how is nitrogenase regulated by oxygen?

FEMS Microbiology Reviews 54 (1988) 111-130 111 Published by Elsevier

FER 00087

How is nitrogenase regulated by oxygen?

Susan Hill

AFRC - IPSR Division of Nitrogen Fixation, University of Sussex, Brighton, U.K.

Received 26 June 1987 Accepted 3 September 1987

Key words: Nitrogen fixation; Aerobic regulation; Protein synthesis

1. SUMMARY

Oxygen can be either beneficial or detrimental for diazotrophy in organisms capable of an aerobic catabolism. It supports the production of a substrate for nitrogenase (ATP), but it can also inhibit the activity and repress the synthesis of this enzyme. Here, aspects of the relevant physiology are reviewed with particular emphasis on those relating to the mechanism of O 2 regulation of nitrogenase synthesis.

2. INTRODUCTION

Two kinds of nitrogenase are known, one hav- ing Mo, the other Va, at or near the N2-binding site. Both catalyse the reduction of dinitrogen to ammonia. Besides a source of electrons and pro- tons, this process, although exergonic, requires a lot of MgATP. (The minimum ATP/N 2 ratio in vitro is 16. [1,2]) Nitrogenases are inactivated by O2, thus anaerobic conditions are needed for activity and the purification of the two component proteins (the Fe protein and the Mo(Va)Fe pro-

Correspondence to: S. Hill, AFRC-IPSR Division of Nitrogen Fixation, University of Sussex, Brighton BN1 9RQ, East Sus- sex, U.K.

tein) [3-5]. The ability to fix N 2 is restricted to prokaryotes but is widely distributed amongst obligate anaerobes, facultative anaerobes and obligate aerobes [6].

The structures and properties of Mo-containing nitrogenases are very similar from all sources so far examined [6-10]. Those of the recently identified Va-containing nitrogenase of Azotobacter chroococcum do not differ markedly [5]. The Fe proteins of Mo nitrogenases are generally more O 2 sensitive (e.g., l l / 2 in air 30 s) than the MoFe proteins (e.g., tl/2 in air 8 rain). On the other hand the VaFe protein of the alternative nitrogenase does not show this relative air-tolerance (tl/2 in air 40 s) [5,11]. An increase in 02 sensitivity of the conventional Fe protein is correlated with MgATP binding and a change in con- formation. This change is required for electron transfer to the MoFe protein which is accom- plished by hydrolysis of the ATP [9].

The 02 sensitivity of the electron-donating processes for nitrogenase activity may differ according to the diazotroph. In the facultative anaerobe Klebsiella pneumoniae an O2-sensitive pyruvate:flavodoxin oxidoreductase is involved [2,7]. In the obligate aerobes the route of electron transfer is less clear, but may involve components of the respiratory chain [2].

The sensitivity of nitrogenase function to 0 2 markedly influences the physiology of aerobic di-

0168-6445/88/$06.65 © 1988 Federation of European Microbiologacal Societies

112

azotrophy. Therefore init ial ly I summarise the mean ing and measurement of the ambien t O 2 status.

3. H O W A R E A E R O B I C C O N D I T I O N S D E F I N E D ?

3.1. 0 2 status of a microbial environment

The O 2 status of a microbial env i ronment depends on the relative rates of 0 2 supply and demand. Excess 0 2 is ma in t a ined unt i l the rate of d e m a n d exceeds the rate of supply, 0 2 then becomes l imi t ing which means that the ambien t concen t ra t ion is near the K m of the highest affin- i ty O2-consuming process. Eventual ly anaerobiosis is reached. Factors inf luencing the rate of supply and d e m a n d [12] are as follows:

3.1a. Supply (i) part ial pressure of 0 2 ( p 0 2 ) in the gas, (ii)

rate of gas supply to the liquid, (iii) rate of t rans-

fer of 02 from the gas to the bulk liquid (medium or buffer in which consuming processes are

bathed), (iv) Bunsen absorpt ion coefficient for 0 2 in the bu lk l iquid (which may be referred to as the activity coefficient).

3. lb. Demand (i) popu la t ion density, (ii) O 2 uptake character-

istics of the consumer, which may depend upon genetic and other env i ronmenta l factors besides 0 2 status, (iii) ambien t dissolved O 2 concentration.

3.2. Methods for measuring 0 2 and controlling supply

The paramagnet ic [13], mass [14] and thermo- conduct iv i ty [15] properties of O 2 are used to

Table 1

Methods for measuring dissolved 02

(M) indicates that probes can or are always separated from the bulk liquid by an O2-permeable membrane. Errors in measurements by membrane-covered probes can arise from 02 uptake within the unstirred layer of bulk liquid adjacent to the membrane even with liquid agitation [34]. With probes bathed in a liquid and separated from the bulk liquid by a membrane the units of measurement should be dissolved 02 tension (pO2), rather than concentration, if the calibration involves the bulk liquid in equilibrium with a known pO 2 in the gas phase [12]. Relating the signal to 0 2 concentration can be achieved by the stoichiometric evolution [29] or consumption [35] of 02 in the bulk liquid, provided that there is no gas phase. In this review, for comparative purposes, levels of dissolved 02 (DOC) are shown as concentrations by assuming, where appropriate, that the small difference between the Bunsen absorption coefficient for 02 in the bulk liquid and in H20 are insignificant.

Principle Signal Sensitivity

0 2 tension 0 2 concentration (kPa) # m ppb

(/~g.1-1)

References and comments

1. Electrochemical a) polarographic (M)

b) galvanic (M)

2. Physical a) mass spectrometry (M)

3. Biological a) haemoglobin

e.g. leghaemoglobin

amps 0.2

amps 0.2

ion current

optical absorption spectrum

b) photobacteria (M) photons - 0.003

2 64

2 64

0.25 8

0.004 0.13

- 0.003 -1.0

Clark principle [29] For microelectrodes see [30] Borkowski and Johnson type [31]

[141

Sensitivity and range depends on the ratio of rate constants for dissociation and association of 02 [20,21] Sensitivity and range depends on the apparent K m of the 02 uptake process [32,33]

measure the partial pressure of 02 in the gas phase. Paramagnetic 02 analysers are used exten- sively to monitor 02 consumption by batch and continuous culture but they are being replaced by mass spectrometry which provides facility for monitoring other gas components.

The methods for measuring 02 in solution together with their sensitivities are shown in Table 1. The maintenance of constant dissolved O 2 by feedback control from the O 2 probe can employ changes in (a) rate of liquid agitation, (b) composition of gas and (c) rate of supply of an oxygenated buffer to the reaction vessel. The first two methods involve a gas phase and are commer- dally available, in conjunction with electrochemical 02 probes, to provide a wide range of 02 supply required for batch growth. Stirrer control is particularly useful in short-term assays requiring a closed vessel for measuring gas components (e.g., CEH 2 reduction assays; [16]). Control of the gas composition, (pO2) , provides a slower rate of change of O 2 input, but is useful in chemostat work [17] and in experiments involving exposure to excess O 2 [18,19]. The controlled addition of an oxygenated buffer to a liquid-filled reaction vessel has been used in conjunction with leghaemoglobin [20,21,22] or electrochemical and photobacterial probes [23]. Extensive physiological studies with symbiotic and free-living diazotrophs (e.g., [24-28]) have been achieved with leghaemoglobin to monitor and thence maintain very low O 2 concentrations where C2H 2 reduction can be moni- tored by degassing samples of effluent liquid [15].

4. WHAT EFFECT DOES 0 2 HAVE ON NITROGENASE ACTIVITY IN VIVO?

Aerobic diazotrophs need an adequate supply of 0 2 for ATP production, but the supply must not exceed their demand for optimum nitrogenase activity. Thus a bell-shaped curve is obtained when nitrogenase activity is plotted against 02 concentration. At suboptimal 02 the system is energy limited. As the O2 supply increases and the optimum is passed nitrogenase activity declines. This inhibition of nitrogenase activity by excess O 2 is reversible in many diazotrophs (see section 4.2.).

113

4.1. 0 2 gradients and other means of keeping 0 2 from nitrogenase

The optimum dissolved 0 2 concentration for nitrogenase activity varies in different diazotrophs, and ranges over 4 orders of magnitude (Table 2). It can be influenced by the O 2 status during growth and in the assay (Table 2). The magnitude of the O 2 gradient between ambient and the site of nitrogenase function, is affected by the rate of O 2 uptake and resistance to O 2 diffusion. In addition, spatial or temporal separation of O 2 metabolism from N 2 fixation is achieved by morphological and biochemical differentiations.

Respiratory activity as a protective device to prevent O 2 inhibition of nitrogenase activity is most highly developed in Azotobacter. This diazotroph can adjust its 02 demand to' satisfy a very wide range of 02 supply (Table 2), and exhibits one of the highest rates of respiration. The concept of respiratory protection of nitrogenase is reviwed elsewhere [36-40]. The probable components contributing to a flexible respiratory activity in response to O 2 supply for diazotrophy are as follows:

(1) Changes in levels and activities of enzymes of carbon metabolism to provide flexibility in the electron pressure for differential flux through the various limbs of the respiratory chain, e.g., (a) sugar catabolism [41-43], (b) tricarboxylic acid cycle components [44-46], and (c) polyhydroxy- butyrate metabolism [41,47].

(2) Changes in the levels and activities of components of a branched respiratory chain to provide flexibility of coupling for adequate 02 consumption [48,49] e.g., (a) dehydrogenases including uptake hydrogenase (b) sites and efficiency of energy transduction and (c) terminal oxidases with different K m values [24,50].

(3) Activity of futile cycles, for the dissipation of unwanted electrical membrane potential or ATP. However, such activity has not yet been correlated with uncoupled respiration in Azoto- bacter (see [44] for discussion) or other diazotrophs.

Many aerobic diazotrophs lack the ability to cope with high rates of O 2 supply and are therefore microaerophilic when fixing N 2. The spatial

114

Table 2

Examples of the optimum dissolved oxygen concentration (DOC) for nitrogenase activity in various diazotrophs, and of the influences of growth and assay conditions on this optimum

Assay conditions are: 1. steady-state diazotrophic growth, 2. C2H 2 reduction without haemoglobin, 3. C2H 2 reduction with haemoglobin ( k 2 / k I 10 -6 M) from (a) soybean (0.04) (b) Sesbania rostra (0,02) (c) sperm whale myoglobin (0.786) (d) French bean.

Diazotroph Growth condition Assay Optimum Refer- Comment condition DOC ence

(/~m)

Anabaena oariabilis high 02 2 300 [66] /

J Plectonema boryanum low 02 2 < 12 [71]

Frankia free-living, high 02 2 150.0 [64] /

J free-living, low O 2 2 3.0 [64]

Gloeothece stationary phase 2 80 [62] } logarithmic phase 2 27 [62] _ see also [72]

Azotobacter high O 2 2 25.0 [69] chroococcum low 02 2 12.0 [69]

Bradyrhizobium soybean bacteroids 2 4.0 [25]~ japonicum 3c 2.0 I2511

3a 0.1 [25]~

Rhizobium ORS571 0.018 [74]~ 0.010 [7411 0.005 [741/

Sesbania bacteroids succinate as sub- lactate strate glucose in 3d

Rhizobium ORS571 free-living high 0 2 1 free-living high 02 3c free-living low 02 3a Sesbania bacteroids 3b

Klebsiella pneumoniae anaerobic or 3a microaerobic

5.9 [70]] 1.0 [28]~ 0.05 [28]~ 0.01-0.03 [28]]

0.02-0.04 [27]

The higher optimum DOC is correlated with the ability to form heterocysts (see also [65])

The higher optimum DOC is correlated with the formation of vesicles

The higher optimum DOC is correlated with a faster rate of 02 uptake

Theoretical aspects of the influence of the quality of the carbon substrate, which affects the rate of 02 uptake, and of the type of haemoglobin, which affects the O 2 supply, are also considered in [74]. The highest activity, found with the homologous haemoglobin, was correlated with the greater rate of ATP production [25].

Possible variations in the respiratory components arising from the 02 status during growth probably affects the ability of the different oxyhaemoglobins to support nitrogenase activity (see [28] [48]).

Nitrogenase activity is not restricted to anaerobiosis

a r r angemen t of respi r ing organ isms can inf luence the local 02 supply , and c lus ter ing p h e n o m e n a are assoc ia ted with N 2 f ixa t ion in mic roaeroph i l i c di- azot rophs . Examples of this p h e n o m e n o n are pel- l icle fo rma t ion in A z o s p i r i l l u m [51] and co lony d i m o r p h i s m [52,53] and, u n d o u b t e d l y the mos t spectacular , the legume nodule [25]. Steep O 2 grad ien ts can develop in such clusters [40,54]. Fea tu re s assoc ia ted with improv ing 02 d i f fus ion into the cluster are reviewed by Bergersen [40].

They inc lude aerotaxis in A z o s p i r i l l u m [55] and the presence of l eghaemoglob in in legume nodules. Leghaemoglobin , a prerequis i te for an effec- t ive symbiosis , funct ions to buffer and faci l i ta te O 9 supply [25,48,56] (Table 2). H a e m o g l o b i n s are also found in some ac t inorhiza l root nodules in which they m a y serve a s imilar func t ion [57]. Legumes and non- legumes car ry the h a e m o g l o b i n genes [58] whereas the haem moie ty is p r o b a b l y of p roka ryo t i c or igin [48]. The d i f ference in the 0 2

association and dissociation kinetics of the various plant haemoglobins may be related to the properties of the respiratory enzymes (e.g., K m of the terminal oxidases) belonging to the resident endo- phyte [48] (Table 2).

Changes in physiology associated with diazotrophy also occur in cyanobacteria and involve the separation of the O2-evolving Photosystem II from nitrogenase. In some filamentous forms the latter is located in the morphological distinct heterocysts (Table 2), which do not contain the 02- evolving Photosystem II [59,60]. In the unicellular forms the two processes are temporally separated (Table 2) [61] and in this nitrogenase is mainly driven by respiration [62].

Striking morphological changes occur under an aerobic N limitation in two diazotrophs. They are the development of heterocysts in cyanobacteria [59,60,63] and the appearance of vesicles in the actinomycete Frankia [57]. The differentiation leading to vesicle formation [64] and the maturation of heterocysts [65] are correlated with an elevation in 02 supply from microaerobic to aerobic conditions (Table 2). The evidence that these structures provide a diffusion barrier for 02 entry comes from studies of cytology [57,59] and of physiology. The latter concerns the comparative response of the differentiated with the undifferen- tiated structures to environmental gas pressure [57,59,65-67]. Diffusion barriers are also associated with particular host cells in legumes [25] and in actinorhizal nodules [57]. In some legume nodules the efficacy of this barrier may be under physiological control [54,68].

4.2. 02 inhibition

Inhibition of nitrogenase activity in vivo by O 2 ranges from slight to complete depending upon the severity of the 02 stress. Upon removal of the 02 stress and in the absence of protein synthesis the inhibition can be completely, partly or not at all reversed. The reversible inhibition, first characterised in Azotobacter [69,75] indicates that nitrogenase can exist in a state which is inactive towards reducible substrate and protected from 02 damage. Additional evidence (see [36] and below) suggests that, in Azotobacter, this protection arises

115

from a protein-protein interaction and is some- times known as a conformational change. Reversi- ble inhibition by 02 is seen in other diazotrophs, including obligate anaerobes (Table 3) but protection by conformational change involving a redox protein may only occur in Azotobacter.

Nitrogenase in crude extracts of Azotobacter sediments as an air-tolerant complex which con- tains a third redox protein (called the Shethna, FeSII, or protective protein). Stability towards 02 involves the FeSII protein and requires Mg 2÷ [36]. The conditions in vitro leading to complex formation are not clear [76,77] but probably require oxidation of the component proteins [78] under controlled redox conditions [79] for maximum stability towards 02. The Fe protein of nitrogenase can reduce the FeSII protein. Hence, changes in electron flux to nitrogenase in vivo might regulate complex formation [80]. Protection from 02

Table 3

Diazotrophs showing reversible inhibition by 02 of nitrogenase activity

The reversibility occurs in the presence of a protein synthesis inhibitor, b Evidence for a complex formation involving nitrogenase is found in crude extracts of X. flavus (by sedimen- tation [100]) but not of Azospirillum, K. pneumoniae, or R. capsulata (by chromatography and immunology) [100a]. How- ever, K. pneumoniae-purified nitrogenase is protected from 02 damage by the FeSII protein isolated from A. vinelandii (Yates, M.G., personal communication), c Inhibition of activity by 0 2 does not lead to a modification of the Fe protein of nitrogenase as occurs during NH~ inhibition in some Azospiri!lum sp. [96].

Diazotroph Reference

Azotobacter chroococcum a [69,75] Azotobacter vinelandii [81 ] Derxia gummosa [52] Xanthobacterflatrus b [95] Azospirillum brasilense a.b,c [961 Azospirillum lipoferum [96] Anabaena spiroides a

(without mature heterocysts) [65] Anabaena sp. strain CA a

(with mature heterocysts) [97] Rhodopseudornonas capsulata b [16] Rhodospirillum rubrum [16] Chromatium vanosum [16] Klebsiella pneumoniae a,b [85,98] Desulfovibrio gigas a [99]

116

damage apparently occurs rapidly in vivo and is independent of the time and magnitude of the 0 2

stress but diminishes during growth at high dissolved 02 concentrations [81]. The FeSII protein may have other functions in vivo, since its synthesis is not regulated by ammonia [82,83] or O 2 [82].

Diazotrophic growth dependent upon the Va nitrogenase in Azotobacter chroococcum is less aerotolerant than that supported by Mo nitrogenase. In crude extracts the Va nitrogenase, unlike the Mo nitrogenase, does not sediment as a complex and is more sensitive to exposure to O 2 [5]. Thus, the Va nitrogenase may not form an 02 stable complex with the FeSII protein. However, this strain of A. chroococcum carries several mutations [84] which may inadvertantly influence FeSII protein synthesis.

In addition to conformational protection of the Mo nitrogenase by the FeSII protein in Azoto- bacters, the reversibility of 02 inhibition probably involves, in this genus and other organisms, the diversion of electrons away from nitrogenase to 02 [2,75]. Such a diversion could account for the simultaneous effect of cyanide on respiration, which is inhibited, and on nitrogenase, which apparently becomes more oxygen tolerant, in Rhodopseudomonas capsulata [61] and Klebsiella pneumoniae [85].

The mechanism and products of 02 inactivation of nitrogenase are not fully characterised [36]. Nitrogenase may generate as well as be damaged by the potential toxic activated 02 species associated with aerobic metabolism [86]. Increased levels of superoxide dismutase, catalase [87-89] and ascorbate peroxidase [90,91] are in some cases correlated with nitrogenase levels. However, mutants with elevated levels of catalase do not exhibit a greater tolerance of diazotrophy to oxygen than the wild-types [92,93].

5. WHAT EFFECT DOES 02 HAVE ON CATABOLISM IN RELATION TO N 2 FIXA- TION?

Estimates of the energy requirement for N 2

fixation, during growth, are based upon compar- ing the efficiency of carbon and energy source

utilisation in N2-fixing and in NH~--assimilating populations. Table 4 compares efficiencies of N incorporation in some heterotrophs grown on various N sources in carbon and energy limited chemostats. (The data for Klebsiella aerogenes grown on NH~- or N O f are included for comparison.) Differences in the energy requirement for processes other than N 2 fixation are minimised in the controlled environment of a chemostat, particularly when the carbon and energy source limits growth.

The data in Table 4 illustrate three points. (1) Efficiencies are generally higher during

aerobic growth than during anaerobic growth due to aerobic respiratory energy transduction (oxida- tive phosphorylation). Although K. pneumoniae fixes N 2 anaerobically, limiting 02 improves catabolism during diazotrophy, which is consistent with a very low concentration (30 nM 02) stimu- lating nitrogenase activity (section 4.1) and synthesis (section 6.1) in this facultative anaerobe.

(2) The efficiency of N incorporation in Azotobacter is influenced by 02 supply. Increasing the O2 supply leads to a lower efficiency of N incorporation in N2-fixing populations, reflecting carbon and energy source used for respiratory protection (section 4.1). However, the magnitude of the response apparently depends upon how the O 2 supply is controlled (note to Table 4). The 02 supply also influences the efficiency of N incorporation in the NH~--assimilating population of A. vinelandii (Table 4). Thus, the mechanisms controlling respiratory protection may not be specific to N2-fixing populations.

(3) The carbon and energy source apparently utilised for N 2 reduction (g carbon and energy source/g N 2 reduced) (Table 4) reflects the energy (ATP and reductant) required for N 2 fixation. A. chroococcum, when grown at a high dilution rate; apparently requires much less energy than do the other aerobes and anaerobes. How- ever, this calculation assumes that the coupling of catabolism to energy transduction is similar in both the NH~- and the N2-fixing populations, and that the reducing equivalents required by nitrogenase are negligible. This is the case during anaerobic growth where ATP is generated from substrate phosphorylation alone and reducing

117

Table 4

Energy requirement for N 2 fixation in heterotrophs grown in chemostats limited by the carbon and energy source

Changes in O 2 supply to chemostats of Azotobacter can be achieved in two ways. In the populations of A. vinelandii, the dissolved oxygen concentration (DOC) was maintained at a predetermined concentration by varying the air feed. The DOC giving the maximum and minimum EN2 are shown (see [17] for additional data). On the other hand, in the population of -4. chroococcum the DOC was not controlled and the chemostat was fed with a constant air flow so that an increase in 02 supply was achieved by decreasing D. The highest EN2 occurs in A. chroococcum growing at the higher dilution rate. Presumably here the 02 supply is just in excess of that needed to support a particular growth rate limited by the steady-state concentration of the carbon and energy source. (It is assumed that the differences in species do not contribute to these effects.) NA, not applicable NR; not reported; PC, personal communication from Professor D. Tempest.

Organism D DOC (/~m) Efficiency of N incor- g carbon and Carbon Reference (h-1) with potation (E) (mgN/ energy source and

g carbon and used for lg N 2 energy energy source reduced (1/EN2310 source used) with

- - I/ENH 2 ) X

N 2 NH~" N 2 NH~

Anaerobic growth C. pasteuranium 0.22 0 0 K. pneumoniae 0.10 0 0

Aerobic growth K. pneumoniae 0.11 < 1 < 1 .4. chroococcum 0.23 5-10 40 A. chroococcum 0.05 5-10 40 A. vinelandii 0.15 2-10 2-10 A. vinelandii 0.15 180 180 Rhizobium ORS571 0.10 2 2 K. aerogenes 0.85 NA NR K. aerogenes 0.28 NR NR

11 22 46 sucrose [101] 8 19 72 glucose [102,98]

15 35 38 glucose [102,98] 33 40 5.3 mannitol [105] 7 42 119 mannitol [105]

16 63 47 sucrose [17,106] 7 38 117 sucrose [17,106]

23 40 19 succinate [107] NA 75 NA glycerol [108] 43 (with NO~- ) 65 8 (for NO 3 glucose PC

reduction)

equivalents are in excess [2,101,102]. On the other hand, during aerobic growth energy coupling depends on many factors (section 4.1) and during diazotrophy there is an additional complication from the recycling of electrons through the uptake hydrogenase from the H 2 evolved by nitrogenase [2,103]. The evidence that such recycling occurs and when it is beneficial is reviwed elsewhere [39,103].

6. HOW IS N I T R O G E N A S E SYNTHESIS REG- U L A T E D BY O27

02 can inhibit nitrogenase activity either re- versibly or irreversibly in vivo (section 4.2). There- fore, measurements of parameters which do not rely on activity are needed to determine the effect

of 0 2 o n nitrogenase synthesis. (One suitable parameter is the O2-stable product, fl-galactosidase, arising from the expression of nif::lac gene fusions.) Such measurements show that excess 0 2 represses the synthesis of nitrogenase in a variety of different diazotrophs (Table 5). Conversely, an 0 2 limitation can curtail nitrogenase synthesis as seen in the obligate aerobe Azotobacter chroococ- curn [109].

Regulation of nitrogenase synthesis by 0 2 is less well understood than that by fixed N for the following reasons: (1) the O 2 lability of nitrogenase, (2) the technical sophistication required to measure and maintain a given dissolved oxygen concentration (section 3), (3) the absence of a straightforward method for selecting oxygen constitutive mutants (section 6.7) and (4) the relative lack of knowledge relating to the molecular

118

Table 5

O 2 repression of nitrogenase synthesis in various diazotrophs

a Gene fusions are of the transcriptional, nif::lac, or of the translational nif-lac, type.

Diazotroph Parameter Reference

Facultative anaerobic heterotrophs Klebsiella pneumoniae

Facultative anaerobic phototrophs Rhodopseudomonas capsulata

Obligate aerobic heterotrophs Bradyrhizobium japonicum Bacteroids (lupin) Rhizobium strain NZP 2257 hgotobacler chroococcum

Obligate aerobic phototrophs Anabaena Gloeothece

nif:: lac a [115] nif mRNA [19,116,117] nitrogenase polypeptides [114,118]

nifH-lac a [119] nitrogenase polypeptides [120]

nitrogenase polypeptides [121,122] nitrogenase polypeptides [123] nitrogenase polypeptides [124]

nifIf mRNA [125] nitrogenase component I [126] polypeptides

biology of oxygen regulation (section 6.6) compared to that of N regulation by the ntr control system [110-112]. Nevertheless, progress has been made, particularly with the facultative anaerobe Klebsiella pneumoniae where extensive knowledge of the nitrogen fixation genes (nif) is an ad- vantage. Therefore, most of the following relates to K. pneumoniae.

The nif gene cluster in K. pneumoniae (reviewed elsewhere [110,111]) occupies 23 kb of the genome and is composed of at least 17 contiguous genes arranged in eight operons. The structural genes (nifHDK) are part of a single operon, but the synthesis of active nitrogenase requires at least five other nif gene products. The products of nifF (a flavodoxin) and of nifJ (pyruvate flavodoxin oxidoreductase) provide reducing power for nitrogenase function and those of the operon nifLA are involved in regulating nif gene expression.

Derepression of nif occurs under N limitation and is associated with anaerobic conditions, although very low 02 concentrations can be beneficial (see below). Expression requires the ntrA product (a sigma factor for RNA polymerase core enzyme) for activation by the ntrC product at the nifL promoter (Fig. 1). Transcription of all other

nif operons is activated by the nifA product. Hence, transcription of the nifLA operon precedes that of nifHDKY and the other nif operons tested [19]. But at least an hour elapses after nifHDKY transcription and translation are detected before nitrogenase activity is seen [19,114]. This lag may reflect the complexity of the processing of the polypeptides.

6.1. Transcriptional regulation by 0 e

Repression of nitrogenase synthesis by oxygen, like that by fixed N, occurs at the level of transcription in K. pneumoniae. These effectors inhibit the derepression of fl-galactosidase synthesis in strains carrying nif::lac transcriptional gene fusions [18,115,127-129], and when nif is dere- pressed they decrease the rate of synthesis of nil mRNA [19,116,117]. Repression by oxygen at the level of mRNA also occurs in Anabaena [125].

In K. pneumoniae expression from the nifL promoter, unlike that from other nif promoters, is insensitive to 6/ tM 02 [115,127]. This differential effect on the expression of nil promoters is similar to that obtained with the intermediate levels of fixed nitrogen (e.g. 3 mM NH~-) [127]. High levels

of fixed nitrogen (40 mM NH~) prevent and repress expression from the nifL promoter [19,127], but the effect of high levels of oxygen (> 6 /~M) is equivocal. Although nifL::lac derepression is 50% inhibited by 60 /~M 02 [115] synthesis of nifLA mRNA is not influenced by this 02 [19].

The elegant technique utilising leghaemoglobin to monitor low dissolved oxygen concentration shows that near the apparent K m of the terminal oxidase (approx. 100 nM 02) nifH::lac expression is 50% inhibited compared to the level under anaerobiosis [26]. On the other hand, at even lower levels (near 30 nM), oxygen can stimulate nifL::lac (Hill, S. unpublished results) nifH:: lac expression [27] and subsequently nitrogenase activity [27]. This stimulation of transcription is correlated with an elevated level of ATP [130]. However, the mechanism of regulation probably differs from that of a specific post-transcriptional control by energy status on anaerobic nitrogenase synthesis in K. pneumoniae [131,132].

6.2. Post-transcriptional regulation by 02

Under anaerobic derepressing conditions nif mRNAs are exceptionally stable [116,133,134]. Treatment with air, as with NH~-, enhances degradation of nifHDKY [116,134], nifUSVM and nifJ mRNA [134] but not that of nifLA mRNA, which resembles his and glnAntrBC mRNA in stability [134]. The relative stabilities of the preformed nif polypeptides to air treatment differ [135]. Notably nifH polypeptide is more stable than nifK and nifD polypeptides in vivo, whereas the reverse is observed with the nitrogenase proteins in vitro (section 2). Taken overall, the most O2-sensitive step leading to the synthesis of active nitrogenase in vivo is probably expression from the nifH promoter. This is because nitrogenase is detected during depression in 100 nM 02 which partially inhibits nifH::lac expression [27].

6.3. Involvement of nifL product in 0 2 regulation

In K. pneumoniae transcription of all nif operons, apart from nifLA, is activated by the nifA gene product [110,111], whereas the function of the nifL product is apparently to inactivate the

119

nifA product (see below). The evidence for the role of nifL in 0 2 regulation of nif expression is based upon the phenotypes of nifL mutants and the apparent stability of the nifA product to 02.

Nif + revertants of certain types of NifA- or of NtrC- mutants [143] carry mutations within the his distal region of the nifLA operon [18,127]. In some, the reversion probably arises from the imprecise excision of Mu from nifh implying that nifL is not necessary for nif expression [144]. One mutation has been physically mapped and carries a 400 base pair deletion in nifL [143]. Such Nif ÷ revertants (referred to here as carrying a nifL mutation) are pleiotropic and show the phenotypes listed below.

(1) After anaerobic growth following removal of excess NH~-, strains carrying a nifL mutation differ from the wild type by showing: (a) faster derepression of nitrogenase [19,127], (b) greater derepression with intermediate levels of fixed nitrogen [127], (c) greater derepression at 37°C [127] (which may be a function of nifA stability [136,145]), (d) uninhibited expression of nifH or nifN::lac in 6 /~m 02 or air [18,127], (e) slower repression of synthesis of nif mRNA [19,117] and of nif polypeptides [18,19] by 02 treatment and (f) less degradation of preformed nif mRNA by either 02 or fixed nitrogen treatment [134].

(2) Transfer of the nifL mutation into a genetic background which is normally Nif-, due to a mutation in the ntrBC region, results in a Nif ÷ phenotype [143]. Such a change probably arises from positive autoregulation by the nifA product.

(3) Cloning of the nifL mutation into a multicopy plasmid and transfer into a strain carrying chromosomal nif results in a Nil ÷ phenotype [143]. This is in contrast to the presence of multiple copies of wild-type nifL which inhibit chromosomal nif expression (see below).

This pleiotrophy can be explained by the nifL product functioning as a negative effector of nif expression, either as a repressor (by binding to nif promoters and blocking transcription) or as an anti-activator (by inactivating the nifA product). The following data are consistent with the latter.

(1) The amino acid sequence of nifL gene product does not reveal a DNA-binding domain [113,138].

120

(2) There is no obvious repressor-binding site in the vicinity of the transcriptional initiation site of nifA-activated promoters [111].

(3) The nifL product inhibits an activator function of the nifA product in vivo. Transcription from nifL, put, or hut promoters is activated by the products of ntrBC. The presence of multiple copies of the wild-type nifL gene does not affect this transcription. However, if the strain is NtrBC- transcription from these promoters is dependent upon the nifA product, then multiple copies of wild-type nifL (cloned separately or with nifA) inhibit this transcription [146,147]. Similarly, in A. oinelandii multiple copies of K. pneumoniae nifL do not inhibit the expression of wild-type nif. However, in regulatory mutants where K. pneumoniae nifA activates nil gene expression multiple copies of nifL inhibit this activation [148].

In addition, the nifL product may influence nif mRNA stability, although this could be accom- plished indirectly if the stability of nif mRNA, under derepressing conditions, involves the nifA product [134].

The mechanism whereby the nifL product is converted between neutral and the anti-activator forms in response to 02 or N status is unknown. Analysis of the DNA sequence of nifL reveals some structural homology with regulatory proteins involved in signal transduction and with the haem-binding site of c-type cytochromes [138]. However, haem is probably not involved in the mechanism of 02 regulation because 02 represses nitrogenase synthesis in a haemA- mutant of Escherichia coli carrying K. pneumoniae nif genes (Smith, Anthony and Hill, unpublished results). Multiple copies of nifL in the presence or absence of multiple copies of nifA inhibit the anaerobic expression of chromosomal nif [139,140]. This may be due to the predominance of the nifL product in the anti-activator form [139]. Conver- sion to the neutral form may involve one or more nif genes because strains that carry multiple copies of the complete nif cluster are Nif + and even derepress nitrogenase faster than those with chromosomal nif [19].

The nifA product is apparently unaffected by 02 in vivo. Air treatment inhibits chromosomal nifH::lac expression in a K. pneumoniae strain

carrying wild-type nif on plasmid pRD1, but not when the strain carries nifA cloned into a constitutive promoter on a high copy number plasmid (pMC71A) instead of pRD1 [136]. Of course, 02 inactivation of nifA product could go undetected if the nifA product were present in excess, so that another gene product limits expression from the nifH promoter, or if an elevated respiratory activity decreases the ambient dissolved oxygen concentration. Nevertheless, expression of K. pneumoniae nifA from pCK1 (a wide host range derivative of pMC71A) overcomes O 2 repression of nitrogenase synthesis in wild-type A chroococcum [137] and complements regulatory mutants which fail to synthesise nitrogenase under normal aerobic conditions [44].

A nifA-like regulatory gene has been identified in Rhizobium meliloti, Rhizobium leguminosarum, Bradyrhizobium japonicum (see [122,141] and references therein), Azotobacter vinelandii, Azoto- bacter chroococcum [124] (Kennedy, C., personal communication) and Rhodopseudomonas capsulata [142]. A nifL-like gene has not been identified in any other diazotroph. In B. japonicum a gene seemingly non-essential for nil (fix) expression is in the same operon and upstream from the nifA- like gene. However, mutations in the former do not lead to relief from 0 2 repression of nitrogenase polypeptide synthesis [122]. In A. chroococcum, a class of Fos- (inability to fix nitrogen with sugar) mutants fail to synthesise nitrogenase in air [44]. This phenotype is corrected by either lowering the atmospheric 0 2 concentration, by supplementing the medium with a carboxylic acid (which lowers the apparent K m for 0 2 uptake) or by introducing K. pneumoniae nifA on a recombinant plasmid (see above). Hence, these mutants appear to be hyper- sensitive to 0 2 repression of nitrogenase synthesis.

6.4. Does the regulation of nif expression by 02 and fixed N share common features?

02 represses synthesis of nifHDK polypeptides in a mutant (Asm-, Glu-) , which makes nitrogenase in the presence of ammonia [114]. Hence, the mechanisms preventing nitrogenase synthesis in response to 02 and N status are apparently different [114]. However, confirmation

is required with defined mutations in ntr and in nifL genes, since the pleiotrophy of NifL- mutants (section 6.3) suggests a common mechanism for the nil specific regulation by 02 and by intermediate levels of fixed N.

6.5. Are there any other nif genes besides nifL involoed in 02 regulation?

Further characterisation of the phenotypes of K. pneumoniae strains, carrying the 400 base pair deletion in nifL (see section 6.3) suggests that the products of the nifHDKY operon have a role in 02 regulation of nitrogenase synthesis. The evidence (Hill, S., unpublished results) rests basically upon the fact that 0 2 treatment inhibits N 2 fixation, thus influencing the N status, so as to regulate nitrogenase synthesis indirectly.

6.6. Does the mechanism of 02 regulation of nif in K. pneumoniae share common features with redox regulation in Enterobacteriaceae?

In the Enterobacteriaceae anaerobic catabolism by fermentation is associated with an incomplete tricarboxylic acid cycle [149]. However, aspects of this catabolism can persist under an O 2 limitation [150] and even when O 2 is in excess, provided that the source of energy does not limit growth [151]. On the other hand components of aerobic catabolism, e.g. respiratory chain components [150,152], are found under anaerobiosis even in the absence of an alternative terminal electron acceptor such as fumarate or nitrate [152]. Biosynthesis is mainly an anaerobic process because during aerobic growth little of the 02 originating in air is found in the biomass [152]. Nevertheless aspects of N metabolism are influenced by 02 status, for example the assimilation of histidine as N source requires aerobic metabolism [153] and in K. pneumoniae N 2 fixation is associated with anaerobiosis although limiting O 2 can be beneficial (section 5).

Rough estimates of random gene fusions and gel electrophoresis show that up to 50 genes may be subject to anaerobic induction in E. coli [154,155] and Salmonella typhimurium [156]. Ex- amples of enzymes known to be induced under anaerobiosis are shown in Table 6. In none is the

121

Table 6

Example of enzymes synthesised predominantly under anaerobiosis in Enterobacteriaceae

References are for recent papers in which other relevant references are quoted.

Enzyme Reference

Catabolic nitrate reductase [169] nitrite reductase [162] fumarate reductase [170] trimethylamine-N-oxide reductase [171] dimethylsulfoxide reductase [172] glycerol-3-phosphate dehydrogenase [173] pyruvate formate lyase [174] formate dehydrogenase (respiratory or fermentative) [159,165] hydrogenase (probably 3 types) [175,176]

Anabolic nitrogenase tripeptide permease [165] aminotripeptidase [177]

mechanism of anaerobic induction or 02 repression fully understood. The catabolic anaerobic reductases, e.g., nitrate and fumarate reductases are also induced by the specific terminal electron acceptor, thus maximising energy yield [152]. Hence, the regulatory signals may involve a redox couple within the cell rather than the 02 molecule. Such a proposal has been made for the regulation of nitrogenase synthesis in K. pneumoniae [157].

Nitrate represses the synthesis of nitrogenase in K. pneumoniae under anaerobic conditions [158]. Such repression requires the anaerobic metabolism of NO~- [157], which functions as a terminal electron acceptor and is reduced to NO 2 and then NH~-. Repression by NO3 occurs in a mutant strain (Asm- Gin-) that synthesises nitrogenase constitutively in the presence o f NH~-. Thus, repression by NO 3- is probably independent of the nitrogen regulatory system [157] encoded by the ntr genes, although confirmation with defined ntr mutants is needed. Fumarate also functions as a terminal electron acceptor under anaerobiosis in K. pneumoniae. It hardly influences nif expression [159] which these authors consider is consistent with a common regulatory mechanism involving redox status for repression by 02 (E~ O2/H20,

122

+0.82 V), NO 3 (E~ NO3-/NO~-, +0.42 V) and fumarate (E~ fumarate/succinate, + 0.031 V).

The best-characterised regulatory locus of anaerobic induction in the Enterobacteriaceae is fnr (nirR or oxrA) [160]. Although expression of fnr is independent of anaerobiosis [161] the product is required for transcriptional regulation of genes encoding anaerobic reductases, e.g., NO 3, NO~- and fumarate reductases (see Table 6 and [162] for references). The amino acid sequence of the fnr product has a presumptive redox sensing site [163], as well as features consistent with a role in transcriptional regulation similar to the cyclic AMP receptor protein [160]. However, E. colifnr- mutants carrying plasmid-borne K. pneumoniae nif genes synthesise nitrogenase [164], so the expression of nil apparently does not require the fnr product.

In S. typhimurium, mutations in oxrC affect anaerobic induction of several enzymes associated with fermentation and a tripeptidase (pepT) [165] (Table 6). The genes under oxrC control are not influenced by fnr and vice versa [162,165]. The oxrC mutation depresses the level of phosphoglu- cose isomerase (PGI); thus, a product of glycolysis may function as an anaerobic regulator [165]. The oxrC mutation prevents glucose fermentation but other sugars, that enter the glycolytic pathway below PGI, suppress the pleiotrophy. To determine the effect of oxrC- on nif expression is complex because an energy limitation specifically inhibits anaerobic nif expression [131]. Neverthe- less, with pyruvate as the carbon energy source, anaerobic expression of K. pneumoniae plasmid- borne nil occurs in a S. typhimurium oxrC- mutant (Postgate, J.R., personal communication).

The degree of supercoiling of the bacterial chromosome may play a role in promoter selectiv- ity [166]. In S. typhimurium aerobic growth is apparently correlated with high topisomerase I activity and the relaxation of the chromosome, whereas anaerobic growth is associated with high DNA gyrase activity [167]. Sub-lethal concentration of antibiotics, which inhibit DNA gyrase apparently selectively curtail anaerobic nif expression in K. pneumoniae and Rhodopseudomonas capsulata. Hence, DNA supercoiling may have a role in 02 regulation of nif expression [119]. How-

ever, Lilley [166] points out that the action of these antibiotics may not be specific in vivo.

An attenuator-like mechanism for 02 regulation is suggested by Buck and Ames [168], who found aerobic growth-dependent tRNA modification in S. typhimurium. A pleiotropic mutation miaA, which prevents this aerobic modification [168] however, fails to influence the expression of three apparent ly O2-regulated loci in S. typhimurium [156]. Expression of nif genes has not been examined in miaA mutants.

6. 7. A model for (9: regulation of nif expression in K. pneumoniae

The current model of nif regulation by fixed N and 02 in K. pneumoniae is shown in Fig. 1. It depicts the two-tier regulation by fixed N at the nifL promoter and other nif promoters. It indicates the three probable interactions of 02 status with nil gene expression. High levels of 02 inhibit expression at the nifL promoter (sections 6.1 and 6.4) possibly involving DNA supercoiling (section 6.6). At other nif promoters 02 regulation in-

l~n]rA I, I ~,~_ __ ~ ~ ~ I glnA i ~ ntrB I ntrC i

i nlfA j nlfL Ip4,, n fK D H

I - ~ [ ~ / 02 ~ I nltr°genose i

I :(~) l n t N I I

I I

Fig. 1. The current model for nif regulation in K. pneumomae. The two-tier regulation by high (hi) and intermediate (int) levels of fixed N (N) are shown, together with the three apparent interactions of 02. An ambient 02 of 60 #M (1) inhibits derepression of nifLA, of 0.1/LM (2) inhibits derepression of other nif operons via the nifL product and of 6 #M (3) inhibits nitrogenase activity, which through N deprivation indi-

rectly prevents nitrogenase synthesis.

volves the nifL product as an anti-activator of nifA function (sections 6.3 and 6.4). The mechanism whereby the nifL product responds to 02 status does not seem to share common features with other O z or redox signal transducing mechanisms in the Enterobacteriaceae (section 6.6). In fact, such signalling could reside in the nil cluster, because it codes for at least four redox proteins (the two nitrogenase components, flavodoxin and pyruvate flavodoxin oxidoreductase). The particular stability of nif mRNA, possibly mediated by nifA product (section 6.3), is destroyed by aerobiosis (section 6.2), but overall the mechanism of transcription is probably more sensitive to 02 than all following events leading to nitrogenase activity (section 6.2). Finally, when diazotrophy is under- way, O 2 can apparently curtail nif expression through an effect on N status (section 6.5).

The apparent interactions of 02, N, and energy status on nif expression could influence the success of lactose-supported growth as a means for selecting O 2 regulatory mutants in strains carrying nif- lac. So far, this selection procedure, performed under anaerobiosis, has only yielded nif promoter mutants in K. pneumoniae [128]. On the other hand, in Rhodopseudomonas capsulata, this procedure yields nif regulatory mutants but only when photosynthesis is active, under anaerobiosis [119].

7. CONCLUSION

Diazotrophy in the facultative anaerobe K. pneumoniae is supported anaerobically by fermentation, but the provision of limiting oxygen be- nefits N 2 fixation, because the synthesis (section 6.1) and the activity of nitrogenase (section 4.1) are enhanced and the efficiency of glucose catabolism for N 2 fixation is improved (see section 5). Nevertheless, N 2 fixation adequate to support growth apparently cannot be sustained by an O2-limited obligate aerobic catabolism (i.e., microaerobic growth on succinate: Hill, S., unpublished results). Perhaps an obligate aerobic catabolism does not provide adequate reducing power for nltrogenase. Preliminary studies in molecular genetics support this possibility. So far a gene homologous to nifJ of K. pneumoniae (en-

123

coding the O2-sensitive pyruvate flavodoxin oxidoreductase) has not been found in obligate aerobes [178]. On the other hand, Rhizobium and Azotobacter carry a gene cluster fixABC [178], which in Rhizobium probably encodes for a membrane-bound protein [179]. Adjacent to this cluster in Rhizobium is fixX,, which encodes for a presumptive ferredoxin [179]. The fixABCX cluster is not found in K. pneumoniae [178,179]. Further- more, the biochemical evidence, as interpreted by Haaker and Klugkist [2], suggests that electron transfer to nitrogenase in obligate aerobes is closely associated with and dependent upon electron transfer to 02 .

Such a fundamental difference in the mechanism of generating electrons for nitrogenase function in facultative anaerobes and obligate aerobes may mean that other aspects of diazotrophic physiology are very different, including the mechanism of 02 regulation of nitrogenase synthesis. In K. pneumoniae this involves nifL (section 6.3). How- ever, a similar gene has not as yet been found in any obligate aerobe (section 6.3).

In obligate aerobes other biochemical and morphological changes besides nitrogenase synthesis are apparently triggered by changes in 02 status as well as in N status, e.g., vesicle formation in Frankia and the maturation of heterocysts in cyanobacter (section 4.1). In addition, the 02 status can influence the composition of the respiratory chain in obligate diazotrophs (section 4.1). How- ever, the effect of N status on this composition is less clear (section 5). Furthermore, aspects of carbon catabolism in obligate aerobes (recently emphasised by the characterisation of Fos- mutants in Azotobacter [44]) may, in addition to 02 and N status, influence the quality and quan- tity of proteins associated with providing adequate O 2 for N 2 fixation in obligate aerobic diazotrophs.

ACKNOWLEDGEMENTS

I thank Professors J.R. Postgate, M.J. Dilworth, Drs. R.A. Dixon, R.R. Eady, C.K. Kennedy, C.J. Pickett, R.L. Robson, and M.G. Yates for stimu- lating discussion and for constructive criticism of the manuscript and Miss B.F. Scutt and Miss M. Farrugla for typing it.

124

R E F E R E N C E S

[1] Mortenson, L.E. and Thorneley, R.N.F. (1979) Struc- ture and function of nitrogenase. Armu. Rev. Biochem. 48, 387-418.

[2] Haaker, H. and Klugldst, J. (1987) The bioenergetics of electron transfer to nitrogenase. FEMS Microbiol. Rev. 46, 57-71.

[3] Eady, R.E. (1980) Methods for studying nitrogenase. In: Methods for Evaluating Biological Nitrogen Fixa- tion (Bergersen, F.J., Ed.), pp. 213-264, John Wiley & Son, Chichester.

[4] Thorneley, R.N.F. and Lowe, D.J. (1983) Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidised iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction. Biochem. J. 215, 393-403.

[5] Eady, R., Robson, R.L., Richardson, T.H., Miller, R.W. and Hawkins, M. (1987) The vanadium nitrogenase of Azotobacter vinelandii. Biochem. J. 244, 197-207.

[6] Postgate, J.R. (1982) The fundamentals of nitrogen fixation. Cambridge University Press, Cambridge.

[7] Early, R.R. (1986) Enzymology in free-riving diazotrophs. In: Nitrogen Fixation. Vol. 4, Molecular Biology (Broughton, W.J. and Piihler, A., Eds.), pp. 1-49, Clarendon Press, Oxford.

[8] Stephens, P.J. (1985) The structure of the iron- molybdenum and the iron protein of the nitrogenase enzyme. In: Molybdenum Enzymes, (Spiro, T.G., Ed.), pp. 117-159, John Wiley & Son, New York.

[9] Thorneley, R.N.F. and Lowe, D.J. (1985) Kinetics and mechanism of the nitrogenase enzyme system. In: Molybdenum Enzymes (Spiro, T.G., Ed.), pp. 221-284, John Wiley & Son, New York.

[10] Burgess, B.K. (1985) Substrate reactions of nitrogenase. In: Molybdenum Enzymes, (Spiro, T.G., Ed.), pp. 161-219, John Wiley & Son, New York.

[11] Yates, M.G. and Planque, K. (1975) Nitrogenase from Azotobacter chroococcum purification and properties of the component proteins. Eur. J. Biochem. 60, 467-476.

[12] Brown, D.E. (1970) Aeration in the submerged culture of microorganisms. In: Methods in Microbiology 2, (Norris, J.R. and Ribbons, D.W., Eds.), pp. 125-174. Academic Press, London.

[13] Elsworth, R. (1970) The measurement of oxygen absorption and carbon dioxide evolution in stirred deep cultures. In: Methods in Microbiology 2, (Norris, J.R. and Ribbons, D.W., Eds.), pp. 213-228, Academic Press, London.

[14] Lloyd, D. and Scott, R.I. (1985) Mass-spectrometric monitoring of dissolved gases. In: Microbial Gas Metabolism (Poole, R.K. and Dow, C.S., Eds.), pp. 239-262, Academic Press, London.

[15] Turner, G.L. and Gibson, A.H. (1980) Measurement of nitrogen fixation by indirect methods. In: Methods for Evaluating Biological Nitrogen Fixation (Bergersen, F.J., Ed.), pp. 118-138, John Wiley & Son, Chichester.

[16] Hochman, A. and Burris, R.H. (1981) Effect of oxygen on acetylene reduction by photosynthetic bacteria. J. Bacteriol. 147, 492-499.

[17] Post, E., Kieiner, D. and Oelze, J. (1983) Whole cell respiration and nitrogenase activities in Azotobacter vinelandii growing in oxygen controlled continuous culture. Arch. Microbiol. 134, 68-72.

[18] Hill, S., Kennedy, K., Kavanagh, E , Goldberg, R.B. and Hanau, R. (1981) Nitrogen fixation gene (hilL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae. Nature 290, 424-426.

[19] Cannon, M., Hill, S., Kavanagh, E. and Cannon, F. (1985) A molecular genetic study of nil expression in Klebsiella pneumoniae at the level of transcription, translation and nitrogenase activity. Mol. Gen. Genet. 198, 198-206.

[20] Bergersen, F.J. and Turner, G.U (1979) Systems utilising oxygenated leghaemogiobin and myoglobin as sources of free dissolved 02 at low concentrations for experiments with bacteria. Anal. Biochem. 96, 165-174.

[21] Bergersen, F.J. and Turner, G.L. (1985) Measurement of components of proton motive force and related parameters in steady microaerobic conditions. J. Micro- biol. Methods. 4, 13-23.

[22] Appleby, C.A. and Bergersen, F.J. (1980) Preparation and experimental use of leghaemoglobin. In: Methods for Evaluating Biological Nitrogen Fixation (Bergersen, F.J., Ed.), pp. 315-335. John Wiley & Son, Chichester.

[23] NoB, T., De Groot, H. and Wisseman, P. (1986) A computer-supported oxystat system maintaining steady-state O 2 partial pressures and simultaneously monitoring O 2 uptake in biological systems. Biochem. J. 236, 765-769.

[24] Bergersen, F.J. and Turner, G.L. (1980) Properties of terminal oxidase systems of bacteroids from root nod- tiles of soybean and cowpea and of N2-fixing bacteria grown in continuous culture. J. Gen. Microbiol. 118, 235-252.

[25] Bergersen, F.J. (1982) Root nodules of legumes: structure and functions. Research Studies Press, John Wiley & Son Ltd., Chichester.

[26] Bergerson, F.J., Kennedy, C. and Hill, S. (1982) In- fluence of low oxygen concentration on derepression of nitrogenase in Klebsieila pneumoniae. J. Gen. Microbiol. 128, 909-915.

[27] Hill, S., Turner, G.L. and Bergersen, F.L. (1984) Synthesis and activity of nitrogenase in Klebsiella pneumoniae exposed to low conc, entrations of oxygen. J. Gen. Microbiol. 130, 1061-1067.

[28] Bergersen, FJ., Turner, G.L., Bogusz, D., Wu, Y-Q. and Appleby, C.A. (1986) Effects of 02 concentration and various haemoglobins on respiration and nitrogenase activity of bacteroids from stem and root nodules of Sesbania rostrata and of the same bacteria from continuous cultures. J. Gen. Microbiol. 131, 3325-3336.

[29] Delieu, T. and Walker, D.A. (1972) An improved cathode for the measurement of photosynthetic oxygen

evolution by isolated chloroplasts. New Phytol. 71, 201-225.

[30] Coombs, J.P. and Peters, A.C. (1985) A simple method for making microelectrodes to measure oxygen. J. Mi- crobiol. Method. 3, 199-204.

[31] Borkowski, J.D. and Johnson, M.J. (1967) Long-lived steam-sterilisable membrane probes for dissolved oxygen measurement. Biotech. Bioeng. 9, 635-639.

[32] Lloyd, D., James, K., Williams, J. and Williams, N. (1981) A membrane-covered photobacterium probe for oxygen measurement in the nanomolar range. Anal. Biochem. 116, 17-21.

[33] Lloyd, D., James, C.J. and Hastings, J.W. (1985) Oxygen affinities of the bioluminescence system of various species of luminous bacteria. J. Gen. Microbiol. 131, 2137-2140.

[34] Lnndsgaard, J.S., Gronlund, J. and Degn, H. (1978) Error in oxygen measurements in open systems owing to oxygen consumption in unstirred layer. Biotech. Bio- eng. 20, 809-819.

[35] Reynafarje, B., Costa, L.E. and Lehninger, A.L. (1985) O 2 solubility in aqueous media determined by a kinetic method. Anal. Bioehem. 145, 406-418.

[36] Robson, R.L. and Postgate, J.R. (1980) Oxygen and hydrogen in biological nitrogen fixation. Annu. Rev. Microbiol. 34, 183-207.

[37] Yates, M.G. and Jones, C.W. (1974) Respiration and nitrogen fixation in Azotobacters. Adv. Microb. Phys- iol. 11, 97-135.

[38] Jones, C.W. (1982) Bacterial respiration and photosynthesis. Aspects of Microbiology 5. T. Nelson & Sons, Ltd., Walton-on-Thames.

[39] Yates, M.G. (1987) The role of oxygen and hydrogen in nitrogen fixation. In SGM Symposium Volume: The Nitrogen and Sulphur Cycles (Cole, J.A. and Ferguson, S., Eds.), Cambridge University Press, Cambridge. In press.

[40] Bergersen, F.J. (1984) Oxygen and the physiology of diazotrophic microorganisms. In: Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W.E., Eds.), pp. 171-180, Nijhoff/Junk, The Hague.

[41] Dawes, E.A. (1985) The effect of environmental oxygen concentration on the carbon metabolism of some aerobic bacteria. In: Environmental Regulation of Microbial Metabolism (Kulaev, I.S., Dawes, E.A. and Tempest, D.W., Eds.), pp. 121-125. Academic Press, London.

[42] Streeter, J.G. and Salminen, S.O. (1985) Carbon metabolism in legume nodules. In: Nitrogen Fixation Research Progress (Evans, H.J., Bottomley, P.J. and Newton, W.E., Eds.), pp. 277-283 Martinus Nijhoff, Dordrecht.

[43] Dilworth, M. and Glenn, A. (1984) How does a legume nodule work? Trends Bioehem. Sci. 9, 519-523.

[44] Ramos, J.L. and Robson, R.L. (1985) Isolation and properties of mutants of Azotobacter chroococcum de- fective in aerobic nitrogen fixation. J. Gen. Microbiol. 131, 1449-1458.

125

[45] Ramos, J.L. and Robson, R.L. (1985) Lesions in citrate synthase that affect aerobic nitrogen fixation by Azoto- bacter chroococcum. J. Bacteriol. 162, 746-751.

[46] Ramos, J. and Robson, R.L. (1987) Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum an enzyme important in aerobic N 2 fixation. Mol. Gen. Genet. 208, 418-484.

[47] Stam, H., Van Verseveld, H.W., De Vries, W. and Stouthamer, A.H. (1986) Utilisation of poly-/~-hydroxy- butyrate in free-hving cultures of Rhizobium ORS571. FEMS Microbiol. Lett. 35, 215-220.

[48] Appleby, C.A. (1984) Leghaemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443-478.

[49] Stouthamer, A.H. (1984) Energy generation and hydrogen metabolism in Rhizobiurn. In: Advances in Nitro- gen Fixation Research (Veeger, C. and Newton, W., Eds.), pp. 189-197, Nijhoff/Junk, The Hague.

[50] McInerney, M.J., Holmes, K.S. and Dervartanian, D. (1982) Effect of 02 limitation on growth and respiration of the wild-type and an ascorbate-tetramethyl-p- phenylene diamine-oxidase negative mutant strain of Azotobacter vinelandii. J. Bioenerg. Biomembr. 14, 451-456.

[51] Okon, Y., Cakmaki, L., Nur, I. and Chet, I. (1980) Aerotaxis and chemotaxis in Azospirillum brasilense. Microb. Ecol. 6, 277-283.

[52] Hill, S. (1971) Influence of oxygen concentration on the colony type of Derxia gummosa grown on nitrogen-free media. J. Gen. Microbiol. 67, 77-83.

[53] Hill, S. (1975) Acetylene reduction by Klebsiella pneumoniae in air related to colony dimorphism on low fixed nitrogen. J. Gen. Microbiol. 91, 207-209.

[54] Witty, J.F., Minchin, F.R., Skot, L. and Sheehy, J.E. (1986) Nitrogen fixation and oxygen in legume root nodules, In: Oxford Surveys of Plant Molecular and Cell Biology 3, (Miflin, B.J., Ed.), pp. 375-314. Oxford University Press, Oxford.

[55] Reiner, O. and Okon, Y. (1986) Oxygen recognition in aerotactic behaviour of Azospirillurn brasilense Cd. Can. J. Microbiol. 32, 829-834.

[56] gheehy, J.E. and Bergersen, F.J. (1986) A simulation study of the functional requirements and distribution of leghaemoglobin in relation to biological nitrogen fixation in legume root nodules. Ann. Bot. 58, 121-136.

[57] Tjepkema, J.D., Scliwintzer, C.R. and Benson, D.R. (1986) Physiology of actinorhizal nodules. Annu. Rev. Plant Physiol. 37, 209-232.

[58] Landsman, J., Dennis, E.S., Higgins, T.J.V., Appleby, C.A., Kortt, A.A. and Peacock, W.J. (1986) Common evolutionary origin of legume and non-legume plant haemoglobins. Nature 324, 166-168.

[59] Stewart, W.D.P. (1980) Some aspects of structure and function in N2-fixing cyanobacteria. Armu. Rev. Mi- crobiol. 34, 497-536.

[60] Haselkorn, R. (1986) Cyanobacterial nitrogen fixation, in Nitrogen Fixation Vol. 4: Molecular Biology, (Broughton, W.J. and Piililer, S., Eds.), pp. 168-193. Clarendon Press, Oxford.

126

[61]

[62]

Walsby, A.E. (1986) Nitrogen fixation, finding a time and a place. Nature 323, 667. Maryan, P.S., Eady, R.R., Chaplin, A.E. and Gallon, J.R. (1986) Nitrogen fixation by Gloeothece sp. PCC6909: Respiration and not photosynthesis supports activity in the light. J. Gen. Microbiol. 132, 789-796.

[63] Wolk, C.P. (1982) Heterocysts. In: The Biology of Blue-Green Algae, (Carr, N.G. and Whitt, B.W, Eds.), pp. 359-386. Blackwells Scientific Publications, Oxford.

[64] Murry, M.A., Zhongze, Z. and Torrey, J.G. (1985) Effect of 02 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCc13 isolated from Casuarina cunninghaniana. Can. J. Mi- crobiol. 31, 804-809.

[65] Rippka, R. and Startler, R.Y. (1978) The effects of anaerobiosis on nitrogenase synthesis and heterocyst development by Nostocacaen cyanobacteria. J. Gen. Microbiol. 105, 83-94.

[66] Jensen, B.B. and Cox, R.P. (1983) Effect of oxygen concentration on dark nitrogen fixation and respiration in cyanobacteria. Arch. Mierobiol. 135, 287-292.

[67] Walsby, A.E. (1985) The permeability of heterocysts to the gases nitrogen and oxygen. Proc. R. Soc. Lond. B 226, 345-366.

[68] Minchin, F.R., Sheehy, J.E. and Witty, J.F. (1985) Factors limiting N 2 fixation by the legumc-Rhizobium symbiosis. In: Nitrogen Fixation Research Progress, (Evans, H.J., Bottomley, P.J. and Newton, W.E., Eds.), pp. 285-291. Martinus Nijhoff, Dordrecht.

[69] Drozd, J. and Postgate, J.R. (1970) Effects of oxygen on acetylene reduction, cytochrome content and respiratory activity of Azotobaeter chroococcurr~ J. Gen. Mi- crobiol. 63, 63-73.

[70] Gebhardt, C., Turner, G.L., Gibson, A.H., Drefus, B.L. and Bergersen, F.J. (1984) Nitrogen-fixing growth in continuous culture of a strain of Rhizobium sp. isolated from stem nodules on Sesbania rostrcL J. Gen. Mi- crobiol. 130, 843-848.

[71] Stewart, W.D.P. and Lex, M. (1970) Nitrogenase activity in the blue-green algae Plectonema boryanum strain 594. Arch. Microbiol. 73, 250-260.

[72] Mitsni, A., Kumazawa, S., Takahashi, A., Ikemoto, H., Cao, S. and Arai, T. (1986) Strategy by which nitrogen- fixing unicellular cyanobacteria grow photoautotrophi- cally. Nature 323, 720-722.

[73] Bergersen, F.J. and Trinchant, J-C. (1985) Flux of 02 affects apparent optimum concentrations of 02 in sus- pensions of bacteria respiring microaerobically. J. Tlieor. Biol. 115, 93-102.

[74] Trinchant, J.D. and Rigaud, J. (1987) Acetylene reduction by bacteroids isolated from stem nodules of Sesbania rostra. Specific role of lactate as an energy- yielding substrate. J. Gen. Microbiol. 133, 37-43.

[75] Yates, M.G. (1970) Control of respiration and nitrogen fixation by oxygen and adenine nucleotides in N2-grown Azotobacter chroococcum. J. Gen. Microbiol. 60, 393-401.

[76] Lough, S., Bums, A. and Watt, G.D. (1983) Redox reaction of the nitrogenase complex from Azotobacter vinelandii. Biochemistry, 22, 4062-4066.

[77] Wang, Z.C., Burns, A. and Watt, G.D. (1985) Complex formation and 02 sensitivity of Azotobacter vinelandii nitrogenase and its component proteins. Biochemistry, 24, 214-221.

[78] Scherings, G., Haaker, H., Wassink, H. and Veeger, C. (1983) On the formation of an oxygen-tolerant three- component nitrogenase complex from Azotobacter vinelandii. Eur. J. BiochenL 135, 591-599.

[79] Simpson, F.B. and Burris, R.H. (1984) Redox-depen- dence of the Shetlma iron-protein II - nitrogenase interaction and the effect of high pN 2 on evolution by nitrogenase. In: Advances in Nitrogen Fixation Re- search (Veeger, C. and Newton, W.E., Eds.), pp. 243. Martinus Nijhoff, The Hague.

[80] Veeger, C., Laane, C., Scherings, G., Matz, L., Haaker, H. and Van Zeeland-Wolbers, L. (1980) Membrane energisation and nitrogen fixation in Azotobacter vinelandii and Rhizobium leguminosarunt In: Nitrogen Fixation Vol. 1: Free-living systems and chemical mod- els (Newton, W.E. and Orme-Johnson, W.H., Eds.), pp. 111-137, University Park Press, Baltimore.

[81] Dingier, C. and O¢lze, J. (1985) Reversible and irre- versible inactivation of cellular nitrogenase upon oxygen stress in Azotobacter vinelandii growing in oxygen-controlled continuous culture. Arch. Microbiol. 141, 80-84.

[82] Robson, R. (1979) O2-repression of nitrogenase synthesis in Azotobacter chroococcum. FEMS Microbiol. Left. 5, 259-262.

[83] Klugkist, J., Haaker, H., Wassink, H. and Veeger, C. (1985) The catalytic activity of nitrogenase in intact Azotobacter vinelandii. Eur. J. Biochem. 146, 509-515.

[84] Robson, R.L. (1986) Nitrogen fixation in strains of Azotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase. Arch. Microbiol. 146, 74-79.

[85] t3oldberg, I., Nadler, V. and Hochman, A. (1987) Mechanism of nitrogenase 'switch off' by oxygen. J. Bacteriol. 169, 874-879.

[86] Youngman, R.J. (1984) Oxygen activation: is the hy- droxyl radical always biologically relevant? Trends Bio- chem. Sci. 9, 280-283.

[87] Puppo, A. and Rigaud, J. (1986) Hypothesis, superoxide dismutase: an essential role in the protection of the nitrogen fixation process? FEBS Lett. 201, 187-189.

[88] Steele, D.B. and Stowers, M.D. (1986) Superoxide dismutase and catalase in Frankia. Canad. J. Microbiol. 32, 409-413.

[89] Tel-or, E., Huflejt, M. and Packer, L. (1986) Hydroper- oxide metabolism in Cyanobacteria. Arch. Biochem. Biophys. 246, 396-402.

[90] Dalton, D.A., Russell, S.A., Hanus, F.J., Pasc.o¢, G.A. and Evans, H.J. (1986) Enzymatic reactions of ascorbate and giutathione that prevent peroxide damage in soybean root nodules. Proc. Natl. Acad. Sci. USA 83, 3811-3815.

127

[91] Dalton, D.A., Hanus, F.J., Russell, S.A. and Evans, H.J. (1987) Purification, properties and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol. 83, 789-794.

[92] Hochman, A., Reich, I. and Nadler, V. (1985) Effect of oxygen on nitrogenase of Rhodopseudomonas capsulata and Klebsiella pneumoniae. In: Nitrogen Fixation Re- search Progress (Evans, H.J., Bottomley, P.J. and New- ton, W.E., Eds.), pp. 442. Martinus Nijhoff, Dordrecht.

[93] Higuti, I.H. and Pedrosa, F.O. (1985) Mutant of Azospirillum brasilense Sp 7 that overproduces catalase. In: Nitrogen Fixation Research Progress (Evans, H.J., Bottomley, P.J. and Newton, W.E., Eds.), pp. 532. Martinus Nijhoff, Dordrecht.

[94] Reference deleted. [95] Yates, M.G. (1977) Physiological aspects of nitrogen

fixation. In: Recent Developments in Nitrogen Fixation (Newton, W.E., Postgate, J.R. and Rodriguez-Barrueco, C., Eds.), pp. 219-270, Academic Press, London.

[96] Hartmann, A. and Burris, R.H. (1987) Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum. J. Bacterioi. 169, 944-948.

[97] Pienkos, P.T., Bodmer, T. and Tahita, F.R. (1983) Oxygen inactivation and recovering nitrogenase activity in cyanobacteria. J. Bacteriol. 153, 182-190.

[98] Hill, S. (1976) Influence of atmospheric oxygen concentration on acetylene reduction and efficiency of nitrogen fixation in intact Klebsiella pneumoniae. J. Gen. Microbiol. 93, 335-345.

[99] Postgate, J.R. and Kent, H.M. (1984) Derepression of nitrogen fixation in Desulfovibrio gigas and its stability to ammonia or oxygen stress in vivo. J. Gen. Microbiol. 130, 2825-2831.

[100] Biggins, D.R. and Postgate, J.R. (1969) Nitrogen fixation by cultures and cell-free extracts of Mycobacterium flavum 301. J. Gen. Microbiol. 56, 181-193.

[100a] Hochman, A., Goldberg, I., Nadler, V. and Hartmann, A. (1987) The mechanism of nitrogenase reversible "switch off" by oxygen in Advanced course on inorganic nitrogen metabolism, June 22-28, 1986, Jaran- dilla, Spain.

[101] Daesch, G. and Mortenson, L.E. (1968) Sucrose catabolism in Clostridium pasteurianum and its relation to N 2 fixation. J. Bacteriol. 96, 346-351.

[102] Hill, S. (1976) The apparent ATP requirement for nitrogen fixation in growing Klebsiella pneumoniae. J. Gen. Microbiol. 95, 297-312.

[103] Stare, H., Stouthamer, A.H. and van Verseveld, H.W. (1987) Hydrogen metabolism and energy costs of nitrogen fixation. FEMS Microbiol. Rev. 46, 73-92.

[104] Reference deleted. [105] Dalton, H. and Postgate, J.R. (1969) Growth and physi-

ology of Azotobacter chroococcum in continuous culture. J. Gen. Microbiol. 56, 307-319.

[106] Kuhla, J., Dingier, C.L. and Oelze, J. (1985) Production of extracellular nitrogen-containing components by

Azotobacter vinelandii fixing dinitrogen in oxygen-controlled continuous culture. Arch. Microbiol. 141, 297-302.

[107] Stam, H., Van Verseveld, H.W., De Vries, W. and Stouthamer, A.H. (1984) Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat culture of Rhizobium ORS511. Arch. Microbiol. 139, 53-60.

[108] Herbert, D. (1976) Stoichiometric aspects of microbial growth. In: Continuous Culture 6, application and new fields, pp. 1-30, Society of Chemical Industry, London.

[109] Postgate, J.R., Eady, R.R., Dixon, R.A., Hill, S., Kahn, D., Kennedy, C., Partridge, P., Robson, R. and Yates, M.G. (1981) Some aspects of the physiology of dinitrogen fixation. In: Biology of Inorganic Nitrogen and Sulfur (Bothe, H. and Trebst, A., Eds.), pp. 103-115. Springer-Verlag, Berlin.

[110] Dixon, R.A. (1984) The genetic complexity of nitrogen fixation. J. Gen. Microbiol. 130, 2745-2755.

[111] Dixon, R. (1987) Genetic regulation of nitrogen fixation. In: The Nitrogen and Sulphur Cycles (Cole, J.A. and Ferguson, S., Eds.), Soc. Gen. Microbiol. Symp. Cambridge University Press, Cambridge. "In press.

[112] Merrick, M.J. (1987) Regulation of nitrogen assimilation by bacteria. In: The Nitrogen and Sulphur Cycles (Cole, J.A. and Ferguson, S., Eds.), Soc. Gen. Micro- biol. Symp. Cambridge University Press, Cambridge. In press.

[113] Kim, Y-M., Ahn, K-J., Beppu, T. and Uozumi, T. (1986) Nucleotide sequence of the nifLA operon of Klebsiella oxytoca NG13 and characterization of the gene products. Mol. Gen. Genet. 205, 253-259.

[114] Eady, R.R., Issack, R., Kennedy, C., Postgate, J.R. and Ratcliff, H. (1978) Nitrogenase synthesis in Klebsiella pneumoniae: comparison of ammonium and oxygen repression. J. Gen. Microbiol. 104, 277-285.

[115] Dixon, R., Eady, R.R., Espin, G., Hill, S., Iaccarino, M., Kahn, D. and Merrick, M. (1980) Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nil) gene cluster with gene fusions. Nature 286, 128-132.

[116] Kaluza, K. and Hennecke, H. (1981) Regulation of nitrogenase messenger RNA synthesis and stability in Klebsiella pneumoniae. Arch. Microbiol. 130, 38-43.

[117] Collins, J.J. and Brill, W.J. (1985) Control of Klebsiella pneumoniae nil mRNA synthesis. J. Bacteriol. 162, 186-190.

[118] St. John, R.T., Shah, V.K. and Brill, W.J. (1974) Regu- lation of nitrogenase synthesis by oxygen in Klebsiella pneumoniae. J. Bacteriol. 119, 266-269.

[119] Kranz, R.G. and Haselkorn, R. (1986) Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. Proc. Natl. Acad. Sci. USA 83, 6805-6809.

[120] Hallenbeck, P.C., Meyer, C.M. and Vignais, P.M. (1982) Regulation of nitrogenase in the photosynthetic bacterium Rhodopseudomonas capsulata as studied by two dimensional gel electrophoresis. J. Bacteriol. 151, 1612-1616.

128

[121] Scott, D.B., Henneeke, H. and Lim, S.T. (1979) The biosynthesis of nitrogenase MoFe protein polypeptide in free-living cultures of Rhizobiumjaponicum. Biochim. Biophys. Acta 565, 365-378.

[122] Fischer, H.M., Alvarez-Morales, A. and Hennecke, H. (1986) The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobiumjaponicum nifA gene involved in control of nif gene expression and formation of determinate symbiosis. EMBO J. 5, 1165-1173.

[123] Shaw, D.B. (1983) Non-coordinate regulation of Rhizobia nitrogenase symbiosis by 02 studies with bacteria for nodulated Lupinus angustifolius. J. Gen. Microbiol. 129, 849-857.

[124] Kennedy, C. and Toukdarian, A. (1987) Genetics of Azotobacters: application to nitrogen fixation and related aspects of metabolism. Annu. Rev. Microbiol. 41, 227-258.

[125] Haselkorn, R., Rice, D., Curtis, S.E. and Robinson, S.J. (1983) Organisation and transcription of. genes important in Anabaena heterocyst differentiation. Annu. Microbiol. (Inst. Pasteur) 134B, 181-193.

[126] Maryan, P.S., Eady, R.R., Chaplin, A.E. and Gallon, J.R. (1986) Nitrogen fixation by the unicellular cyano- bacterium Gloeothece. Nitrogenase synthesis is only transiently repressed by oxygen. FEMS Microbiol. Lett. 34, 251-255.

[127] Merrick, M., Hill, S., Hennecke, H., Hahn, M., Dixon, R. and Kennedy, C. (1982) Repressor properties of the nifL gene product in Klebsiella pneumoniae. Mol. Gen. Genet. 185, 75-81.

[128] MacNeil, D., Zhu, J. and Brill, W. (1981) Regulation of nitrogen fixation in Klebsiella pneumoniae isolation and characterisation of strains with nif-lac fusion. J. Bacteriol. 145, 348-357.

[129] Kong, Q-T., Wu, Q-L., Ma, Z-F. and Shen, S-C. (1986) Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae. J. Bacteriol. 166, 353-356.

[130] Hill, S., Turner, G.L. and Bergersen, F.J. (1984) Synthe- sis and activity of nitrogenase in Klebsiella pneumoniae exposed to low concentrations of oxygen. In: Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W.E., Eds.), pp. 260. Nijhoff/Junk, The Hague.

[131] Jensen, J.S. and Kennedy, C. (1982) Pieiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae. EMBO J. 1, 197-204.

[132] Jensen, J.S. and Kennedy, C. (1987) Regulation of nitrogenase synthesis in histidine auxotrophs of Klebsiella pneumoniae with altered levels of adenylate nucleotides. J. Bacteriol. in press.

[133] Kahn, D., Hawkins, M. and Eady, R.R. (1982) Meta- bolic control of Klebsiella pneumoniae mRNA degradation by the availability of fixed nitrogen. J. Gen. Micro- biol. 130, 38-43.

[134] Collins, J.J., Roberts, G.P. and Brill, W.J. (1986) Post- transcriptional control of Klebsiella pneumoniae nil mRNA stability by the nifL product. J. Bacteriol. 168, 173-178.

[135] Roberts, G.P. and Brill, W.J. (1980) Gene product

relationships of nil regulon of Klebsiella pneumoniae. J. Bacteriol. 144, 210-216.

[136] Buchanan-Wollaston, V., Cannon, M.C., Beynon, J.L. and Cannon, F.C. (1981) Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294, 776-778.

[137] Kennedy, C. and Robson, R.L. (1983) Activation of nil gene expression in Azotobacter by the nifA gene product of Klebsiella pneumoniae. Nature 301, 626-628.

[138] Drummond, M.H. and Wootton, J.C. (1987) Sequence of nifL from Klebsiella pneumoniae: mode of action and relationship to two families of regulatory proteins. Mol. Microbiol. 1, 37-44.

[139] Buchanan-Wollaston, V., Cannon, M.C. and Cannon, F.C. (1981) The use of cloned nil (nitrogen fixation) DNA to investigate transcriptional regulation of nil expression in Klebsiella pneumoniae. Mol. Gen. Genet. 184, 102-106.

[140] Riedel, G.E., Brown, S.E. and Ausubel, F.M. (1983) Nitrogen fixation in Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmdds. J. Bacteriol. 153, 45-56.

[141] Gussin, G.N., Ronson, C.W. and Ausubel, F.M. (1986) Regulation of nitrogen fixation genes. Annu. Rev. Genet. 20, 567-591.

[142] Haselkorn, R. (1986) Organisation of the genes for nitrogen fixation in Photosynthetic bacteria and cyanobacteria. Annu. Rev. Microbiol. 40, 525-547.

[143] Filser, M., Merrick, M. and Cannon, F. (1983) Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae. Mol. Gen. Genet. 191, 485-491.

[144] MacNeil, T., MacNeil, D., Roberts, G.P., Supiano, M. and BriU, W.J. (1978) Fine-structure mapping and com- plementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae. J. Bacteriol. 136, 253-266.

[145] Zhu, J. and Bfill, W.J. (1981) Temperature sensitivity of the regulation of nitrogenase synthesis by Klebsiella pneumoniae. J. Bacteriol. 145, 1116-1118.

[146] Dixon, R., Alvarez-Morales, A., Clements, J., Drum- mond, M., Filser, M. and Merrick, M. (1983) Regu- lation of transcription of the nitrogen fixation operons. In: Advances in Gene Technology: Molecular Genetics of Plants and Animals (Voellmy, R., Downey, K., Ahmad, F. and Schultz, J., Eds.), pp. 223-232. Academic Press, London.

[147] Buchanan-Wollaston, V. and Cannon, F. (1984) Regu- lation of nil transcription in Klebsiella pneumoniae. In: Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W.E., Eds.), pp. 732. Nijhoff/Junk, The Hague.

[148] Kennedy, C. and Drummond, M.H. (1985) The use of cloned nil regulatory elements from Klebsiella pneumoniae to examine nil regulation in Azotobacter vinelandii. J. Gen. Microbiol. 131, 1787-1795.

[149] Gest, H. (1981) Evolution of the citric acid cycle and respiratory energy conversion in prokaryotes. FEMS Microbiol. Lett. 12, 209-215.

[150] Harrison, D.E.F. (1972) Physiological effects of dis-

solved oxygen tension and redox potential on growing populations of microorganism. J. App. Chem. Biotech- nol. 22, 417-440.

[151] Tempest, D.W. and Neijssel, O.M. (1980) Growth yield values in relation to respiration. In: Diversity of Bacterial Respiration (Knowles, C.J., Ed.), pp. 2-31. CRC Press, Boca Raton.

[152] Morris, J.G. (1984) Changes in oxygen tension and the microbial metabolism of organic acids. In: Aspects of Microbial Metabolism and Ecology, (Codd, G.A., Ed.), pp. 59-96. Academic Press, London.

[153] Goldberg, R.B. and Hanau, R. (1980) Regulation of Klebsiellapneumoniae hut operon by oxygen. J. Bacteriol. 141,745-750.

[154] Smith, M.W. and Neidhardt, F.C. (1983) Proteins induced by anaerobiosis in Escherichia coli. J. Bacteriol. 154, 336-343.

[155] Clark, D.P. (1984) The number of anaerobically regulated genes in Escherichia coli. FEMS Microbiol. Lett. 24, 251-254.

[156] Alibadi, Z., Warren, F., Mya, S. and Foster, J.W. (1986) Oxygen-regulated stimulons of Salmonella typhimurium identified by Mud(Aplac) operon fusions. J. Bacteriol. 165, 780-786.

[157] Horn, S.S.M., Hennecke, H. and Shanmugam, K.T. (1980) Regulation of nitrogenase biosynthesis in Klebsiella pneumoniae: effect of nitrate. J. Gen. Micro- biol. 117, 169-179.

[158] Tubb, R.S. and Postgate, J.R. (1973) Control of nitrogenase synthesis in Klebsiella pneumoniae. J. Gen. Microbiol. 79, 103-117.

[159] Pecher, A., Zinoni, F., Jatisatienr, C., Wirth, R., Hen- necke, H. and B~Sck, A. (1983) On the redox control of synthesis of anaerobically induced enzymes in enterobacteriaceae. Arch. Microbiol. 136, 131-136.

[160] Shaw, D.J., Rice, D.W. and Guest, J.R. (1982) Ho- mology between CAP and Fur, a regulator of anaerobic respiration in Escherichia co& J. Mol. Biol. 166, 241-247.

[161] Pascal, M-C., Bonnefoy, V., Fons, M. and Chippanx, M. (1986) Use of gene fusions to study the expression of fnr, the regulatory gene of anaerobic electron transfer in Escherichia coli. FEMS Microbiol. Lett. 36, 35-39.

[162] Cole, J.A. (1987) Assimilatory and dissimilatory nitrate reduction to ammonia. In: The Nitrogen and Sulphur Cycles (Cole, J.A. and Ferguson, S., Eds.), Soc. Gen. Microbiol. Symp. Cambridge University Press, Cam- bridge, in press.

[163] Uden, G. and Guest, J.R. (1985) Isolation and characterisation of the Fnr proteins, the transcriptional regulation of anaerobic electron transport in Escherichia coli. Eur. J. Biochem. 146, 193-199.

[164] Hill, S. (1985) Redox regulation of enteric nil expression is independent of the fnr gene product. FEMS Microbiol. Lett. 29, 5-9.

[165] Jamieson, D.J. and Higgins, C.F. (1986) Two geneti- cally distinct pathways for transcriptional regulation of anaerobic gene expression in Salmonella typhimurium. J. Bacteriol. 168, 389-397.

129

[166] LiUey, D.M. (1986) The genetic control of DNA supercoiling, and vice versa. In: Regulation of Gene Expres- sion 25 years on, 39th Symposium of the Society of Gene Microbiology (Booth, I.R. and Higgins, C.F., Eds.), pp. 105-126. Cambridge University Press, Cam- bridge.

[167] Yamamoto, N. and Droffner, M.L. (1985) Mechanisms determining aerobic or anaerobic growth in the faculta- rive anaerobe Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 82, 2077-2081.

[168] Buck, M. and Ames, B.N. (1984) A modified nucleoside in tRNA as a possible regulator of aerobiosis: Synthesis of cis-2-methyl thioribosyl-zeatin in the tRNA of Salmonella. Cell 36, 523-531.

[169] Bonnefoy, V., Pascal, M-C., Ratouch-Niak, J. and Chip- paux, M. (1986) Alteration by mutation of the control by oxygen of the nar operon in Escherichia coll. Mol. Gen. Genet. 205, 349-352.

[170] Jones, H.M. and Gunsalus, R.P. (1985) Transcription of the Escherichia coli fumarate reductas¢ genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. J. Bacteriol. 164, 110-1109.

[171] Pascal, M-C., Burini, J.F. and Chippaux, M. (1984) Regulation of the trimethylamine L-oxide (TMNO) reductase in Escherichia coil analyses of tor::Mudl operon fusion. Mol. Gen. Genet. 195, 351-355.

[172] Bilous, P.T. and Weiner, J.H. (1985) Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101. J. Bacteriol. 162, 1151-1155.

[173] Kuritzkes, D.R., Zhang, X-Y. and Lin, E.C.C. (1984) Use of 4' (glp-lac) in studies of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB). J. Bacteriol. 157, 591-598.

[174] Pecher, A., Blaschkowski, H.P., Knappe, K. and B~Sck, A. (1982) Expression of pyruvate formate,lyase of Escherichia coil from the cloned structural gene. Arch. Microbiol. 132, 365-371.

[175] Zinoni, F., Beier, A., Pecher, A., Wirth, R. and BiSck, A. (1984) Regulation of the synthesis of hydrogenase (formate hydrogen-lyase linked) of E. coll. Arch. Microbiol. 139, 299-304.

[176] Jamieson, D.J., Sawers, R.G., Rucman, P.A., Boxer, D.H. and Higgins, C.F. (1986) Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoen- zyme composition in Salmonella typhimurium. J. Bacteriol. 168, 405-411.

[177] Stranch, K.L., Lenk, J.B., Gamble, B.L. and Miller, C.G. (1985) Oxygen regulation in Salmonella typhimurium. J. Bacteriol. 161, 673-680.

[178] Gubler, M. and Hennecke, H. (1986) fixA, B and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett. 200, 186-192.

[179] Earl, C.D., Ronson, C.W. and Ausubel, F.M. (1987) Genetic and structural analysis of the Rhizobium meliloti fix.A, fixB, f ixC and f i xX genes. J. Bacteriol. 169, 1127-1136.

how is nitrogenase regulated by oxygen?

Documents