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Utilization of carbon monoxide by aerobes: recent advances
FEMS MicrobiologyReviewsS7 (1990) 253-260 Publishedby Elsevier
253
FEMSRE00199
Utilization of carbon monoxide by aerobes: recent advances Ortwin Meyer, Kurt Frunzke, Dilip Gadkari, Susanne Jacobitz, Iris Hugendieck and Maria Kraut Lehrstuhlf~r Mikrobiologie, Universit&Bayreuth, Bayreuth, F..R.G. Key words: Carboxydotrophic bacteria; Genetics; Plasmids; CO dehydrogenase; Hydrogenase; CO 2 fixation; Denitrification; Nitrate reduction; Cytochrome 1. SUMMARY The fist of carboxydotrophic bacteria is constantly growing and we have found that burning charcoal piles harbor an especially rich CO-oxidizing microflora. The newly isolated Streptomyces thermoautotrophicus UBT1 is particularly interesting as it is thermophilic, capable of chemolithoautotrophic growth with CO or H~ plus CO 2 and incapable of using organic substrates. Molybdenum is essential for CO-autotrophic growth. Some species of carboxydotrophic bacteria can denitrify under heterotrophic conditions yielding N 2 (e.g. Pseudomonas carboxydoflava) or N20 (e.g. Pseudomonas carboxydohydrogena); others perform nitrate respiration (e.g. Azomonas B1). P. carboxydohydrogena could grow at the expense of H 2 plus CO 2 using nitrate as electron acceptor. In intact cells of Pseudomonas carboxydovorans, CO dehydrogenase has the ability of dissociating from and rebinding to the cytoplasmic membrane. That process can be simulated in vitro by removing CO dehydrogenase from cytoplasmic membranes and rebinding it to depleted membranes. Reconstitution ¢~f the enzyme onto depleted membranes requiring di- or trivalent cations, was
specific for membranes from CO-grown bacteria and led to reactivation of respiratory activities with CO. A complex consisting of 1 molecule of CO dehydrogenase and 2 molecules of cytochrome b561 could be isolated from cytoplasmic membranes of P. carboxydovorans sohibilized with dodecyl ~-D-maltoside. Within the complex as wel! as h~ a~ays containing purified CO dehydrogenase and cytochrome b561 the latter could serve as an electron acceptor. CO dehydrogenase had hydrogenase activity, and its KM of only 5 mM H 2 suggested a role in the formation of H2. P. carbox.vdovorans OM5 contaL~,~ the 128-kilobase pairs (kb) plasmid pHCG3 which is essential for CO- and H2-fithoautoh-ophic growth. Evidence for the existence of pHCG3-coded structural genes of CO dehydrogenase waz obtained from dot blot hybridizations employing synthetic ofigodeoxynucleotides as heterologous probes for the detection of the S- and M-subunit genes. Employing appropriate probe genes encoding membrane-bound hydrogenase, ribulose bisphosphate carboxylase and phosphoribulokinase were also identified on plasmid pHCG3.
2. INTRODUCTION Correspondence to: O. Meyer, Lehrstuhl ffir Mikrobiologie, UniversitatBayreuth,Universiflttsstrasse30, D-8580 Bayreuth, F.R.G.
CO-oxidizing (carboxydotrophic) bacteria are characterized by the utilization of CO as a sole
0168-6445/90/$03.50 © 1990 Federationof European MicrobiologicalSocieties
254
source of energy and carbon. Their microbiology, physiology and biochemistry have been dealt with in numerous recent reviews [1-7]. With only very few exceptions, carboxydotrophs can use H 2 plus CO2 as well, indicating the presence of two different capacities for chemolithoautotr0phic life. Until now, their metabolism has been considered strictly aerobic but we report here on nitrate respiration and denitrification by some of them. CO oxidation is catalyzed by CO dehydrogenase. The CO2 formed is subsequently assimilated in the reductive pentosephosphate cycle. CO dehydrogenase has been found to be a new molybdo-iron-sulfur flavoprotcin containing bactopterin in its molybdenum cofactor [8-11]. Depending on the growth phase, CO dehydrogenase occurs in the cytoplasm or in association with the cytoplasmic membrane of Pseudomonas carboxy. dovorans. The basis of the process is not yet understood. However, we have recently achieved the in vitro removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes and rebinding of the enzyme to depleted membranes [12]. Since the detection of plasmids in carboxydotrophic bacteria [6,13-15], plasmid-cured and deletion mutants were obtained [3,15] the properties of Which suggested the residence of structural and/or regulatory genes of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase on plasmid pHCG3 of Pseudomonas carboxy. dovorans [15]. Recently, CO dehydrogenase structural genes have been located on plasmid pHCG3 [161. This paper presents the results of those studies and includes a brief account on the properties of a newly isolated CO-oxidizing streptomyeete.
3. ISOLATION OF AN APPARENTLY OBLIGATELY CHEMOLITHOAUTOTROPHIC THERMOPHILIC CO- AND H2-OXIDIZING STREtrrOMYCETE Recently we became interested in the microflora of burning charcoal piles of the Bavarian Fichtelgebirge and Frankenwald mountains. Among the many new carboxydotrophic isolates
which were obtained was the thermophilic Streptomyces UBT1 which grew in the form of a characteristic white, dry, hydrophobic pellicle entirely covering the surface of unshaken liquid media. The isolate is briefly characterized as follows: colonies are only very loosely attached to agar surfaces. After sporulation the color of the peilicle turns from white to gray. Vegetative cells form short branching mycelia, and 2-8 spores of oval shape are arranged in a chain. The optimal growth temperature was 65°C; no growth occurred below 40 o C. Growth was best around pH 7-7.5. A peculiarity of Streptomyces UBT1 is the exclusive utilization of CO or H 2 plus CO2 as growth substrates under chemolithoautotrophic conditions. In contrast, gtreptomyces G26 can not utilize H 2 plus CO 2 [17]. An interesting characteristic of (326 is the ability of CO dehydrogenase to reduce low-potential electron acceptors such as methyl- and benzyl viologen in addition to those acceptors u s ~ by the enzymes from other sources [18]. Organic substrates supporting growth of Streptomyces UBT1 under aerobic conditions are not known. The isolate is apparently obligate chemolithotrophic. As known from other carboxydotrophic bacteria [4], the utilization of CO by Streptomyces UBT1 required molybdate.
4. NITRATE REDUCTION A N D DENITRIF1CATION
4.1. Conditions of denitrification Until now, the metabolism of carboxydotrophic bacteria was considered to be strictly aerobic [2,4,5]. On the other hand, calculations of free energy changes of nitrate respiration or denitrifi. cation with CO, H 2 or organic substrates as electron donors did not exclude the existence of these metabolic types [7]. Examination of the currently available carboxydotrophic bacteria revealed the capability of some of them to use oxidized nitrogen compounds as electron acceptors [19]. Generally, denitrifyin$ growth was fairly slow (t d > 20 h). The most rapidly proliferating bacterium was P. carboxydoflava (td = 11 h). The capability of carboxydotrophic bacteria for chemolithoautotrophic denitrification or nitrate
255 respiration is very limited. Only P. carboxydohydrogena grew anaerobically with H 2 plus CO 2
carboxydohydrogena, P. gazotropha and P. compransoris N20 was the final product of dcnitrifica-
as electron donor and carbon source and nitrate as electron acceptor [19]. Again growth was very slow (td=40 h). So far, we have not been able to demonstrate denitrification or nitrate respiration with CO under chemolithoautotrophic conditions. With CO or H 2 as electron donors some carboxydotrophic bacteria, however, performed a reduction of nitrate to nitrite which did not support growth. The nitrite was accumulated in the medium. Such strains were Acinetobacter JC1, Arthrobacter l l / x , P. carboxydohydrogena, P. carboxydovorans OM3, P. gazotropha and P.
tion.
carboxydoflava. 4.2. Kinetics, intermediates and products of denitrification Heterotrophic growth of P. carboxydoflava with ,Jtrate led to the formation of nitrite and N 2 in that order (Fig. 1). NO and N20 were not detected as intermediates. Nitrite was accumulated quantitatively before onset of N 2 evolution. N 2 was formed from nitrate or nitrite, respectively, at the theoretically expected ratio of 1 : 2 (Fig. 1). Denitrification by P. carboxydoflava was insensitive towards 02 since it occurred at same rates in shaken cultures exposed to 20% ( v / v ) 02. With P.
3.2 1.6
"~-o
• •
,
,
/_
g
_
0.8 '~ o.4 o.2 o.1 "7"--'~---°--'~ -'" ~ 40 60 Time (h)
"lO
Fig. 1. Kineticsof denitrificationby P. carboxydoflava.Bacteria were grown in stoppered li-Erlenmeycrflasks containing 11 of mineral medium supplemented with 4 g Na-pyruvate and 15 mmol Na-nitrate. Gases evolvedby the growing culture were sampled in glass cyfindersunder 50 mM aqueous Ba(OH)2, pH 11, in order to trap CO2. A436, nitrate and nitrite were dctermincd from 1-ml samples. Symbols: Nitrate. O; nitrite, D; Na, I1; A4~s 0 (from [19]).
4.3. Effect of CO on denitrification As already mentioned, CO ui=l not support denitrifying g]awth of the c;- ;~oxydotrophic bacteria examined. CO at a conc: ltration of 50% ( v / v ) increased the doubling time of heterotrophicaily denitrifying P. carboxydoflava from 11 to 28 h but had no effect on the growth yields. CO had also no effect on the activities of nitrate and nitrite reductases of carboxydotrophic bacteria. However, it was an inhibitor of N20 reductase. Consequently, in the presence of CO, N20 appeared as an intermediate in resting cell suspensions of denitrifying P. carboxydoflava, Pa. denitrificans and P. stutzeri. Similar observations have been made by others employing noncarboxydotrophic denitrifying bacteria [20,21].
5. R E M O V A L O F CO D E H Y D R O G E N A S E F R O M PSEUDOMONAS CARBOXYDOVOR/INS CYTOPLASMIC M E M B R A N E S A N D R E B I N D I N G TO D E P L E T E D M E M B R A N E S Depending on the growth phase, CO dehydrogenase in P. carboxydovorans can occur in the cytoplasm or attached to the inner aspect of the cytoplasmic membrane [7,22,23]. The enzyme was shown to associate with cytoplasmic membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low-ionicstrength buffer [12]. The tightly bound pool of the enzyme could be differentially soluhilized under conditio~ls leaving the electron transport system intact employing the nondenaturing zwitteronic detergent 3-[(3-cholamidopropyl)]dimethylammonio)l-propanesulfonate acid (CHAPS) and the nonionic detergent dodecyi ~-D-maltoside [24]. In vitro reconstitution of depleted membranes with CO dehydrogenas¢ led to binding of the enzyme and to reactivation of respiratory activities with CO (Table 1). Reconstitution required cations in order of effectiveness trivalent: (Cr 3+, La 3+) > divalent (Mg e+, Mn 2+) > monovalent
256
Table 1 Aff'mityof cytoplasmicmembranesfrom lithotrophicallyand heterotrophicallygrown Pseudomonascarboxydovoransfor CO dehydrogenase Growth substrate CO H2 +CO2 Pymvate/NB
Reconstitution activitya CO--, MB b
CO-, O, c
505 (ND) 36(76) 29(51)
12.3( N D ) 0 (0) 0 (0.3)
c o dehydrogenasewas removedfrom cytoplasmicmembranes by solubilizafionwith CHAPS as described [24.25]. Reconstituted activitiesof untreated membranesare given in brackets. a Reconstltutionwas in the presenceof 15 mM LaC!3 using cytoplasmic fractions of CO-grown Pseudomonas carboxydovorans. b Activitiesare in nmol methyleneblue reduced per rain and m8 protein. c Activitiesare in nmol O2 per rain and mg protein. NB, nutrientbroth; ND, not determined.(From [24].)
(Li+). The reconstitution of CO dehydrogenase onto depleted cytoplasmic membranes was specific for CO-grown P. carboxydovorans. Membranes from bacteria grown with H 2 plus CO2 or organic substrates were devoid of binding sites for CO dehydrogenase [12,24].
6. CO DEHYDROGENASE GENASE ACTIVITY
HAS HYDRO-
That CO dehydrogenase has hydrogenase activi t y was obvious from the ability of the electrophoreticaUy homogeneous enzyme to catalyze the oxidation of H 2 [12,24]: H 2 + A(ox)---~AH2(red). The two electrons and protons formed can be transferred to oxidized artificial 2-electron acceptors (A). The electron acceptors used are similar to those, of -'--k~cCO-oxidation reaction. Growth of P. carboxydovorans with H 2 requires the induction of a separate membrane-bound uptake hydrogenase, since the hydrogenase activity of CO dehydrogenase is incapable of supporting growth under these conditions. This agrees with the affinities of hydrogenase and CO dehydrogenase for H2, which were 77 ~tM and only 5 mM, respectively [12,24].
The low K M of CO dehydrogenase for H 2 and the absence of Ni would suggest a role in the formation rather than the uptake of H 2. That CO dehydrogenase is capable of using H 2 explains why Aicaligenes carboxydus, which does not grow with H 2, has hydrogenase activity [4].
7. CYTOCHROME b561 IS THE IN VIVO ELECTRON ACCEPTOR O F CO DEHYDROGENASE Solubilization of cytoplasmic membranes from CO-grown P. carboxydovorans with dodecyl ~-Vmaltoside yielded a complex consisting of CO dehydrogenase and cytochrome bs6~ at a 1 : 2 molar ratio. The complex could be isolated and purified. Cytochrome b561, as dissociated from the complex, was a monomeric 55 kDa protein containing 0.8 heme b per purified cytochrome molecule. The heme content of the complex of CO dehydrogenase and cytochrome was 1.9 to 2.2 per molecule of CO dehydrogenase. When the complex of CO dehydrogenase and cytochrome bs6~ was incubated with CO, reduction of the cytochrome could be observed spectroscopically. Cytochrome b561 was also reduced by CO dehydrogenase in th e presence of CO in assays containing the purified proteins (Fig. 2). Addition of ultrafiltrate containing flavins and an unknown pterin stimulated the reduction of cytochrome bs61, presumably by complementation of cofactor-deficient CO dehydrogenase molecules. The components of the ultrafiltrate itself were incapable of accepting electrons from CO dehydrogenase. Hydrogenase could not transfer electrons to cytochrome b561, and ubiquinone is not an electron acceptor of CO dehydrogenase. On the basis of these result cytochrome b561 was considered the in vivo electron acceptor of CO dehydrogenase [24].
8. GENETICS O F CARBOXYDOTROPHIC CO OXIDATION
8.1. Plasmids Of the currently available carboxydotrophic bacteria (about 21 species and strains) eight
257
3°
1
s~
o
cured mutants of P. carboxydovorans OM5 (OM512, OM5-17, OM5-23, OM5-27, OM5-31, OM5-32, OM5-43, OM5-53, OM5-61) obtained so far, as well as the deletion mutant OM5-29, were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. Consequently, they lost the ability to grow chemolithotrophicaily with CO or H 2 plus CO 2. Mutant OM5-24 carried a 15-kb deletion on plasmid pHCG3 and retained the ability to grow with CO, but could not utilize H 2 plus CO e and was devoid of hydrogenase. It was thus concluded that structural a n d / o r regulatory genes encoding CO dehydrogenase, hydrogenase or ribulose bisphosphate carboxylase would reside on plasmid pHCG3 [15].
8.2. Location of CO dehydrogenase structural genes CO dehydrogenases are composed of 17-(S), 30-(M) and 87-(L) kDa subunits. The enzymes were purified from P. carboxydovorans OM5, P.
carboxydohydrogena and P. carboxydoflava, their
soo
5so
600
WAVELENGTH (rim)
Fig. 2. Reduction of cytochrome/1561 by CO dehydrogenase. Reactions were followed by redox difference spectroscopy at room temperature in assays containing the purified proteins. For this purpose serum-stopperedcuvettescontaining0.4 ml of 20 mM Hepes buffer, pH 7.0. were flushed for 5 rain with pure CO or air, respectively. After the addition of CO dehydrogenase (29/tg of protein) and 10 ~tl of ultrafiitrate (see text) reactions were initiated by injecting210 #g of cytochromeb56] into both cuvettes. Reduction of the cytochrome, as obvious from the absorption increase in the a-resion, was followed with time. Spectrum D shows the dithionite-reduced minus air-oxidized difference spectrum of cytochrome b~l in the same assay.
harbored plasmids between 45 and 558 kb in size (Table 2). The plasmids p H C G I - a and p H C G I - b of Alcaligenes carboxydus were megaplasmids (Table 2). Restriction digest patterns of plasmids from different carboxydotrnphic bacteria were dissimilar [15]. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of P. carboxydovorans were very much the same. All
subunits prepared and analyzed for N-terminal amino acid sequences [16]. The N-terminal sequences of corresponding subunits from the different bacteria showed distinct homologies. Dot blot hybridization employing synthetic oligudeoxynucleotides as heterologous probes, the sequences of which were derived from the N-terminal amino acid sequences of the S-subunit of P. carboxydovorans OM5 and the M-subunit of P. carboxydohydrogena, and D N A (total D N A and purified plasmid D N A ) of the plasmid-containing CO-oxidizing carboxydotrophs, indicated that all genes encoding these subunits reside on plasmids (Table 2). That P. carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing D N A from the cured mutant strain OM5-12 (Table 2). CO dehydrogenase struc~aral genes could be identified on the chromosome of all plasmid-free carboxydotrophic bacteria examined. There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on a plasmid. It has thus been generalized that CO dehydrogenase structural genes reside on plasmids in plasmid-harboring carboxydotrophs [161.
258 Table 2 Plasmids in earboxydotrophic bacteria, presence and lu:atinn of structural genes of chemolithoautotrophic key enzymes Strain
Plasmid designation
Size (kb)
Locutionof CO dehydro- Presence of structural genes genase structural genes a Rubisco b PRK b HydroPlasChromogenase b mid some
pHCG4 pHCG5-a pHCG5-b pHCG3 AlpHCG3 A2pHCG3 -
128 158 128 128 113 74 -
+ +
Pseudomonascarboxydoflava
pHCG2-a pHCG2-b
110 45
Alcaligenescarboxydus
pHCGI-a pHCGI-b pHCG1-c
558 428 129
Azomonas B1
pHCG6-a pHCO6-b
254 200
Aeomonas C2
pHCG7
AzotobacterAl
pHCG8
Actinomyces UBTI; Arthrobactcrl l / x Bacillusschlegelii MA48; P. carboxydovoransOM3; P. carboxydohydrogena Acinetobacter JC1; Bacillus OMT2; BacillusschlegeliiOMT1, OMT4, OMTT; P. compransoris;P. gacotropha; P. thermocarboxydovorans;strain GKS.
Pseudomonascarboxydovorans OM2 OM4 OM5 OM5-24 OM5-29 OM5-12
ND c ND
+ +
+ +
+ +
(+ )d (+ )
+ +
+ +
(+ ) (+)
+
+
(+ )
+
ND
+
+
(+ )
+
ND
+
+
+
+
ND
+
+
+
3.5
ND
ND
ND
ND
ND
3.5
ND
ND
ND
ND
ND
no plasnfid
-
-
+
+
+
(+)
no plasmid
-
ND
ND
ND
ND
ND
Results are as according to [6,12,14,15]. b DOt blot hybridisation employing oligonucleotide probes derived from the M- or S-subunits of CO dehydrogenase. Genes encoding ribulose bisphosphate carboxylase (Rubisco) were identified employing a l.l-kb BamH1 fragment containing the cfxLp gene 0arge subunit) of AIcafigeneseutrophus H16. Phosphoribulokinase (PRK) genes were identified by means of a 1-kb Kpnl/Pstl fragment carrying major parts of the cfxPp gene of AIcafigenes eutrophus H16. Both probes were kindly donated by B. Bowien (University of Ggtfingen). Particulate hydrogenase genes were identified employing two different probes originating from Alcaligenes eutrophus H16. One was a 2-kb BamHl fragment carrying the hoxK gene (small subunit). The other was a 2.25-kb XhoI fragment carryL'nghoxLO (large subunit) and parts of hoxLK. The latter two probes were kindly made available by B. Friedrich (Free University, Berlin). All probes were used for dot blot hybridisation as detailed by Kraut et ai. [16]. c ND, not determined. o Brackets indicate hybridization under non-stringent conditions.
8.3. Presence of structural genes encoding chemolithoautotrophic key enzymes other than CO dehydrogenase E m p l o y i n g heterologous gene p r o b e s encoding ribulose b i s p h o s p h a t e c a r b o x y l a s e (Rubisco), phosphoribulokinase (PRK) and membrane-bound
h y d r o g e n a s e we have e x a m i n e d the occurrence o f these genes in c a r b o x y d o t r o p h i c bacteria (Table 2). R u b i s c o a n d P R K structural genes could be identified u n d e r stringent conditions (hybridization a n d w a s h e s at 5 5 ° C ) in all strains of c a r b o x y d o t r o p h i c bacteria m e n t i o n e d in Table 2.
259 The results obtained with the hydrogenase probes revealed the presence of a gene similar to hoxG in drthrobacter l l / x , Azomonas B1 and Alcaligenes carboxydus (Table 2). Although all other carboxydotrophic bacteria examined showed some homology with hoxG when hybridizations were at 40 ° C, further experiments are required to reach a final conclusion on the presence of the gene in these carboxydotrophs (Table 2). The hoxK probe was positive with the control D N A (Alcaligenes eutrophus H16) but did not hybridize with any bacterial D N A mentioned in Table 2. Southern blots employing total D N A from P. carboxydovorans OM5 wild type or its plasmid-cured mutant OM5-12 revealed hybridization with cfxLp and cfxPp gene probes. This refers to duplicated genes of Rubisco and P R K located on the plasmid pHCG3 and the chromosome. The results of Table 2 show a considerable degree of homology in structural genes encoding the key enzymes of chemolithoautotrophic metabolism in carboxydotrophic (CO dehydrogenase, Rubisco, PRK, membrane-bound hydrogenase) and Knallgas bacteria (Rubisco, PRK, membranebound hydrogcnase), Thus, the possibility of parallel evolution from common ancestral proteins or the spread of genes or plasmids encoding enzymes involved in chemolithoautotrophic metabolism is a plausible explanation for this homology.
9. C O N C L U D I N G R E M A R K S One important conclusion of our study is that
P. carboxydovorans OM5 and other carboxydotrophic bacteria are amenable to molecular biology. We have provided evidence that the structural genes encoding the M- or S-subunits of P. carboxydovorans CO dehydrogenase reside on plasmid pHCG3 exclusively. The plasmid also carries genes coding for ribulose bisphosphat¢ carboxylase, phosphoribulokinase and presumably hydrogenase. It now seems likely that we have established the basis for an extensive molecular analysis of P. carboxydovorans and other carboxydotrophic bacteria.
ACKNOWLEDGEMENTS We are grateful to the Deutsche Forschungsgesellschaft and the Fonds der Chemischen Industrie for financial support.
N O T E A D D E D IN P R O O F The structure of the organic component of the C O d e h y d r o g e n a s e m o l y b d e n u m cofactor (bactopterin) has been identified as molybdopterin cytosine dinucleotide (MCD) (Johnson, J.L., Rajagopalan, K.V. and Meyer, O. (1990) Isolation and characterisation of a second molybdopterin dinucleotide: molybdopterin cytosine nucleotide. Arch. 8iochem. Biophys., in press).
REFERENCES [I] Meyer, O. (1988) Biology and biotechnology of aerobic carbon monoxide-oxidizing bacteria, in Biotechnology Focus 1 (Finn, R.K. et al., Eds.), pp. 3-51 Hanser. Munich. [2] Meyer, O. (1989) Aerobic carbon monoxide-oxidizing bacteria, in Autotrophic Bacteria (SchlegeL H.G. and Bowien, B., Eds.), pp. 331-350. Springer-Verlag,Berlin. [3] Meyer, O. (1985) Metabolism of aerobic carbon monoxide-utiliziog bacteria, in Microbial Gas MetabolismMechanistic, Metabolic and Biotechnological Aspects (Poole, R.K. and Dow, C.S., Eds.), pp. 131-151. Academic Press, London. [4] Meyer, O. and Scblegel, H.G. (1983) Biologyof aerobic carbon monoxide-oxidizingbacteria. Annu. Rev. Microbiol. 37, 277-310. [5] Meyer, O., Jacobitz, S. and Krtiger, B. (1986) Biochemistry and physiology of aerobic carbon monoxide-oxidizing bacteria. FEMS MicrobioLRev. 39,161-179. [6] Park, Y.L and Hegeman, G.D. (1984) The oxidation of carbon monoxide by bacteria, in Microbial Chemoautotrophy (Strohi, W.R. and Tuovinen, O.H., Eds.), pp. 211-218. Ohio State University,Columbus. [7] Meyer, O. and Rohde, M. (1984) Enzymologyand bioenergetics of carbon monoxide-oxidizingbacteria, in Microbial Growth on C 1 Compounds (Crawford, R.L. and Hanson, R.S., Eds.), pp. 26-33. American Society for Microbiology,Washington. [8] Meyer, O. (1982~ Chemical and spectral properties of carbon monoxide: methyleneblue oxidoreductase.J. Biol. Chem. 257,1333-341. [9] Krfiger, B. and Meyer, O. (1986) The pterin of carbon monoxide dehydrogenase from Pseudomonascarboxydo~lava.Eur. J. Biochem.157,121-128.
26O [10] Krfiser, B. and Meyer, O. (1987) Structural elements of bactopterln from Pseudomonas carboxydoflava carbon monoxide dehydrogenase. Biochim. Biophys. Acta 912, 357-364. [11] Meyer, O. and Rajagopalan, K.V. (1984) Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. J. Baateriol. 157, 843-848. [12] Jacobitz. S. and Meyer, O. (1989) Removal of CO dehydrogenase from Pseudomonas carboxydovoruns cytoplasmic membranes, rebinding to depleted membranes, and restoration of respiratory activities. J. Bacteriol. 171, 6294-6299. ~3] Gerstenberg, C., Friedrich, B. and Schlegei, H.G. (1982), Physical evidence for plasmids in autotrophic, especially hydrogen-oxidizing bacteria. Arch. Microbiol. 133, 90~96. [14] Kwon, M.O. and Kim, Y.M. (1985) Relationship between carbon monoxide dehydrogenase and a small plasmid of Pseudomonas carbox.vdovorans. FEMS Micrebiol. Lett. 29, 155-159. [15] graut, M. and Meyer, O. (1988) Plasmids in carboxydotrophic bacteria: physical and restriction analysis. Arch. Microbiol. 149, 540-546. [16] K.rant, M., Hugendieck, I., Herwi8, S. and Meyer, O. (1989) Homology and distribution of CO dehydrogenese structural genes in carboxydotrophic bacteria. Arch. Microbiol. 152, 335-341. [17] Williaras, E. and Colby, J. (1936) Biotcclmological applications of carboxydotrophic bacteria. Microbiol. Sci. 3, 149-153.
[18] 1~11, J.M., Williams, E. and Colby, J. (1985) Carbon monoxide oxidoreductese from thermophilic carboxydobacteria, in Microbial Gas Metabolism (Poole, R.K. and Dow, C.S., Eds.), pp. 153-160. Academic Press, London [19] Frunzke, K. and Meyer, O. (1990) Nitrate respiration, denitrification and utilization of nitrogen sources by aerobic carbon monoxide-oxidizingbacteria. Arch. Microbiol. 154,168-174. [20] Matsubara, T. and Mori, T. (1968) Studies on denitrification IX: Nitrous oxide, its production and reduction to nitrogen. J. Biocbem. 64, 863-871. [21] Kxistjansson, J.K. and Hollocher, T.C. (1980) First practical essay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization. J. Biol. Chem. 255, 704-707. [22] Rohde, M., Mayer, F. and Meyer, O. (1984) lmmunocytochemical localization of carbon monoxide oxidas© in Pseudomonas carboxydovorans. The enzyme is attached to the inner aspect of the cytoplasmic membrane. J. Biol. Chem. 259,14788-14792. [23] Rohde, M., Mayer, F., Jacobitz, S. and Meyer, O. (1985) Attachment of CO dehydrogenas¢ to the cytoplasmic membrane is limiting the respiratory rate of Pseudomonas carboxydovorans. FEMS Microbiol. Lett. 28,141-144. [24] Jacobitz, S. (1989) Isolierung und Charakterisierung von Cytochrom bs61 aus Pseudomonas carboxydovorans und Identifizicrung als physiologischem Elektronenakzeptor yon CO-Dehydroganase. Ph.D. thesis, University of Bayreuth.
253
FEMSRE00199
Utilization of carbon monoxide by aerobes: recent advances Ortwin Meyer, Kurt Frunzke, Dilip Gadkari, Susanne Jacobitz, Iris Hugendieck and Maria Kraut Lehrstuhlf~r Mikrobiologie, Universit&Bayreuth, Bayreuth, F..R.G. Key words: Carboxydotrophic bacteria; Genetics; Plasmids; CO dehydrogenase; Hydrogenase; CO 2 fixation; Denitrification; Nitrate reduction; Cytochrome 1. SUMMARY The fist of carboxydotrophic bacteria is constantly growing and we have found that burning charcoal piles harbor an especially rich CO-oxidizing microflora. The newly isolated Streptomyces thermoautotrophicus UBT1 is particularly interesting as it is thermophilic, capable of chemolithoautotrophic growth with CO or H~ plus CO 2 and incapable of using organic substrates. Molybdenum is essential for CO-autotrophic growth. Some species of carboxydotrophic bacteria can denitrify under heterotrophic conditions yielding N 2 (e.g. Pseudomonas carboxydoflava) or N20 (e.g. Pseudomonas carboxydohydrogena); others perform nitrate respiration (e.g. Azomonas B1). P. carboxydohydrogena could grow at the expense of H 2 plus CO 2 using nitrate as electron acceptor. In intact cells of Pseudomonas carboxydovorans, CO dehydrogenase has the ability of dissociating from and rebinding to the cytoplasmic membrane. That process can be simulated in vitro by removing CO dehydrogenase from cytoplasmic membranes and rebinding it to depleted membranes. Reconstitution ¢~f the enzyme onto depleted membranes requiring di- or trivalent cations, was
specific for membranes from CO-grown bacteria and led to reactivation of respiratory activities with CO. A complex consisting of 1 molecule of CO dehydrogenase and 2 molecules of cytochrome b561 could be isolated from cytoplasmic membranes of P. carboxydovorans sohibilized with dodecyl ~-D-maltoside. Within the complex as wel! as h~ a~ays containing purified CO dehydrogenase and cytochrome b561 the latter could serve as an electron acceptor. CO dehydrogenase had hydrogenase activity, and its KM of only 5 mM H 2 suggested a role in the formation of H2. P. carbox.vdovorans OM5 contaL~,~ the 128-kilobase pairs (kb) plasmid pHCG3 which is essential for CO- and H2-fithoautoh-ophic growth. Evidence for the existence of pHCG3-coded structural genes of CO dehydrogenase waz obtained from dot blot hybridizations employing synthetic ofigodeoxynucleotides as heterologous probes for the detection of the S- and M-subunit genes. Employing appropriate probe genes encoding membrane-bound hydrogenase, ribulose bisphosphate carboxylase and phosphoribulokinase were also identified on plasmid pHCG3.
2. INTRODUCTION Correspondence to: O. Meyer, Lehrstuhl ffir Mikrobiologie, UniversitatBayreuth,Universiflttsstrasse30, D-8580 Bayreuth, F.R.G.
CO-oxidizing (carboxydotrophic) bacteria are characterized by the utilization of CO as a sole
0168-6445/90/$03.50 © 1990 Federationof European MicrobiologicalSocieties
254
source of energy and carbon. Their microbiology, physiology and biochemistry have been dealt with in numerous recent reviews [1-7]. With only very few exceptions, carboxydotrophs can use H 2 plus CO2 as well, indicating the presence of two different capacities for chemolithoautotr0phic life. Until now, their metabolism has been considered strictly aerobic but we report here on nitrate respiration and denitrification by some of them. CO oxidation is catalyzed by CO dehydrogenase. The CO2 formed is subsequently assimilated in the reductive pentosephosphate cycle. CO dehydrogenase has been found to be a new molybdo-iron-sulfur flavoprotcin containing bactopterin in its molybdenum cofactor [8-11]. Depending on the growth phase, CO dehydrogenase occurs in the cytoplasm or in association with the cytoplasmic membrane of Pseudomonas carboxy. dovorans. The basis of the process is not yet understood. However, we have recently achieved the in vitro removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes and rebinding of the enzyme to depleted membranes [12]. Since the detection of plasmids in carboxydotrophic bacteria [6,13-15], plasmid-cured and deletion mutants were obtained [3,15] the properties of Which suggested the residence of structural and/or regulatory genes of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase on plasmid pHCG3 of Pseudomonas carboxy. dovorans [15]. Recently, CO dehydrogenase structural genes have been located on plasmid pHCG3 [161. This paper presents the results of those studies and includes a brief account on the properties of a newly isolated CO-oxidizing streptomyeete.
3. ISOLATION OF AN APPARENTLY OBLIGATELY CHEMOLITHOAUTOTROPHIC THERMOPHILIC CO- AND H2-OXIDIZING STREtrrOMYCETE Recently we became interested in the microflora of burning charcoal piles of the Bavarian Fichtelgebirge and Frankenwald mountains. Among the many new carboxydotrophic isolates
which were obtained was the thermophilic Streptomyces UBT1 which grew in the form of a characteristic white, dry, hydrophobic pellicle entirely covering the surface of unshaken liquid media. The isolate is briefly characterized as follows: colonies are only very loosely attached to agar surfaces. After sporulation the color of the peilicle turns from white to gray. Vegetative cells form short branching mycelia, and 2-8 spores of oval shape are arranged in a chain. The optimal growth temperature was 65°C; no growth occurred below 40 o C. Growth was best around pH 7-7.5. A peculiarity of Streptomyces UBT1 is the exclusive utilization of CO or H 2 plus CO2 as growth substrates under chemolithoautotrophic conditions. In contrast, gtreptomyces G26 can not utilize H 2 plus CO 2 [17]. An interesting characteristic of (326 is the ability of CO dehydrogenase to reduce low-potential electron acceptors such as methyl- and benzyl viologen in addition to those acceptors u s ~ by the enzymes from other sources [18]. Organic substrates supporting growth of Streptomyces UBT1 under aerobic conditions are not known. The isolate is apparently obligate chemolithotrophic. As known from other carboxydotrophic bacteria [4], the utilization of CO by Streptomyces UBT1 required molybdate.
4. NITRATE REDUCTION A N D DENITRIF1CATION
4.1. Conditions of denitrification Until now, the metabolism of carboxydotrophic bacteria was considered to be strictly aerobic [2,4,5]. On the other hand, calculations of free energy changes of nitrate respiration or denitrifi. cation with CO, H 2 or organic substrates as electron donors did not exclude the existence of these metabolic types [7]. Examination of the currently available carboxydotrophic bacteria revealed the capability of some of them to use oxidized nitrogen compounds as electron acceptors [19]. Generally, denitrifyin$ growth was fairly slow (t d > 20 h). The most rapidly proliferating bacterium was P. carboxydoflava (td = 11 h). The capability of carboxydotrophic bacteria for chemolithoautotrophic denitrification or nitrate
255 respiration is very limited. Only P. carboxydohydrogena grew anaerobically with H 2 plus CO 2
carboxydohydrogena, P. gazotropha and P. compransoris N20 was the final product of dcnitrifica-
as electron donor and carbon source and nitrate as electron acceptor [19]. Again growth was very slow (td=40 h). So far, we have not been able to demonstrate denitrification or nitrate respiration with CO under chemolithoautotrophic conditions. With CO or H 2 as electron donors some carboxydotrophic bacteria, however, performed a reduction of nitrate to nitrite which did not support growth. The nitrite was accumulated in the medium. Such strains were Acinetobacter JC1, Arthrobacter l l / x , P. carboxydohydrogena, P. carboxydovorans OM3, P. gazotropha and P.
tion.
carboxydoflava. 4.2. Kinetics, intermediates and products of denitrification Heterotrophic growth of P. carboxydoflava with ,Jtrate led to the formation of nitrite and N 2 in that order (Fig. 1). NO and N20 were not detected as intermediates. Nitrite was accumulated quantitatively before onset of N 2 evolution. N 2 was formed from nitrate or nitrite, respectively, at the theoretically expected ratio of 1 : 2 (Fig. 1). Denitrification by P. carboxydoflava was insensitive towards 02 since it occurred at same rates in shaken cultures exposed to 20% ( v / v ) 02. With P.
3.2 1.6
"~-o
• •
,
,
/_
g
_
0.8 '~ o.4 o.2 o.1 "7"--'~---°--'~ -'" ~ 40 60 Time (h)
"lO
Fig. 1. Kineticsof denitrificationby P. carboxydoflava.Bacteria were grown in stoppered li-Erlenmeycrflasks containing 11 of mineral medium supplemented with 4 g Na-pyruvate and 15 mmol Na-nitrate. Gases evolvedby the growing culture were sampled in glass cyfindersunder 50 mM aqueous Ba(OH)2, pH 11, in order to trap CO2. A436, nitrate and nitrite were dctermincd from 1-ml samples. Symbols: Nitrate. O; nitrite, D; Na, I1; A4~s 0 (from [19]).
4.3. Effect of CO on denitrification As already mentioned, CO ui=l not support denitrifying g]awth of the c;- ;~oxydotrophic bacteria examined. CO at a conc: ltration of 50% ( v / v ) increased the doubling time of heterotrophicaily denitrifying P. carboxydoflava from 11 to 28 h but had no effect on the growth yields. CO had also no effect on the activities of nitrate and nitrite reductases of carboxydotrophic bacteria. However, it was an inhibitor of N20 reductase. Consequently, in the presence of CO, N20 appeared as an intermediate in resting cell suspensions of denitrifying P. carboxydoflava, Pa. denitrificans and P. stutzeri. Similar observations have been made by others employing noncarboxydotrophic denitrifying bacteria [20,21].
5. R E M O V A L O F CO D E H Y D R O G E N A S E F R O M PSEUDOMONAS CARBOXYDOVOR/INS CYTOPLASMIC M E M B R A N E S A N D R E B I N D I N G TO D E P L E T E D M E M B R A N E S Depending on the growth phase, CO dehydrogenase in P. carboxydovorans can occur in the cytoplasm or attached to the inner aspect of the cytoplasmic membrane [7,22,23]. The enzyme was shown to associate with cytoplasmic membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low-ionicstrength buffer [12]. The tightly bound pool of the enzyme could be differentially soluhilized under conditio~ls leaving the electron transport system intact employing the nondenaturing zwitteronic detergent 3-[(3-cholamidopropyl)]dimethylammonio)l-propanesulfonate acid (CHAPS) and the nonionic detergent dodecyi ~-D-maltoside [24]. In vitro reconstitution of depleted membranes with CO dehydrogenas¢ led to binding of the enzyme and to reactivation of respiratory activities with CO (Table 1). Reconstitution required cations in order of effectiveness trivalent: (Cr 3+, La 3+) > divalent (Mg e+, Mn 2+) > monovalent
256
Table 1 Aff'mityof cytoplasmicmembranesfrom lithotrophicallyand heterotrophicallygrown Pseudomonascarboxydovoransfor CO dehydrogenase Growth substrate CO H2 +CO2 Pymvate/NB
Reconstitution activitya CO--, MB b
CO-, O, c
505 (ND) 36(76) 29(51)
12.3( N D ) 0 (0) 0 (0.3)
c o dehydrogenasewas removedfrom cytoplasmicmembranes by solubilizafionwith CHAPS as described [24.25]. Reconstituted activitiesof untreated membranesare given in brackets. a Reconstltutionwas in the presenceof 15 mM LaC!3 using cytoplasmic fractions of CO-grown Pseudomonas carboxydovorans. b Activitiesare in nmol methyleneblue reduced per rain and m8 protein. c Activitiesare in nmol O2 per rain and mg protein. NB, nutrientbroth; ND, not determined.(From [24].)
(Li+). The reconstitution of CO dehydrogenase onto depleted cytoplasmic membranes was specific for CO-grown P. carboxydovorans. Membranes from bacteria grown with H 2 plus CO2 or organic substrates were devoid of binding sites for CO dehydrogenase [12,24].
6. CO DEHYDROGENASE GENASE ACTIVITY
HAS HYDRO-
That CO dehydrogenase has hydrogenase activi t y was obvious from the ability of the electrophoreticaUy homogeneous enzyme to catalyze the oxidation of H 2 [12,24]: H 2 + A(ox)---~AH2(red). The two electrons and protons formed can be transferred to oxidized artificial 2-electron acceptors (A). The electron acceptors used are similar to those, of -'--k~cCO-oxidation reaction. Growth of P. carboxydovorans with H 2 requires the induction of a separate membrane-bound uptake hydrogenase, since the hydrogenase activity of CO dehydrogenase is incapable of supporting growth under these conditions. This agrees with the affinities of hydrogenase and CO dehydrogenase for H2, which were 77 ~tM and only 5 mM, respectively [12,24].
The low K M of CO dehydrogenase for H 2 and the absence of Ni would suggest a role in the formation rather than the uptake of H 2. That CO dehydrogenase is capable of using H 2 explains why Aicaligenes carboxydus, which does not grow with H 2, has hydrogenase activity [4].
7. CYTOCHROME b561 IS THE IN VIVO ELECTRON ACCEPTOR O F CO DEHYDROGENASE Solubilization of cytoplasmic membranes from CO-grown P. carboxydovorans with dodecyl ~-Vmaltoside yielded a complex consisting of CO dehydrogenase and cytochrome bs6~ at a 1 : 2 molar ratio. The complex could be isolated and purified. Cytochrome b561, as dissociated from the complex, was a monomeric 55 kDa protein containing 0.8 heme b per purified cytochrome molecule. The heme content of the complex of CO dehydrogenase and cytochrome was 1.9 to 2.2 per molecule of CO dehydrogenase. When the complex of CO dehydrogenase and cytochrome bs6~ was incubated with CO, reduction of the cytochrome could be observed spectroscopically. Cytochrome b561 was also reduced by CO dehydrogenase in th e presence of CO in assays containing the purified proteins (Fig. 2). Addition of ultrafiltrate containing flavins and an unknown pterin stimulated the reduction of cytochrome bs61, presumably by complementation of cofactor-deficient CO dehydrogenase molecules. The components of the ultrafiltrate itself were incapable of accepting electrons from CO dehydrogenase. Hydrogenase could not transfer electrons to cytochrome b561, and ubiquinone is not an electron acceptor of CO dehydrogenase. On the basis of these result cytochrome b561 was considered the in vivo electron acceptor of CO dehydrogenase [24].
8. GENETICS O F CARBOXYDOTROPHIC CO OXIDATION
8.1. Plasmids Of the currently available carboxydotrophic bacteria (about 21 species and strains) eight
257
3°
1
s~
o
cured mutants of P. carboxydovorans OM5 (OM512, OM5-17, OM5-23, OM5-27, OM5-31, OM5-32, OM5-43, OM5-53, OM5-61) obtained so far, as well as the deletion mutant OM5-29, were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. Consequently, they lost the ability to grow chemolithotrophicaily with CO or H 2 plus CO 2. Mutant OM5-24 carried a 15-kb deletion on plasmid pHCG3 and retained the ability to grow with CO, but could not utilize H 2 plus CO e and was devoid of hydrogenase. It was thus concluded that structural a n d / o r regulatory genes encoding CO dehydrogenase, hydrogenase or ribulose bisphosphate carboxylase would reside on plasmid pHCG3 [15].
8.2. Location of CO dehydrogenase structural genes CO dehydrogenases are composed of 17-(S), 30-(M) and 87-(L) kDa subunits. The enzymes were purified from P. carboxydovorans OM5, P.
carboxydohydrogena and P. carboxydoflava, their
soo
5so
600
WAVELENGTH (rim)
Fig. 2. Reduction of cytochrome/1561 by CO dehydrogenase. Reactions were followed by redox difference spectroscopy at room temperature in assays containing the purified proteins. For this purpose serum-stopperedcuvettescontaining0.4 ml of 20 mM Hepes buffer, pH 7.0. were flushed for 5 rain with pure CO or air, respectively. After the addition of CO dehydrogenase (29/tg of protein) and 10 ~tl of ultrafiitrate (see text) reactions were initiated by injecting210 #g of cytochromeb56] into both cuvettes. Reduction of the cytochrome, as obvious from the absorption increase in the a-resion, was followed with time. Spectrum D shows the dithionite-reduced minus air-oxidized difference spectrum of cytochrome b~l in the same assay.
harbored plasmids between 45 and 558 kb in size (Table 2). The plasmids p H C G I - a and p H C G I - b of Alcaligenes carboxydus were megaplasmids (Table 2). Restriction digest patterns of plasmids from different carboxydotrnphic bacteria were dissimilar [15]. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of P. carboxydovorans were very much the same. All
subunits prepared and analyzed for N-terminal amino acid sequences [16]. The N-terminal sequences of corresponding subunits from the different bacteria showed distinct homologies. Dot blot hybridization employing synthetic oligudeoxynucleotides as heterologous probes, the sequences of which were derived from the N-terminal amino acid sequences of the S-subunit of P. carboxydovorans OM5 and the M-subunit of P. carboxydohydrogena, and D N A (total D N A and purified plasmid D N A ) of the plasmid-containing CO-oxidizing carboxydotrophs, indicated that all genes encoding these subunits reside on plasmids (Table 2). That P. carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing D N A from the cured mutant strain OM5-12 (Table 2). CO dehydrogenase struc~aral genes could be identified on the chromosome of all plasmid-free carboxydotrophic bacteria examined. There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on a plasmid. It has thus been generalized that CO dehydrogenase structural genes reside on plasmids in plasmid-harboring carboxydotrophs [161.
258 Table 2 Plasmids in earboxydotrophic bacteria, presence and lu:atinn of structural genes of chemolithoautotrophic key enzymes Strain
Plasmid designation
Size (kb)
Locutionof CO dehydro- Presence of structural genes genase structural genes a Rubisco b PRK b HydroPlasChromogenase b mid some
pHCG4 pHCG5-a pHCG5-b pHCG3 AlpHCG3 A2pHCG3 -
128 158 128 128 113 74 -
+ +
Pseudomonascarboxydoflava
pHCG2-a pHCG2-b
110 45
Alcaligenescarboxydus
pHCGI-a pHCGI-b pHCG1-c
558 428 129
Azomonas B1
pHCG6-a pHCO6-b
254 200
Aeomonas C2
pHCG7
AzotobacterAl
pHCG8
Actinomyces UBTI; Arthrobactcrl l / x Bacillusschlegelii MA48; P. carboxydovoransOM3; P. carboxydohydrogena Acinetobacter JC1; Bacillus OMT2; BacillusschlegeliiOMT1, OMT4, OMTT; P. compransoris;P. gacotropha; P. thermocarboxydovorans;strain GKS.
Pseudomonascarboxydovorans OM2 OM4 OM5 OM5-24 OM5-29 OM5-12
ND c ND
+ +
+ +
+ +
(+ )d (+ )
+ +
+ +
(+ ) (+)
+
+
(+ )
+
ND
+
+
(+ )
+
ND
+
+
+
+
ND
+
+
+
3.5
ND
ND
ND
ND
ND
3.5
ND
ND
ND
ND
ND
no plasnfid
-
-
+
+
+
(+)
no plasmid
-
ND
ND
ND
ND
ND
Results are as according to [6,12,14,15]. b DOt blot hybridisation employing oligonucleotide probes derived from the M- or S-subunits of CO dehydrogenase. Genes encoding ribulose bisphosphate carboxylase (Rubisco) were identified employing a l.l-kb BamH1 fragment containing the cfxLp gene 0arge subunit) of AIcafigeneseutrophus H16. Phosphoribulokinase (PRK) genes were identified by means of a 1-kb Kpnl/Pstl fragment carrying major parts of the cfxPp gene of AIcafigenes eutrophus H16. Both probes were kindly donated by B. Bowien (University of Ggtfingen). Particulate hydrogenase genes were identified employing two different probes originating from Alcaligenes eutrophus H16. One was a 2-kb BamHl fragment carrying the hoxK gene (small subunit). The other was a 2.25-kb XhoI fragment carryL'nghoxLO (large subunit) and parts of hoxLK. The latter two probes were kindly made available by B. Friedrich (Free University, Berlin). All probes were used for dot blot hybridisation as detailed by Kraut et ai. [16]. c ND, not determined. o Brackets indicate hybridization under non-stringent conditions.
8.3. Presence of structural genes encoding chemolithoautotrophic key enzymes other than CO dehydrogenase E m p l o y i n g heterologous gene p r o b e s encoding ribulose b i s p h o s p h a t e c a r b o x y l a s e (Rubisco), phosphoribulokinase (PRK) and membrane-bound
h y d r o g e n a s e we have e x a m i n e d the occurrence o f these genes in c a r b o x y d o t r o p h i c bacteria (Table 2). R u b i s c o a n d P R K structural genes could be identified u n d e r stringent conditions (hybridization a n d w a s h e s at 5 5 ° C ) in all strains of c a r b o x y d o t r o p h i c bacteria m e n t i o n e d in Table 2.
259 The results obtained with the hydrogenase probes revealed the presence of a gene similar to hoxG in drthrobacter l l / x , Azomonas B1 and Alcaligenes carboxydus (Table 2). Although all other carboxydotrophic bacteria examined showed some homology with hoxG when hybridizations were at 40 ° C, further experiments are required to reach a final conclusion on the presence of the gene in these carboxydotrophs (Table 2). The hoxK probe was positive with the control D N A (Alcaligenes eutrophus H16) but did not hybridize with any bacterial D N A mentioned in Table 2. Southern blots employing total D N A from P. carboxydovorans OM5 wild type or its plasmid-cured mutant OM5-12 revealed hybridization with cfxLp and cfxPp gene probes. This refers to duplicated genes of Rubisco and P R K located on the plasmid pHCG3 and the chromosome. The results of Table 2 show a considerable degree of homology in structural genes encoding the key enzymes of chemolithoautotrophic metabolism in carboxydotrophic (CO dehydrogenase, Rubisco, PRK, membrane-bound hydrogenase) and Knallgas bacteria (Rubisco, PRK, membranebound hydrogcnase), Thus, the possibility of parallel evolution from common ancestral proteins or the spread of genes or plasmids encoding enzymes involved in chemolithoautotrophic metabolism is a plausible explanation for this homology.
9. C O N C L U D I N G R E M A R K S One important conclusion of our study is that
P. carboxydovorans OM5 and other carboxydotrophic bacteria are amenable to molecular biology. We have provided evidence that the structural genes encoding the M- or S-subunits of P. carboxydovorans CO dehydrogenase reside on plasmid pHCG3 exclusively. The plasmid also carries genes coding for ribulose bisphosphat¢ carboxylase, phosphoribulokinase and presumably hydrogenase. It now seems likely that we have established the basis for an extensive molecular analysis of P. carboxydovorans and other carboxydotrophic bacteria.
ACKNOWLEDGEMENTS We are grateful to the Deutsche Forschungsgesellschaft and the Fonds der Chemischen Industrie for financial support.
N O T E A D D E D IN P R O O F The structure of the organic component of the C O d e h y d r o g e n a s e m o l y b d e n u m cofactor (bactopterin) has been identified as molybdopterin cytosine dinucleotide (MCD) (Johnson, J.L., Rajagopalan, K.V. and Meyer, O. (1990) Isolation and characterisation of a second molybdopterin dinucleotide: molybdopterin cytosine nucleotide. Arch. 8iochem. Biophys., in press).
REFERENCES [I] Meyer, O. (1988) Biology and biotechnology of aerobic carbon monoxide-oxidizing bacteria, in Biotechnology Focus 1 (Finn, R.K. et al., Eds.), pp. 3-51 Hanser. Munich. [2] Meyer, O. (1989) Aerobic carbon monoxide-oxidizing bacteria, in Autotrophic Bacteria (SchlegeL H.G. and Bowien, B., Eds.), pp. 331-350. Springer-Verlag,Berlin. [3] Meyer, O. (1985) Metabolism of aerobic carbon monoxide-utiliziog bacteria, in Microbial Gas MetabolismMechanistic, Metabolic and Biotechnological Aspects (Poole, R.K. and Dow, C.S., Eds.), pp. 131-151. Academic Press, London. [4] Meyer, O. and Scblegel, H.G. (1983) Biologyof aerobic carbon monoxide-oxidizingbacteria. Annu. Rev. Microbiol. 37, 277-310. [5] Meyer, O., Jacobitz, S. and Krtiger, B. (1986) Biochemistry and physiology of aerobic carbon monoxide-oxidizing bacteria. FEMS MicrobioLRev. 39,161-179. [6] Park, Y.L and Hegeman, G.D. (1984) The oxidation of carbon monoxide by bacteria, in Microbial Chemoautotrophy (Strohi, W.R. and Tuovinen, O.H., Eds.), pp. 211-218. Ohio State University,Columbus. [7] Meyer, O. and Rohde, M. (1984) Enzymologyand bioenergetics of carbon monoxide-oxidizingbacteria, in Microbial Growth on C 1 Compounds (Crawford, R.L. and Hanson, R.S., Eds.), pp. 26-33. American Society for Microbiology,Washington. [8] Meyer, O. (1982~ Chemical and spectral properties of carbon monoxide: methyleneblue oxidoreductase.J. Biol. Chem. 257,1333-341. [9] Krfiger, B. and Meyer, O. (1986) The pterin of carbon monoxide dehydrogenase from Pseudomonascarboxydo~lava.Eur. J. Biochem.157,121-128.
26O [10] Krfiser, B. and Meyer, O. (1987) Structural elements of bactopterln from Pseudomonas carboxydoflava carbon monoxide dehydrogenase. Biochim. Biophys. Acta 912, 357-364. [11] Meyer, O. and Rajagopalan, K.V. (1984) Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria. J. Baateriol. 157, 843-848. [12] Jacobitz. S. and Meyer, O. (1989) Removal of CO dehydrogenase from Pseudomonas carboxydovoruns cytoplasmic membranes, rebinding to depleted membranes, and restoration of respiratory activities. J. Bacteriol. 171, 6294-6299. ~3] Gerstenberg, C., Friedrich, B. and Schlegei, H.G. (1982), Physical evidence for plasmids in autotrophic, especially hydrogen-oxidizing bacteria. Arch. Microbiol. 133, 90~96. [14] Kwon, M.O. and Kim, Y.M. (1985) Relationship between carbon monoxide dehydrogenase and a small plasmid of Pseudomonas carbox.vdovorans. FEMS Micrebiol. Lett. 29, 155-159. [15] graut, M. and Meyer, O. (1988) Plasmids in carboxydotrophic bacteria: physical and restriction analysis. Arch. Microbiol. 149, 540-546. [16] K.rant, M., Hugendieck, I., Herwi8, S. and Meyer, O. (1989) Homology and distribution of CO dehydrogenese structural genes in carboxydotrophic bacteria. Arch. Microbiol. 152, 335-341. [17] Williaras, E. and Colby, J. (1936) Biotcclmological applications of carboxydotrophic bacteria. Microbiol. Sci. 3, 149-153.
[18] 1~11, J.M., Williams, E. and Colby, J. (1985) Carbon monoxide oxidoreductese from thermophilic carboxydobacteria, in Microbial Gas Metabolism (Poole, R.K. and Dow, C.S., Eds.), pp. 153-160. Academic Press, London [19] Frunzke, K. and Meyer, O. (1990) Nitrate respiration, denitrification and utilization of nitrogen sources by aerobic carbon monoxide-oxidizingbacteria. Arch. Microbiol. 154,168-174. [20] Matsubara, T. and Mori, T. (1968) Studies on denitrification IX: Nitrous oxide, its production and reduction to nitrogen. J. Biocbem. 64, 863-871. [21] Kxistjansson, J.K. and Hollocher, T.C. (1980) First practical essay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization. J. Biol. Chem. 255, 704-707. [22] Rohde, M., Mayer, F. and Meyer, O. (1984) lmmunocytochemical localization of carbon monoxide oxidas© in Pseudomonas carboxydovorans. The enzyme is attached to the inner aspect of the cytoplasmic membrane. J. Biol. Chem. 259,14788-14792. [23] Rohde, M., Mayer, F., Jacobitz, S. and Meyer, O. (1985) Attachment of CO dehydrogenas¢ to the cytoplasmic membrane is limiting the respiratory rate of Pseudomonas carboxydovorans. FEMS Microbiol. Lett. 28,141-144. [24] Jacobitz, S. (1989) Isolierung und Charakterisierung von Cytochrom bs61 aus Pseudomonas carboxydovorans und Identifizicrung als physiologischem Elektronenakzeptor yon CO-Dehydroganase. Ph.D. thesis, University of Bayreuth.