ribulosebis-phosphate carboxylase during carbon limitation of Alcaligeneseutrophus. J. Bacteriol. 149:203-210.

11. Friedrich, C. G., and B. Friedrich. 1983. Regulation of hydroge-nase formation is temperature sensitive and plasmid coded inAlcaligenes eutrophus. J. Bacteriol. 153:176-181.

12. Friedrich, C. G., B. Friedrich, and B. Bowien. 1981. Formationof enzymes of autotrophic metabolism during heterotrophicgrowth of Alcaligenes eutrophus. J. Gen. Microbiol. 122:69-78.

13. Lowe, R. H., and H. J. Evans. 1964. Preparation and someproperties of a soluble nitrate reductase from Rhizobiumjaponi-cum. Biochim. Biophys. Acta 85:377-389.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

15. Ohi, K., N. Takada, S. Komemushi, M. Okazaki, and Y. Miura.1979. A new species of hydrogen-utilizing bacterium. J. Gen.Appl. Microbiol. 25:53-58.

16. Pfitzner, J. 1974. Ein Beitrag zum H2-02-Oxidoreduktasesystemvon Hydrogenomonas eutropha Stamm H16. Hydrogenase-defekte Mutanten. Zentralbl. Bakteriol. Parasitenkd. Infek-tionskr. Hyg. Abt. 1 Orig. Reihe A 228:121-127.

17. Schink, B., and H. G. Schlegel. 1978. Mutants of Alcaligeneseutrophus defective in autotrophic metabolism. Arch. Micro-biol. 117:123-129.

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