- Email: [email protected]
Oxygen, hydrogen and nitrogen fixation in Azotobacter
SoilBio/.Biochem.Vol. 29, No. 516,pp. 863-869,1997 0 1997ElsevierScience Ltd. Al1rights reserved Printed in Gnat Britain PH: !30038-0717(%)00213-1 oo38-0717/97~17.00+ 0.00
OXYGEN,
HYDROGEN AND NITROGEN FIXATION IN AZOTOBACTER
M. G. YATES*t, E. M. DE SOUZA$ and J. H. KAHINDQ BBSRC, IPSR, Nitrogen Fixation Laboratory, Sussex University, Brighton BNl 9RQ, U.K. (Accepted 1 July 1996) Snmmary-This paper discusses four aspects of the Hz-uptake hydrogenase (Hup) - dependent respiration in Azotobacter. (1): Competition between isogenic Hup+ and Hup- strains of Azofobacterchroococcum containing the vanadium nitrogenase showed that the Hup+ strain dominated under carbon-, phosphate- or intrinsic Ns-limited conditions but not under Os-, sulphate- or Fe-limitation. (2): A Cydmutant of Azofobacter vinelundiiwas capable of catalysing Os-dependent H3H uptake, indicating that Hz-dependent respiration linked to a high affinity (Oz) oxidase system. (3): High-Os-adapted A. chroococcum showed lower specific nitrogenase activity at low ambient Os and 16°C than did low-Os-adapted A. chroococcum, suggesting that the high-affinity oxidases were inactivated at high ambient Os. (4): A second region of DNA, distinct from the main hup gene region, involved in Hz-dependent respiration is described. 0 1997 Elsevier Science Ltd
Aerobic diazotrophs must regulate the oxygen supply both to provide ATP and protect nitrogenase against oxygen damage at the same time. Diazotrophs have developed many strategies for limiting oxygen access to nitrogenase, from growth in micro-aerobic conditions to morphological changes, such as legume nodules, heterocysts in cyanobacteria, vesicles in non legumes and the variable oxygen banier in nodule cortex cells. Perhaps the best understood of al1 protection mechanisms are the two proposed by Dalton and Postgate (1969): respiratory and conformational protection in Azotobacter, the most aero-tolerant of al1 diazotrophs. An aspect of respiratory protection is the recycling of H2 produced by nitrogenase via the respiratory chain. This review wil1 discuss aspects of Hz-dependent respiration, including competition experiments between hydrogenase-positive (Hup+) and hydrogenase-negative (Hup-) strains of Azotobacter chroococcum to determine conditions under which Hz recycling benefits diazotrophic growth. Hoch et al. (1957) first reported Hz production by soybean nodules. However, Burns and Hardy (1975), having failed to find Hz production by Nz*Present
address:
Department
of
Biochemistry,
Universidade Federal do Parana, C. Postal 19046, CEP 81531-990, Curitiba, PR, Brazil. tAuthor for correspondence: Fax: 55 41 266 2042; email: [email protected]. $Present address: Department of Biochemistry, Universidade Federal do Parana, C. Postal 19046, CEP 81531990, Curitiba, PR, Brazil. address: §Present Department of Microbiology, Nairobi University, Nairobi, Kenya.
fixing Azotobacter vinelandii suggested it might be an in vitro artefact of isolated nitrogenase activity. These doubts were removed when it was shown that both symbiotic and asymbiotic systems produced Hz by nitrogenase in vivo (Schubert and Evans, 1976; Smith et al., 1976). The Hz-uptake hydrogenases of aerobic diazotrophs are dimeric (a/?) nickel-iron, membrane-bound proteins which catalyse Hz oxidation via the respiratory chain to produce ATP in vivo. In pure cultures the Hz derives solely from nitrogenase activity and may benefit the organism by providing ATP, contributing to respiratory protection and removing a potential inhibitor of N2 fixation (Hz) from the vicinity of the nitrogenase site (Dixon, 1972). Evans et al. (1985a) reported that a Hup+ Bradyrhizobium japonicurn strain compared with an isogenic Hup- B. japonicum strain as inoculum for soybeans resulted in a 5.2% increase in seed N, a 11% increase in plant N and a 9% increase in total biomass. However, some workers failed to establish that inoculating legumes with hydrogenase-positive strains of rhizobia was beneficial to plant growth. Evans et af. (1985b) estimated that more than half the experiments reported indicated that legumes benefited from Hz recycling but contradictory results were attributed to non-standard design (Evans et al., 1987) and major field variables (Dilworth and Glenn, 1984). These contradictions were resolved, in part, by adopting a model system of competition between isogenic Hup+ and Hupstrains of A. chroococcum under controlled conditions in a chemostat. Yates and Campbell (1989) showed that the presence of the Hz-uptake hydrogenase, which recycled al1 the Hz produced by nitro-
863
864
M. Cì. Yates el al
genase (Fig. l), was beneficial or otherwise according to the culture conditions. At pH 7.0 and 30°C in carbon-limited medium containing a mixed population of Mo-dependent, nitrogen-fixing Hup+ and Hup- A. chroococcum, the Hup+ strain dominated: from 1% to 100% in 4 days at a dilution rate of 0.1 h-‘. Phosphate-limited mixed populations also became Hup+ at about half the rate of the carbonlimited cultures, whereas under Oz-, sulphate- or iron-limited conditions the Hup- strain slowly dominated. Hz recycling would be advantageous under carbon limitation by providing extra electrons for ATP synthesis and by providing respiratory protection for nitrogenase. Two additional observations lend support to this conclusion: (1) Hup- mutants have a considerably longer lag phase than the Hup+ wild-type in Nz-fixing batch cultures (Aguilar et al., 1985) and (2) hydrogenase conveys no advantage in NHZ -dependent mixed populations (Yates and Campbell, 1989). It is unlikely that preventing HZ inhibiting Nz reduction plays a significant part in these populations, otherwise the Hup+ strain would dominate under al1 conditions. Linkerhagner and Oelze (1995) compared diazotrophic growth of Azotobacter vinelandii wild-type strain DJ with a hydrogenase negative (HoxKG-) strain and could find no evidente that cellular energy was benefitted by recycling Hz produced by nitrogenase in glucose-limited continuous culture. They suggested that Hz produced by the mutant would be used by the wild-type, a point considered by Yates and Campbell (1989) who calculated that a mixed population of sucrose-limited A. chroococcum, eventually dominated by the Hup’, at 7% Hup+ consumed 10% of the evolved HZ.
Phosphate-limited Nz-fixing A. chroococcum is extremely Oz-sensitive (Dalton and Postgate, 1969) and a contribution to respiratory protection would advantage the Hup+ population if, as suggested by Ackrell et al. (1972) and others, substrate oxidation was the rate-limiting step in respiration. Oz-limited Hup+ populations, on the other hand, would be disadvantaged by competition between Hz and NADH oxidation if Hz oxidation yielded less energy than that of NADH. Again, this cannot be a major factor in OZ-limited A. chroococcum populations otherwise the Hupstrain would have dominated more rapidly. Sulphur and iron are constituents of both nitrogenase and hydrogenase. Hup- strains might gain advantage under sulphate- or iron-limited conditions due to lack of competition for enzyme synthesis (Yates and Campbell, 1989). From these observations it is clear that H2 recycling can be advantageous to Nz-fixing cultures. We have extended these experiments to investigate the vanadium nitrogenase system in A. chroococcum. Vanadium nitrogenase is less efficient at fixing Nz than Mo nitrogenase: the accepted stoichiometries are that Mo nitrogenase loses a minimum of 25% of its electrons and energy to H2 production, whereas the V nitrogenase loses approximately 50% (Bishop and Eady, 1985) (Fig. 2). It was therefore possible that HZ recycling would benefit V-dependent more than Mo-dependent N2 fixation, and that this would be reflected in a wider range of Hup+ dominante in mixed populations. However, a detailed examination of competition between isogenie strains of Hup+ and Hup- V-dependent A. yielded a similar pattern of dominante chroococcum
r
500
2? 300 0 6 Z- 200 1 E = 100
0
0
10
20
30
40
40
Time (min) Time (min)
Fig.
1. Hz
produced by carbon-limited, Nz-fixing strain MCDl (Hup’); A, under
Azotobacter chroococcum:
air;
?? , under
Ar:02 (411): strain MCD122 (Hup-): under air; 0 under Ar:02 (4:l). D = 0.1 h-‘.
n
Fig. 2. Comparison of Mo and V nitrogenases in vivo. Hz production by Nz-fixing, Oz-limited Hup- Azotobacter chroococcum strains MCDlO5 (Nif+) and MCD1155 (Nif-, Vnf+). 0. MCDlO5 under air: ?? . under Ar:O, (4:l); A, MkDÏl55 under air; ?? , uide; Ar:02 (4:lc D = 0.06 h-‘.
02, Hz and N2 fixation Azobacter Table 1. Competition beween Hup- and isogenic Hup+ strains of Azotobocter chroococcum containing either Mo (Nif)- or V (Vnf)dependent nitrogenase in N&ing continuous culture
865
oxidases in A. vinelandii, using the deoxygenation of oxyleghaemoglobin and myoglobin as probes. They concluded that A. vinelandii may have three termNutrient Dominant strain Rate of inal oxidases: the cytochrome bd type, which has a Limitation dominante low affinity for Oz (K,,, for 02 of 4.5 PLM),a second Nif Vnf oxidase of high affinity (Km for 02 of 0.33 PM) disCarbon Hup + Hup+ Fast tinguished by the deoxygenation kinetics of myogloPhosphate Hup+ Hup+ Fast HupHupSlow 02 bin, and a third oxidase of very high affinity (K,,, Sulphate HupHupSIOW for Oz of 0.013-0.019ptM) identified by following HupFe SIOW Hupthe deoxygenation kinetics of oxyleghaemoglobin. HupHup+ Fast N2 Kelly et al. (1990) isolated a cytochrome d mutant (Cyd-) of A. vinelandii. This mutant (MK5) in carbon-, phosphate-, Oz-, sulphate- and iron-limand strain MK8, a mutant which over-produced ited cultures as observed for Mo-dependent nitrocytochrome d, together with the wild-type A. vinegen fixation. Only under intrinsic Nz limitation was landii (UW136) were tested for hydrogenase activity a response differente observed: in V-dependent using methylene blue, which accepts electrons populations the Hup+ strain dominated, whereas in directly from hydrogenase, or Oz, which accepts Mo-dependent cultures the Hup- strain was domielectrons via the respiratory chain, as the terminal nant (J.H. Kahindi, unpublished D. Phil thesis, electron acceptors. Al1 three strains expressed both University of Sussex, 1994) (Table 1). methylene blue- and Oz-dependent hydrogenase activity, that of strain MK5 was equivalent to that of the wild-type, whereas that of MK8, which overTHE Hz-DEPENDENT RESPIRATORY CHAIN produced cytochrome d, consistently expressed approximately one-third of that activity. This clearly The one certain component of the Hz-dependent indicates that A. vinelundii is capable of oxidising respiratory chain in Azotobacter is hydrogenase. Hz via the respiratory chain in the absente of cytoWong and Maier (1984, 1985), using Hz-reduced VS chrome d. Although the cytochrome d-overproduOz-oxidised absorption spectra indicated the involcing strain MK8 produced less hydrogenase activity vement of cytochromes b, c and d, but not cytothan the Cyd- mutant or the wild-type, it apparchrome o in Hz oxidation by A. vinelundii. Low ently enhanced Hz-dependent respiration inasmuch sensitivity to cyanide or chlorpromazine confirmed as the ratio of Oz-dependent to methylene bluethe non-contribution of cytochrome o in Hz-dependependent H3H uptake was highest in MKS and dent respiration. Inhibition by 2-n-heptyl-4-hydrolowest in MK5, the Cyd- mutant. Whether the Hzxyquinoline-N-oxide (HQNO) and also by uv dependent respiration in MK5 was via the third irradiation treatment, which was partly restored by oxidase system or cytochrome o was not deteradding co-enzyme QS, indicated that ubiquinone mined; D’Mello et af. (1994) could not distinguish was als0 a component. whether cytochrome o or the third oxidase system A possibly important conclusion arises from in A. vinelandii had the higher affinity for OZ. these observations: that hydrogenase-linked respiration is a separate entity, involving a single terminal oxidase, distinct from the branched chain linked to POSSIBLE OXYGEN REGULATION OF HIGH AFFINITY NADH-, malate- and succinate-dependent respirOXIDASES ation (Jones and Redfearn, 1967). These obserThe Cyd- mutant of A. vinelundii could not grow vations are consistent with the data on competition between Hup+ and Hup- strains of A. chroococcum diazotrophically under air, but did so under 2% Oz reviewed above. Cytochrome d oxidase synthesis is in nitrogen, thus clearly demonstrating that cytoincreased under high stirring rates (Drozd and chrome d is necessary for aerobic N2 fixation (Kelly Postgate, 1970) and might, therefore, be expected to et al., 1990). The Cyd- mutant also grew poorly be higher under carbon- or phosphate limitation, when ammonium acetate was added to the medium, when the medium contains dissolved oxygen, than unless a high inoculum was used, indicating that under Oz-limited conditions when the medium has the high cel1 density protected against inhibition of no discernible dissolved Oz. However, Nz-fixing, OZ-sensitive systems (Kelly et al., 1990). OZ-limited cultures of A. chroococcum do not evolve Drozd and Postgate (1970) compared Oz-depenHz, indicating that hydrogenase is recycling the Hz dent nitrogenase activity in high- and low-Ozproduced by nitrogenase in Oz-limited cultures. adapted A. chroococcum cultures and showed that Clearly, there is either sufficient cytochrome d oxithe Oz optimum for acetylene reduction by the dase present in such cultures to recycle Hz or high-OZ-adapted cells was considerably broader and another terminal oxidase is present which accepts at a higher ambient O2 than that of the low-Ozadapted culture. This is consistent with the subelectrons from Hz-dependent respiration. D’Mello et al. (1994) determined the OZ affinities of terminal sequent observations of McInery et al. (1984) and
M. G. Yates et al.
866
96 oxygen in argon
Fig. 3. Hz production by high- and low-Oz-adjusted, Na*. . fixing contmuous cultures 01 A.?OIODac1er Chroococcum strain MCDlO5 (Hup-) under a range of Ar:Os ratios at 30°C. 0, high-0,adapted; ?? , low-Oz-adapted. Aliquots (100 ml) were taken from two Oz-limited continuous cultures grown at 30°C and pH 7.0, stirred at 100 (low-Osadapted) and 300 (high-Oz-adapted) rev min-‘. respectively. The O.D. was adjusted to equality with medium before transferring aliquöts (10 ml) to subs-sealed flasks (25 ml) containing a range of Ar:Os ratios. The flasks were
shaken for 45 min at 30°C. Hz production was measured by a thermal conductivity gas chromatograph. references therein) that the high-affinity (Oz) branch of the A. vinelandii respiratory chain, containing cytochromes c4, cs and o is unlikely to be involved in energy conservation under non oxygen-limited growth conditions. However, Drozd and Postgate (1970) also showed that, although the high-Ozadapted culture had more cytochrome d, the amounts of cytochromes c4 and c5 were similar in both cultures. They did not measure amounts of cytochrome o nor did they comment on the apparent greater efficiency of nitrogenase activity in the low-Oz-adapted cultures at low ambient 02 concentrations. The data suggested either that high-Oz-affinity oxidases were not functioning in the high-Ozadapted culture, despite the presence of cytochromes c4 and ~5, or that ATP synthesis was uncoupled by adaptation to high concentrations of 02.
An attempt to confirm the above observation at 30°C using cells from continuous cultures grown as described under Fig. 3 at a dilution rate of 0.1 h-‘, was unsuccessful: the high-Oz-adapted cells had a broad 02 optimum for maximum rates of nitrogenase activity (Hz production) and were as efficient, in terms of the specific activity of nitrogenase, as the low-Oz-adapted cells at low ambient 02 concentrations (Fig. 3). However, when both culture types were cooled rapidly in ice to 16°C and the experiment repeated at 16°C the specific activity of the nitrogenase in the low-02-adapted cultures was con-
% oxygen in argon Fig. 4. Hz production by high- and low-Oz-adjusted, Nzfixing Azorobacter chroococcum, strain MCD105 (Hup-) under a range of Ar:02 ratios at 16°C. 0, high-Oz, low-Oz-adapted. Aliquots (10ml) were taken adapted; ?? from two continuous cultures as described in Fig. 3 and cooled rapidly in ice to 16°C before transferring 1 ml aliquots to suba-sealed serum bottles (8 ml) under a range of Ar:Oa ratios. The flasks were shaken for 45 minutes at 16°C. The data are representative of three separate experiments.
sistently higher at low ambient 02 than was that of the high-Os-adapted culture (Fig. 4). The data suggest that some component of the high-affinity respiratory chain is repressed or inactivated by 02 which is not resynthesised or reactivated within the time of the experiment (45 min) at 16°C.
MOLECULAR GENETICS OF A. CHROOCOCCUM HYDROGENASE
The hup gene region, responsible for hydrogenase expression in A. chroococcum was isolated by Tibelius et al. (1987) and has since been sequenced and partly characterised (see Du et af., 1994). It contains 16 open reading frames in, at least, two operons (Fig. 5). The first two genes hupX code for the two structural subunits of hydrogenase (Ford et al., 1990) while hupZ codes for a membrane protein involved in Hz-dependent respiration (Du et al., 1994). The HupM protein may play a role in attachment of hydrogenase to the membrane, HupN is a metallo-protein of unknown function; Hup0 is another processing protein and HupQ may be involved in nickel insertion into hydrogenase. HupR is a rubredoxin-type protein, presumably involved in electron transport, but whether it is involved in Ha-dependent respiration or in hydrogenase synthesis is not known. HupT is of unknown function, while HupV may be a scaffolding protein for the nickel cofactor assembly. The hupABYC gene region is homologous to the hypABC of Escherichia coli, excepting hupY, which
Oz, Hz and Nz fixation Azobacter
8
L
ZY
Y
VAB
I Ilnfí
II I
CD
B
II I I
1978). A second effect of this mutation was that the culture pH of MCD124 at the end of growth in either Burk’s medium or in rich medium (RM; Robson et al., 1984) was usually half a pH unit higher than that of the wild-type strain MCDl. The phenotype of this mutant was designated HupP- (P for particulate).
Fig. 5. The main hup gene region of Azotobacterchroococcum: adapted from the data of Du et al. (1994).
has no homologous gene in the E. coli hyp genes operon (Tibehus et al., 1993). HupA has a cysteinerich region near the centre which may act to bind metals and HupB has both cysteine- and histidinerich regions and is involved in nickel metabolism (Du and Tibelius, 1994). HupY is a large protein of 755 amino acid residues and contains metal-binding motifs, while HupC has no known function. The last two genes, hupD and hupE code for metalloand membrane proteins respectively (Du et al., 1992).
A SECOND
DNA REGION INVOLVED RFSPIRATION
IN HrDEPENDENT
Yates and Robson (1985) isolated four classes of hydrogenase mutants of A. chroococcum by Nmethyl-N’-nitro-N-nitrosoguanidine mutagenesis, identified by their inability to catalyse OZ-dependent H3H uptake. One class of mutant, containing only one member strain, MCD124, was not complemented by DNA from the main hup gene region, described above, in the wide-host-range plasmid pRK290, whereas al1 the mutants in the other three classes were complemented to restore activity commensurate with that of the wild-type (Tibelius and Yates, 1989). This mutant, MCD124, had a distinctive phenotype: it had very low activity with methylene blue at pH 8.0, the optimum pH for the membrane-bound enzyme, and very low activity with Oz as the terminal electron acceptor, indicating that it could not support Hz-dependent respiration. However, it had substantial activity with methylene blue as the electron acceptor at pH 5.5, the pH optimum of solubilised hydrogenase (Van der Werf and Yates,
I
G Intergenic Amino MW
acids
PI Class Function Homology
ISOLATION
THE HUP2 GENE PRODUCT
The hup2 gene starts 16 bases downstream from the structural genes coding for the hydrogenase subunits. A mutation in hupZ (Du et al., 1994)
hup&
2
531 177 20125 10.95 ? E.coli
0296 0121
= P-ribosyl-cAMP
3
399 -1
OF THE HUJ’f GENFS
The hupP genes were isolated by complementing the mutant MCD124 by conjugation, using a gene library of A. chroococcum DNA in the wide-hostrange plasmid, pLAFR1 together with the helper plasmid pRK2013. Approximately 2000 tetracyclinresistant colonies were screened for Oz-dependent H3H uptake by growing individual colonies on Ucavity plates and exposing to Hz plus H3H in air for 20 min. Three transconjugants were isolated, each with a 25 kb insert which restricted to three EcoRI fragments of 3.0, 7.4 and 15 kb, respectively. The 7.4 kb fragment complemented both phenotypes, whereas a 2.7 kb EcoRI-BamHI fragment restored hydrogenase activity to the leve1 and pattem of the wild-type but did not affect the pH shift phenotype. A sequence analysis of the 2.7 kb EcoRI-BamHI fragment, together with a further 0.5 kb downstream from the BumHI site, revealed five open reading frames Gembank accession number U48404, the first, orfl, being incomplete (Table 2). An analysis of this DNA, using kanamycin cassettes in suitable restriction sites in orfs 2, _I and 5 and transposon mutagenesis in orf4 and orf5, revealed that the mutation in orf4 produced both the HupP- and the pH shift phenotypes, whereas a mutation in orfs produced only the pH shift phenotype. That orf4 was responsible for the HupP- phenotype was confirmed by restoring wild-type activity by introducing, in the wide-host-range vector, pLAFR3, hupP DNA containing only orf4 as a complete orf.
of the hupP region of Azorobacfer chroococcum DNA (PRcH
Table 2. Proper&
ORF
867
51
133 15416 6.71
110 11909 5.01
PRcH PRcH
PRcH PRcH
5
579
330 -8
hydrolase)
4
738 27
192 20931 7.78 HupP E. di 0261 0131
246 26959 6.51 Hydrophobic Membrane protein E. di 0154
M. G. Y ates et al.
868
Bishop P. E. and Eady R. R. (1985) Nitrogen fixation by produced an identical phenotype to that of the a nifHDK deletion strain of Azotobacter vinelandii. In HupP- mutant, MCD124: high hydrogenase activity Nitrogen Fixation Research Progress (H. J. Evans, P. J. at pH 5.5; low activity at pH 8.0 with methylene Bottomly and W. E. Newton, Eds), p. 622. Martinus blue as the electron acceptor and low activity with Nijhof, Dordrecht. Burns R. C. and Hardy R. W. F. (1975) In Nitrogen O2 as the electron acceptor. A double mutation, Fixation in Bacteria and Higher Plan&. Springer, New obtained by introducing the mutated hupZ gene York. into the chromosome of MCD124, gave a similar Dalton H. and Postgate J. R. (1969) Effect of oxygen on activity pattern to that of the two single mutants. the growth of Azotobacter chroococcum in continuous However, the mutation in hupZ did not affect the culture. Journal of General Microbiology 54, 463473. pH of the culture during growth in the manner of a Dilworth M. J. and Glenn A. (1984) How does a legume nodule work?. Trends in Biochemical Sciences 9, 519hupP mutation. Moreover, the putative proteins 523. expressed by hupZ and orf4 are very different: Dixon R. 0. D. (1972) Hydrogenase in legume root HupZ has four pronounced hydrophobic domains nodule bacteroids: occurrence and properties. Archiv fur and is a typical membrane-type protein, possibly Mikrobiologie 85, 193-20 1. containing a b-type cytochrome (Du et al., 1994). D’Mello R., Hill S. and Poole R. K. (1994) Determination of the oxygen affinities of terminal oxidases in On the other hand, ORF4 of the hupP region Azorobacter vinelandii using the deoxygenation of oxyleshows only weak hydrophobicity in two regions at ghaemoglobin and myoglobin: cytochrome bd is a low the N- and C-terminals, respectively and shows affinity oxidase. Microbiology 140, 1395-1402. Dross F., Geisler V., Lenger R., Theis F., Krafft T., some homology to proteins that interact with Fahrenholz F., Kojro E., Duchene A., Tripier D., DNA. Finally, to emphasise the differente between Juvenal K. and Kroger A. (1992) The quinone-reactive these two genes, they do not cross-complement: the Ni/Fe hydrogenase of Wolinella succinogenes. European hupZ gene does not complement the HupP- mutant Journal of Biochemistry 206, 93-102. and the hupP region does not restore activity to the Drozd J. W. and Postgate J. R. (1970) Effect of oxygen on acetylene reduction, cytochrome content and respiratory hupZ- mutant. activity of Azotobacter chroococcum. Journal of Genera1 Since neither of these two mutations affects other Microbiology 63, 63-73. substrate reductions: NADH-, malate-, succinateDu L., Stejskahl F. and Tibelius K. H. (1992) and lactate-dependent respiration activities were Characterisation of two genes (hupD and hupE) required similar in both mutants to those in the wild-type, for hydrogenase activity in Azotobacier chroococcum. FEMS Microbio1og.y Letters 96, 93-102. these two proteins must be regarded as specific components of Hz-dependent respiration. HupZ is a Du L., Tibelius K. H., Sousa E. M., Garg R. P. and Yates M. G. (1994) Sequences, organisation and analytypical membrane protein and is, therefore, a likely sis of the hupZMNOQRTV genes of the Azotobacter component of the Hz-dependent respiratory chain. chroococcum hydrogenase gene cluster. Journal of Indeed, it shows considerable homology to HydC Molecular Biology 242, 5499557. (Du et al., 1994) a protein subunit necessary for N? Du L. and Tibelius K. H. (1994) The hupB gene of the Azotobacter chroococcum hydrogenase gene cluster is vitro activity of the purified Wolinella succinogenes involved in nickel metabolism. Current Microbiology 28, nickel-iron hydrogenase (Dross et al., 1992). 21-24. Whether ORF4 is a membrane protein or acts in a Evans H. J., Hanus F. J., Haugland R. A., Cantrell M. processing or regulatory role is not known at preA., Xu L.-S., Russell S. A., Lambert G. R. and Harker A. R. (1985a). Hydrogen recycling in nodules affects sent. nitrogen fixation and growth of soybeans. In World This review has indicated some progress in the Soybean Research Conference HI (R. Shibles, Ed.), pp. understanding of Hz-dependent respiration in 935-942. Westview Press, London. Azotobacter: it is clearly beneficial to the Nz-fixing Evans H. J., Hanus F. J., Russell S. A., Harker A. R., Lambert G. R. and Dalton D. A. (1985b). Biochemical organism under carbon- or phosphate-limiting concharacterisation, evolution and genetics of Hz recycling ditions; it is not dependent solely on cytochrome d in Rhizobium. In Nitrogen Fixation and CO, as the terminal cytochrome oxidase, but can use an Merabolism (P. W. Ludden and J. E. Burris, Eds), pp: alternative oxidase or oxidases and we have ident3-11. Elsevier, New York. ified two proteins associated specifically with HZ- Evans H. J., Zuber M. and Dalton D. A. (1987) Some processes related to nitrogen fixation in nodulated dependent respiration, in addition to those of the legumes. Philosophical Transaciions of the Royal Society hydrogenase enzyme itself 317, 143-160.
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159, 348-
Wong T.-Y. and Maier R. J. (1985) Chlorpromazine inhibition of electron transport in Azofobacter vinelandii membranes. Biochimica et Biophysica Acts 807, 320323.
Yates M. G. and Campbell F. 0. (1989) The effect of nutrient limitation on the competition between an Hzuptake, hydrogenase-positive recombinant strain of Azotobacter chroococcum and the Hup- mutant parent in mixed populations. Journal of Genera1 Microbiology 135, 221-226.
Yates M. G. and Robson
R. L. (1985) Mutants
of
Azofobacfer chroococcum defective in hydrogenase activity. Jounal of Genera1 Microbiology 131, 1459-1466.
OXYGEN,
HYDROGEN AND NITROGEN FIXATION IN AZOTOBACTER
M. G. YATES*t, E. M. DE SOUZA$ and J. H. KAHINDQ BBSRC, IPSR, Nitrogen Fixation Laboratory, Sussex University, Brighton BNl 9RQ, U.K. (Accepted 1 July 1996) Snmmary-This paper discusses four aspects of the Hz-uptake hydrogenase (Hup) - dependent respiration in Azotobacter. (1): Competition between isogenic Hup+ and Hup- strains of Azofobacterchroococcum containing the vanadium nitrogenase showed that the Hup+ strain dominated under carbon-, phosphate- or intrinsic Ns-limited conditions but not under Os-, sulphate- or Fe-limitation. (2): A Cydmutant of Azofobacter vinelundiiwas capable of catalysing Os-dependent H3H uptake, indicating that Hz-dependent respiration linked to a high affinity (Oz) oxidase system. (3): High-Os-adapted A. chroococcum showed lower specific nitrogenase activity at low ambient Os and 16°C than did low-Os-adapted A. chroococcum, suggesting that the high-affinity oxidases were inactivated at high ambient Os. (4): A second region of DNA, distinct from the main hup gene region, involved in Hz-dependent respiration is described. 0 1997 Elsevier Science Ltd
Aerobic diazotrophs must regulate the oxygen supply both to provide ATP and protect nitrogenase against oxygen damage at the same time. Diazotrophs have developed many strategies for limiting oxygen access to nitrogenase, from growth in micro-aerobic conditions to morphological changes, such as legume nodules, heterocysts in cyanobacteria, vesicles in non legumes and the variable oxygen banier in nodule cortex cells. Perhaps the best understood of al1 protection mechanisms are the two proposed by Dalton and Postgate (1969): respiratory and conformational protection in Azotobacter, the most aero-tolerant of al1 diazotrophs. An aspect of respiratory protection is the recycling of H2 produced by nitrogenase via the respiratory chain. This review wil1 discuss aspects of Hz-dependent respiration, including competition experiments between hydrogenase-positive (Hup+) and hydrogenase-negative (Hup-) strains of Azotobacter chroococcum to determine conditions under which Hz recycling benefits diazotrophic growth. Hoch et al. (1957) first reported Hz production by soybean nodules. However, Burns and Hardy (1975), having failed to find Hz production by Nz*Present
address:
Department
of
Biochemistry,
Universidade Federal do Parana, C. Postal 19046, CEP 81531-990, Curitiba, PR, Brazil. tAuthor for correspondence: Fax: 55 41 266 2042; email: [email protected]. $Present address: Department of Biochemistry, Universidade Federal do Parana, C. Postal 19046, CEP 81531990, Curitiba, PR, Brazil. address: §Present Department of Microbiology, Nairobi University, Nairobi, Kenya.
fixing Azotobacter vinelandii suggested it might be an in vitro artefact of isolated nitrogenase activity. These doubts were removed when it was shown that both symbiotic and asymbiotic systems produced Hz by nitrogenase in vivo (Schubert and Evans, 1976; Smith et al., 1976). The Hz-uptake hydrogenases of aerobic diazotrophs are dimeric (a/?) nickel-iron, membrane-bound proteins which catalyse Hz oxidation via the respiratory chain to produce ATP in vivo. In pure cultures the Hz derives solely from nitrogenase activity and may benefit the organism by providing ATP, contributing to respiratory protection and removing a potential inhibitor of N2 fixation (Hz) from the vicinity of the nitrogenase site (Dixon, 1972). Evans et al. (1985a) reported that a Hup+ Bradyrhizobium japonicurn strain compared with an isogenic Hup- B. japonicum strain as inoculum for soybeans resulted in a 5.2% increase in seed N, a 11% increase in plant N and a 9% increase in total biomass. However, some workers failed to establish that inoculating legumes with hydrogenase-positive strains of rhizobia was beneficial to plant growth. Evans et af. (1985b) estimated that more than half the experiments reported indicated that legumes benefited from Hz recycling but contradictory results were attributed to non-standard design (Evans et al., 1987) and major field variables (Dilworth and Glenn, 1984). These contradictions were resolved, in part, by adopting a model system of competition between isogenic Hup+ and Hupstrains of A. chroococcum under controlled conditions in a chemostat. Yates and Campbell (1989) showed that the presence of the Hz-uptake hydrogenase, which recycled al1 the Hz produced by nitro-
863
864
M. Cì. Yates el al
genase (Fig. l), was beneficial or otherwise according to the culture conditions. At pH 7.0 and 30°C in carbon-limited medium containing a mixed population of Mo-dependent, nitrogen-fixing Hup+ and Hup- A. chroococcum, the Hup+ strain dominated: from 1% to 100% in 4 days at a dilution rate of 0.1 h-‘. Phosphate-limited mixed populations also became Hup+ at about half the rate of the carbonlimited cultures, whereas under Oz-, sulphate- or iron-limited conditions the Hup- strain slowly dominated. Hz recycling would be advantageous under carbon limitation by providing extra electrons for ATP synthesis and by providing respiratory protection for nitrogenase. Two additional observations lend support to this conclusion: (1) Hup- mutants have a considerably longer lag phase than the Hup+ wild-type in Nz-fixing batch cultures (Aguilar et al., 1985) and (2) hydrogenase conveys no advantage in NHZ -dependent mixed populations (Yates and Campbell, 1989). It is unlikely that preventing HZ inhibiting Nz reduction plays a significant part in these populations, otherwise the Hup+ strain would dominate under al1 conditions. Linkerhagner and Oelze (1995) compared diazotrophic growth of Azotobacter vinelandii wild-type strain DJ with a hydrogenase negative (HoxKG-) strain and could find no evidente that cellular energy was benefitted by recycling Hz produced by nitrogenase in glucose-limited continuous culture. They suggested that Hz produced by the mutant would be used by the wild-type, a point considered by Yates and Campbell (1989) who calculated that a mixed population of sucrose-limited A. chroococcum, eventually dominated by the Hup’, at 7% Hup+ consumed 10% of the evolved HZ.
Phosphate-limited Nz-fixing A. chroococcum is extremely Oz-sensitive (Dalton and Postgate, 1969) and a contribution to respiratory protection would advantage the Hup+ population if, as suggested by Ackrell et al. (1972) and others, substrate oxidation was the rate-limiting step in respiration. Oz-limited Hup+ populations, on the other hand, would be disadvantaged by competition between Hz and NADH oxidation if Hz oxidation yielded less energy than that of NADH. Again, this cannot be a major factor in OZ-limited A. chroococcum populations otherwise the Hupstrain would have dominated more rapidly. Sulphur and iron are constituents of both nitrogenase and hydrogenase. Hup- strains might gain advantage under sulphate- or iron-limited conditions due to lack of competition for enzyme synthesis (Yates and Campbell, 1989). From these observations it is clear that H2 recycling can be advantageous to Nz-fixing cultures. We have extended these experiments to investigate the vanadium nitrogenase system in A. chroococcum. Vanadium nitrogenase is less efficient at fixing Nz than Mo nitrogenase: the accepted stoichiometries are that Mo nitrogenase loses a minimum of 25% of its electrons and energy to H2 production, whereas the V nitrogenase loses approximately 50% (Bishop and Eady, 1985) (Fig. 2). It was therefore possible that HZ recycling would benefit V-dependent more than Mo-dependent N2 fixation, and that this would be reflected in a wider range of Hup+ dominante in mixed populations. However, a detailed examination of competition between isogenie strains of Hup+ and Hup- V-dependent A. yielded a similar pattern of dominante chroococcum
r
500
2? 300 0 6 Z- 200 1 E = 100
0
0
10
20
30
40
40
Time (min) Time (min)
Fig.
1. Hz
produced by carbon-limited, Nz-fixing strain MCDl (Hup’); A, under
Azotobacter chroococcum:
air;
?? , under
Ar:02 (411): strain MCD122 (Hup-): under air; 0 under Ar:02 (4:l). D = 0.1 h-‘.
n
Fig. 2. Comparison of Mo and V nitrogenases in vivo. Hz production by Nz-fixing, Oz-limited Hup- Azotobacter chroococcum strains MCDlO5 (Nif+) and MCD1155 (Nif-, Vnf+). 0. MCDlO5 under air: ?? . under Ar:O, (4:l); A, MkDÏl55 under air; ?? , uide; Ar:02 (4:lc D = 0.06 h-‘.
02, Hz and N2 fixation Azobacter Table 1. Competition beween Hup- and isogenic Hup+ strains of Azotobocter chroococcum containing either Mo (Nif)- or V (Vnf)dependent nitrogenase in N&ing continuous culture
865
oxidases in A. vinelandii, using the deoxygenation of oxyleghaemoglobin and myoglobin as probes. They concluded that A. vinelandii may have three termNutrient Dominant strain Rate of inal oxidases: the cytochrome bd type, which has a Limitation dominante low affinity for Oz (K,,, for 02 of 4.5 PLM),a second Nif Vnf oxidase of high affinity (Km for 02 of 0.33 PM) disCarbon Hup + Hup+ Fast tinguished by the deoxygenation kinetics of myogloPhosphate Hup+ Hup+ Fast HupHupSlow 02 bin, and a third oxidase of very high affinity (K,,, Sulphate HupHupSIOW for Oz of 0.013-0.019ptM) identified by following HupFe SIOW Hupthe deoxygenation kinetics of oxyleghaemoglobin. HupHup+ Fast N2 Kelly et al. (1990) isolated a cytochrome d mutant (Cyd-) of A. vinelandii. This mutant (MK5) in carbon-, phosphate-, Oz-, sulphate- and iron-limand strain MK8, a mutant which over-produced ited cultures as observed for Mo-dependent nitrocytochrome d, together with the wild-type A. vinegen fixation. Only under intrinsic Nz limitation was landii (UW136) were tested for hydrogenase activity a response differente observed: in V-dependent using methylene blue, which accepts electrons populations the Hup+ strain dominated, whereas in directly from hydrogenase, or Oz, which accepts Mo-dependent cultures the Hup- strain was domielectrons via the respiratory chain, as the terminal nant (J.H. Kahindi, unpublished D. Phil thesis, electron acceptors. Al1 three strains expressed both University of Sussex, 1994) (Table 1). methylene blue- and Oz-dependent hydrogenase activity, that of strain MK5 was equivalent to that of the wild-type, whereas that of MK8, which overTHE Hz-DEPENDENT RESPIRATORY CHAIN produced cytochrome d, consistently expressed approximately one-third of that activity. This clearly The one certain component of the Hz-dependent indicates that A. vinelundii is capable of oxidising respiratory chain in Azotobacter is hydrogenase. Hz via the respiratory chain in the absente of cytoWong and Maier (1984, 1985), using Hz-reduced VS chrome d. Although the cytochrome d-overproduOz-oxidised absorption spectra indicated the involcing strain MK8 produced less hydrogenase activity vement of cytochromes b, c and d, but not cytothan the Cyd- mutant or the wild-type, it apparchrome o in Hz oxidation by A. vinelundii. Low ently enhanced Hz-dependent respiration inasmuch sensitivity to cyanide or chlorpromazine confirmed as the ratio of Oz-dependent to methylene bluethe non-contribution of cytochrome o in Hz-dependependent H3H uptake was highest in MKS and dent respiration. Inhibition by 2-n-heptyl-4-hydrolowest in MK5, the Cyd- mutant. Whether the Hzxyquinoline-N-oxide (HQNO) and also by uv dependent respiration in MK5 was via the third irradiation treatment, which was partly restored by oxidase system or cytochrome o was not deteradding co-enzyme QS, indicated that ubiquinone mined; D’Mello et af. (1994) could not distinguish was als0 a component. whether cytochrome o or the third oxidase system A possibly important conclusion arises from in A. vinelandii had the higher affinity for OZ. these observations: that hydrogenase-linked respiration is a separate entity, involving a single terminal oxidase, distinct from the branched chain linked to POSSIBLE OXYGEN REGULATION OF HIGH AFFINITY NADH-, malate- and succinate-dependent respirOXIDASES ation (Jones and Redfearn, 1967). These obserThe Cyd- mutant of A. vinelundii could not grow vations are consistent with the data on competition between Hup+ and Hup- strains of A. chroococcum diazotrophically under air, but did so under 2% Oz reviewed above. Cytochrome d oxidase synthesis is in nitrogen, thus clearly demonstrating that cytoincreased under high stirring rates (Drozd and chrome d is necessary for aerobic N2 fixation (Kelly Postgate, 1970) and might, therefore, be expected to et al., 1990). The Cyd- mutant also grew poorly be higher under carbon- or phosphate limitation, when ammonium acetate was added to the medium, when the medium contains dissolved oxygen, than unless a high inoculum was used, indicating that under Oz-limited conditions when the medium has the high cel1 density protected against inhibition of no discernible dissolved Oz. However, Nz-fixing, OZ-sensitive systems (Kelly et al., 1990). OZ-limited cultures of A. chroococcum do not evolve Drozd and Postgate (1970) compared Oz-depenHz, indicating that hydrogenase is recycling the Hz dent nitrogenase activity in high- and low-Ozproduced by nitrogenase in Oz-limited cultures. adapted A. chroococcum cultures and showed that Clearly, there is either sufficient cytochrome d oxithe Oz optimum for acetylene reduction by the dase present in such cultures to recycle Hz or high-OZ-adapted cells was considerably broader and another terminal oxidase is present which accepts at a higher ambient O2 than that of the low-Ozadapted culture. This is consistent with the subelectrons from Hz-dependent respiration. D’Mello et al. (1994) determined the OZ affinities of terminal sequent observations of McInery et al. (1984) and
M. G. Yates et al.
866
96 oxygen in argon
Fig. 3. Hz production by high- and low-Oz-adjusted, Na*. . fixing contmuous cultures 01 A.?OIODac1er Chroococcum strain MCDlO5 (Hup-) under a range of Ar:Os ratios at 30°C. 0, high-0,adapted; ?? , low-Oz-adapted. Aliquots (100 ml) were taken from two Oz-limited continuous cultures grown at 30°C and pH 7.0, stirred at 100 (low-Osadapted) and 300 (high-Oz-adapted) rev min-‘. respectively. The O.D. was adjusted to equality with medium before transferring aliquöts (10 ml) to subs-sealed flasks (25 ml) containing a range of Ar:Os ratios. The flasks were
shaken for 45 min at 30°C. Hz production was measured by a thermal conductivity gas chromatograph. references therein) that the high-affinity (Oz) branch of the A. vinelandii respiratory chain, containing cytochromes c4, cs and o is unlikely to be involved in energy conservation under non oxygen-limited growth conditions. However, Drozd and Postgate (1970) also showed that, although the high-Ozadapted culture had more cytochrome d, the amounts of cytochromes c4 and c5 were similar in both cultures. They did not measure amounts of cytochrome o nor did they comment on the apparent greater efficiency of nitrogenase activity in the low-Oz-adapted cultures at low ambient 02 concentrations. The data suggested either that high-Oz-affinity oxidases were not functioning in the high-Ozadapted culture, despite the presence of cytochromes c4 and ~5, or that ATP synthesis was uncoupled by adaptation to high concentrations of 02.
An attempt to confirm the above observation at 30°C using cells from continuous cultures grown as described under Fig. 3 at a dilution rate of 0.1 h-‘, was unsuccessful: the high-Oz-adapted cells had a broad 02 optimum for maximum rates of nitrogenase activity (Hz production) and were as efficient, in terms of the specific activity of nitrogenase, as the low-Oz-adapted cells at low ambient 02 concentrations (Fig. 3). However, when both culture types were cooled rapidly in ice to 16°C and the experiment repeated at 16°C the specific activity of the nitrogenase in the low-02-adapted cultures was con-
% oxygen in argon Fig. 4. Hz production by high- and low-Oz-adjusted, Nzfixing Azorobacter chroococcum, strain MCD105 (Hup-) under a range of Ar:02 ratios at 16°C. 0, high-Oz, low-Oz-adapted. Aliquots (10ml) were taken adapted; ?? from two continuous cultures as described in Fig. 3 and cooled rapidly in ice to 16°C before transferring 1 ml aliquots to suba-sealed serum bottles (8 ml) under a range of Ar:Oa ratios. The flasks were shaken for 45 minutes at 16°C. The data are representative of three separate experiments.
sistently higher at low ambient 02 than was that of the high-Os-adapted culture (Fig. 4). The data suggest that some component of the high-affinity respiratory chain is repressed or inactivated by 02 which is not resynthesised or reactivated within the time of the experiment (45 min) at 16°C.
MOLECULAR GENETICS OF A. CHROOCOCCUM HYDROGENASE
The hup gene region, responsible for hydrogenase expression in A. chroococcum was isolated by Tibelius et al. (1987) and has since been sequenced and partly characterised (see Du et af., 1994). It contains 16 open reading frames in, at least, two operons (Fig. 5). The first two genes hupX code for the two structural subunits of hydrogenase (Ford et al., 1990) while hupZ codes for a membrane protein involved in Hz-dependent respiration (Du et al., 1994). The HupM protein may play a role in attachment of hydrogenase to the membrane, HupN is a metallo-protein of unknown function; Hup0 is another processing protein and HupQ may be involved in nickel insertion into hydrogenase. HupR is a rubredoxin-type protein, presumably involved in electron transport, but whether it is involved in Ha-dependent respiration or in hydrogenase synthesis is not known. HupT is of unknown function, while HupV may be a scaffolding protein for the nickel cofactor assembly. The hupABYC gene region is homologous to the hypABC of Escherichia coli, excepting hupY, which
Oz, Hz and Nz fixation Azobacter
8
L
ZY
Y
VAB
I Ilnfí
II I
CD
B
II I I
1978). A second effect of this mutation was that the culture pH of MCD124 at the end of growth in either Burk’s medium or in rich medium (RM; Robson et al., 1984) was usually half a pH unit higher than that of the wild-type strain MCDl. The phenotype of this mutant was designated HupP- (P for particulate).
Fig. 5. The main hup gene region of Azotobacterchroococcum: adapted from the data of Du et al. (1994).
has no homologous gene in the E. coli hyp genes operon (Tibehus et al., 1993). HupA has a cysteinerich region near the centre which may act to bind metals and HupB has both cysteine- and histidinerich regions and is involved in nickel metabolism (Du and Tibelius, 1994). HupY is a large protein of 755 amino acid residues and contains metal-binding motifs, while HupC has no known function. The last two genes, hupD and hupE code for metalloand membrane proteins respectively (Du et al., 1992).
A SECOND
DNA REGION INVOLVED RFSPIRATION
IN HrDEPENDENT
Yates and Robson (1985) isolated four classes of hydrogenase mutants of A. chroococcum by Nmethyl-N’-nitro-N-nitrosoguanidine mutagenesis, identified by their inability to catalyse OZ-dependent H3H uptake. One class of mutant, containing only one member strain, MCD124, was not complemented by DNA from the main hup gene region, described above, in the wide-host-range plasmid pRK290, whereas al1 the mutants in the other three classes were complemented to restore activity commensurate with that of the wild-type (Tibelius and Yates, 1989). This mutant, MCD124, had a distinctive phenotype: it had very low activity with methylene blue at pH 8.0, the optimum pH for the membrane-bound enzyme, and very low activity with Oz as the terminal electron acceptor, indicating that it could not support Hz-dependent respiration. However, it had substantial activity with methylene blue as the electron acceptor at pH 5.5, the pH optimum of solubilised hydrogenase (Van der Werf and Yates,
I
G Intergenic Amino MW
acids
PI Class Function Homology
ISOLATION
THE HUP2 GENE PRODUCT
The hup2 gene starts 16 bases downstream from the structural genes coding for the hydrogenase subunits. A mutation in hupZ (Du et al., 1994)
hup&
2
531 177 20125 10.95 ? E.coli
0296 0121
= P-ribosyl-cAMP
3
399 -1
OF THE HUJ’f GENFS
The hupP genes were isolated by complementing the mutant MCD124 by conjugation, using a gene library of A. chroococcum DNA in the wide-hostrange plasmid, pLAFR1 together with the helper plasmid pRK2013. Approximately 2000 tetracyclinresistant colonies were screened for Oz-dependent H3H uptake by growing individual colonies on Ucavity plates and exposing to Hz plus H3H in air for 20 min. Three transconjugants were isolated, each with a 25 kb insert which restricted to three EcoRI fragments of 3.0, 7.4 and 15 kb, respectively. The 7.4 kb fragment complemented both phenotypes, whereas a 2.7 kb EcoRI-BamHI fragment restored hydrogenase activity to the leve1 and pattem of the wild-type but did not affect the pH shift phenotype. A sequence analysis of the 2.7 kb EcoRI-BamHI fragment, together with a further 0.5 kb downstream from the BumHI site, revealed five open reading frames Gembank accession number U48404, the first, orfl, being incomplete (Table 2). An analysis of this DNA, using kanamycin cassettes in suitable restriction sites in orfs 2, _I and 5 and transposon mutagenesis in orf4 and orf5, revealed that the mutation in orf4 produced both the HupP- and the pH shift phenotypes, whereas a mutation in orfs produced only the pH shift phenotype. That orf4 was responsible for the HupP- phenotype was confirmed by restoring wild-type activity by introducing, in the wide-host-range vector, pLAFR3, hupP DNA containing only orf4 as a complete orf.
of the hupP region of Azorobacfer chroococcum DNA (PRcH
Table 2. Proper&
ORF
867
51
133 15416 6.71
110 11909 5.01
PRcH PRcH
PRcH PRcH
5
579
330 -8
hydrolase)
4
738 27
192 20931 7.78 HupP E. di 0261 0131
246 26959 6.51 Hydrophobic Membrane protein E. di 0154
M. G. Y ates et al.
868
Bishop P. E. and Eady R. R. (1985) Nitrogen fixation by produced an identical phenotype to that of the a nifHDK deletion strain of Azotobacter vinelandii. In HupP- mutant, MCD124: high hydrogenase activity Nitrogen Fixation Research Progress (H. J. Evans, P. J. at pH 5.5; low activity at pH 8.0 with methylene Bottomly and W. E. Newton, Eds), p. 622. Martinus blue as the electron acceptor and low activity with Nijhof, Dordrecht. Burns R. C. and Hardy R. W. F. (1975) In Nitrogen O2 as the electron acceptor. A double mutation, Fixation in Bacteria and Higher Plan&. Springer, New obtained by introducing the mutated hupZ gene York. into the chromosome of MCD124, gave a similar Dalton H. and Postgate J. R. (1969) Effect of oxygen on activity pattern to that of the two single mutants. the growth of Azotobacter chroococcum in continuous However, the mutation in hupZ did not affect the culture. Journal of General Microbiology 54, 463473. pH of the culture during growth in the manner of a Dilworth M. J. and Glenn A. (1984) How does a legume nodule work?. Trends in Biochemical Sciences 9, 519hupP mutation. Moreover, the putative proteins 523. expressed by hupZ and orf4 are very different: Dixon R. 0. D. (1972) Hydrogenase in legume root HupZ has four pronounced hydrophobic domains nodule bacteroids: occurrence and properties. Archiv fur and is a typical membrane-type protein, possibly Mikrobiologie 85, 193-20 1. containing a b-type cytochrome (Du et al., 1994). D’Mello R., Hill S. and Poole R. K. (1994) Determination of the oxygen affinities of terminal oxidases in On the other hand, ORF4 of the hupP region Azorobacter vinelandii using the deoxygenation of oxyleshows only weak hydrophobicity in two regions at ghaemoglobin and myoglobin: cytochrome bd is a low the N- and C-terminals, respectively and shows affinity oxidase. Microbiology 140, 1395-1402. Dross F., Geisler V., Lenger R., Theis F., Krafft T., some homology to proteins that interact with Fahrenholz F., Kojro E., Duchene A., Tripier D., DNA. Finally, to emphasise the differente between Juvenal K. and Kroger A. (1992) The quinone-reactive these two genes, they do not cross-complement: the Ni/Fe hydrogenase of Wolinella succinogenes. European hupZ gene does not complement the HupP- mutant Journal of Biochemistry 206, 93-102. and the hupP region does not restore activity to the Drozd J. W. and Postgate J. R. (1970) Effect of oxygen on acetylene reduction, cytochrome content and respiratory hupZ- mutant. activity of Azotobacter chroococcum. Journal of Genera1 Since neither of these two mutations affects other Microbiology 63, 63-73. substrate reductions: NADH-, malate-, succinateDu L., Stejskahl F. and Tibelius K. H. (1992) and lactate-dependent respiration activities were Characterisation of two genes (hupD and hupE) required similar in both mutants to those in the wild-type, for hydrogenase activity in Azotobacier chroococcum. FEMS Microbio1og.y Letters 96, 93-102. these two proteins must be regarded as specific components of Hz-dependent respiration. HupZ is a Du L., Tibelius K. H., Sousa E. M., Garg R. P. and Yates M. G. (1994) Sequences, organisation and analytypical membrane protein and is, therefore, a likely sis of the hupZMNOQRTV genes of the Azotobacter component of the Hz-dependent respiratory chain. chroococcum hydrogenase gene cluster. Journal of Indeed, it shows considerable homology to HydC Molecular Biology 242, 5499557. (Du et al., 1994) a protein subunit necessary for N? Du L. and Tibelius K. H. (1994) The hupB gene of the Azotobacter chroococcum hydrogenase gene cluster is vitro activity of the purified Wolinella succinogenes involved in nickel metabolism. Current Microbiology 28, nickel-iron hydrogenase (Dross et al., 1992). 21-24. Whether ORF4 is a membrane protein or acts in a Evans H. J., Hanus F. J., Haugland R. A., Cantrell M. processing or regulatory role is not known at preA., Xu L.-S., Russell S. A., Lambert G. R. and Harker A. R. (1985a). Hydrogen recycling in nodules affects sent. nitrogen fixation and growth of soybeans. In World This review has indicated some progress in the Soybean Research Conference HI (R. Shibles, Ed.), pp. understanding of Hz-dependent respiration in 935-942. Westview Press, London. Azotobacter: it is clearly beneficial to the Nz-fixing Evans H. J., Hanus F. J., Russell S. A., Harker A. R., Lambert G. R. and Dalton D. A. (1985b). Biochemical organism under carbon- or phosphate-limiting concharacterisation, evolution and genetics of Hz recycling ditions; it is not dependent solely on cytochrome d in Rhizobium. In Nitrogen Fixation and CO, as the terminal cytochrome oxidase, but can use an Merabolism (P. W. Ludden and J. E. Burris, Eds), pp: alternative oxidase or oxidases and we have ident3-11. Elsevier, New York. ified two proteins associated specifically with HZ- Evans H. J., Zuber M. and Dalton D. A. (1987) Some processes related to nitrogen fixation in nodulated dependent respiration, in addition to those of the legumes. Philosophical Transaciions of the Royal Society hydrogenase enzyme itself 317, 143-160.
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Ford C. M., Garg N., Garg R. P., Tibelius K. H., Yates M. G., Arp D. J. and Seefeldt L. C. (1990) The identification, characterisation, sequencing and mutagenesis of the genes (hupSL) encoding the smal1 and large subunits of the Hz-uptake hydrogenase of Azotobacter chroococcum. Molecular Microbiology 4, 999-1008.
Hoch G. E., Little H. N. and Burris R. H. (1957) Hydrogen evolution from soybean root nodules. Narure, London 179,430-43
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Jones C. W. and Redfearn E. R. (1967) The cytochrome system of Azotobacter vinelandii. Biochimica et Biophysica Acts 143, 340-353.
Oz, Hz and Nz fixation Azobacter Kelly M. J. S., Poole R. K., Yates M. G. and Kennedy C. (1990) Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air. Journal of Bacreriology172, 6010-6019. Linkerhagner K. and Oelze J. (1995) Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation. Journal of Bacteriology 177, 6018-6020. Mclnerney M. J., Holmes K. S., Hoffman P. and Dervartanyan D. V. (1984) Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d. European Journal of Biochemistry
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Robson R. L., Cheshyre K., Wheeler K., Jones R., Woodley P. and Postgate J. R. (1984) Genome size and complexity in Azotobacter chroococcum. Journal of General Microbiology 130, 1603- 1612. Schubert K. R. and Evans H. J. (1976) Hydrogen evolution: a major factor affecting the efficiency of nodulated symbionts. Proceedings of the National Academy of Sciences of the United Stak of America 73, 1207-12il._ Smith L. A., Hill S. and Yates M. G. (1976) Inhibition by acetylene of conventional hydrogenase in nitrogen-fixing bacteria. Nature, London 262, 209-210. Tibelius K. H.. Du L.. Tito D. and Steiskal F. (1993). The Azotobacter chroococcum hydrogenase gene chrster: sequences and genetic analysis of four accessory genes, hupA. hupB, hupY and hupC. Gene 127, 53-61. Tibelius K. H., Robson R. L. and Yates M. G. (1987) Cloning and characterisation of hydrogenase genes from
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Azotobacter chroococcum. Genetics 206, 285-290.
Molecular
and
General
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