Biosynthesis of the Iron-Molybdenum Cofactor and the Molybdenum

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However, the effect of cystine on the molybdenum requirement could not be ..... pneumoniae UN'. Molybdate. % Ethylene. %S *ra added (nM) formed. 0.4. lOOb.
Vol. 164, No. 3

JOURNAL OF BACTERIOLOGY, Dec. 1985, p. 1081-1087

0021-9193/85/121081-07$02.00/0 Copyright C 1985, American Society for Microbiology

Biosynthesis of the Iron-Molybdenum Cofactor and the Molybdenum Cofactor in Klebsiella pneumoniae: Effect of Sulfur Source RODOLFO A. UGALDE,t JUAN

IMPERIAL,t

VINOD K.

SHAH,§

AND WINSTON J.

BRILL¶T

Department of Bacteriology and Center for Studies of Nitrogen Fixation, University of Wisconsin, Madison, Wisconsin 53706 Received 14 June 1985/Accepted 10 September 1985

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and' metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the samne effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli Ch1D- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molibdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of ipolybdateb This stlggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high. It is not known how sulfur is incorporated into FeMo-co. An interaction between sulfate and molybdate has been found in some cases. Transport of MoO42 in Clostridium pasteurianum and Aspergillus niger (24) is competed with by sulfate. ATP sulfurylase, the first enzyme in the pathway of sulfate metabolism (23), recognizes molybdate as a-substrate (1, 30). The utilization of sulfate is tightly regulated in E. coli and Salmonella typhimurium by cysteine, the final product in the assimilation pathway (13, 17, 18, 23, 27). Cysteine feedback inhibits transport and utilization of sulfate (6, 13, 18, 23, 28). Cysteine also is thought to be the sulfur donor for the synthesis of iron-sulfur clusters in E. coli (29). In this paper we describe an interaction between the pathways of utilization of molybdenum and sulfur for the biosynthesis of FeMo-co and Mo-co in K. pneumoniae.

Nitrogenase (21), nitrate reductase (14), and a number of other enzymes (11) require molybdenum for their activity. Molybdenum is part of small-molecular-weight cofactors in molybdoenzymes. Two different molybdenum cofactors have been described: iron-molybdenum cofactor (FeMo-co), which is found only in nitrogenase (20), and molybdenum cofactor (Mo-co), which is found in other molybdoenzymes (11). FeMo-co is a cluster containing Mo, Fe, and S (1 Mo:8 Fe:6 S) with 2 Mo per roolecule of nitrogenase component I (20). Molybdenum in Mo-co is bound to a substituted pteridin containing no iron (8, 11). Molybdate can be utilized by bacteria as molybdenum source for the synthesis of these cofactors. Little is known about the reactions required for the transformation of molybdate into an active cofactor (21). Mutants deficient in the synthesis of these cofactors have been described and characterized (19, 21, 22). Some of these mutations can be phenotypically suppressed by the addition of high levels of molybdate- to' the medium. In Klebsiella pneumoniae two types of mutants requiring high molybdate for nitrogenase activity have been describeoI, NifQ- (9) ard Mol- (10). Whereas nifQ mutations affect FeMo-co synthesis alone (9), mol mutations pleiotropically affect the synthesis of both FeMo-co and Mo-co and are equivMent to chID mutations in Escherichia coli (10). The effects of nifQ 'and mol-mutations on nitrogenase are very similar (9, 10).

MATERIALS AND METHODS

Media and chemicals. K and KN media (9) were used for growth of K. pneumoniae. Glucose was substituted for sucrose for growth of E. coli. L-Cysteine and L-cystine were obtained from Sigma Chemical Co., St. Louis, Mo. Carrier-free Na2'MoO4 was purchased from Centichem, Inc., Tuxedo, N.Y. Na235SO4 (413.6 mCi mmol-1) was obtained from New England Nuclear Corp., Boston, Mass. Bacteriai strains. Wild-type K. pneumoniae UN and UN900 are independent wild-type strains from cultures of strain M5al provided by P. W. Wilson. Strain UN900 is nitrate reductase positive, whereas strain UN shows very low level of nitrate reductase activity. K. pneumoniae UN2454 (nifQ5027::Mu) and UN2458 (nifQ5031::Mu) (9) and UN5075 (mol4O18: :Mu) and UN5076 (mol4O19: :Mu) (10) have been described previously. UN5105 (cysA4007) and

* Corresponding author. t Present address: Instituto de Investigaciones Bioquimicas, Fundacion Campomar, 1405 Buenos Aires, Argentina. t Present address: Departamento de Microbiologia, Facultad de Biologia, Universidad de Barcelona, 08071 Barcelona, Spain. § Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706. Present address: Agracetus, Middleton, WI 53562.

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13ACTERIOL.

acid, filtered through glass fiber, and counted. Adenosine5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate synthesis from 35S042- was studied in crude extracts (26). Other techniques. 99Mo labeling (9), determination of ethanol-insoluble 9Mo in cell extracts (25), and protein assays (25) were performed as described previously. RESULTS

4~ 0

-

X

E 0

E

0

120

40

£~ OO 120 ~

MO added (r_____ d d A0131410

800

FIG. 1. Effect of the sulfur source on the molybdate requirement for nitrogenase activity of K. pneumoniae. A, Sulfate (0.8 mM); B, cystine (0.2 mM). Symbols: *, wild type; A, UN2458 (nifQ::Mu); O, UN5076 (mol:: Mu). Activities were assayed in 1-ml cultures with 15 min of incubation as described previously (3).

UN5099 (mol4003::Mu cysA4001) are sulfate-permease mu-

tants selected from UN and UN5076, respectively, by resistance to chromate (17; D. MacNeil, Ph.D. thesis, University of Wisconsin, Madison, 1979). E. coli RK4353 (1AlacU169

araD139 rspL gyrA non) and its derivative RK5202 (AlacU169 araD139 rpsL gyrA non chlD202::Mu) have been described previously (22). Growth and derepression. Cells were grown and derepressed for nitrogenase (9) and nitrate reductase (10) as described previously. Enzymne assays. Acetylene reduction (3) and nitrate reduction (10) were assayed in whole cells. Cysteine sulfhydrylase was assayed in crude extracts (13 ). 35s042- metabolism studies. 3s042 transport was studied in the presence of 50 ,ug of chloramphenicol per ml by filtration (15). 3S04- accumulation in whole cells was measured after a 10-min incubation of a washed cell suspension (A^,= 8) in complete medium containing 68 ,uM Na23S04 Incorporation of so4 into cell protein was determined as follows: cells (1 ml) were derepressed for nitrogenase in the presence of 0.1 mM ptus Na2SO4 Na2,5S04 (30 lMuC); after derepression 0.9 ml was added to 5 ml of boiling ethanol, dried, suspended in 5) trichloroacetic

Effect of cysteine on molybdenum requirement for nitrogenase activity. The dependence of acetylene reduction activity on the concentration of molybdate added to derepressing cultures of K. pneumoniae UN, UN2458 (nifQ::Mu), and UN5076 (mol::Mu) was studied in minimal K medium containing different sulfur sources (Fig. 1). The molybdenum requirement for maximum nitrogenase activity in UN2458 (nifQ::Mu) and UN5076 (mol::Mu) derepressed in sulfate (0.8 mM) as the only sulfur source was 3 to 10 times higher than that of the wild-type strain UN, as previously reported (9, 10). Molybdate concentrations higher than 400 ,uM resulted in inhibition of the acetylene-reduction activity of all three strains. When sulfate was substituted by cystine (0.4 mM sulfur) as the sulfur source in the medium for derepression of the wild type, higher activities were obtained, but the molybdenum requirement for maximumn nitrogenase activity was not affected (Fig. 1). Cystine significantly lowered the requirement of molybdenum for maximum acetylene reduction activity in UN2458 (nifQ::Mu) and UN5076 (mol::Mu) to levels similar to those necessary for the wild type (Fig. 1). Thus, the use of cystine as sulfur source abolished the effects of nifQ and mol mutations on nitrogenase activity. Cysteine had the same effect (data not shown); however, cystine was routinely used to avoid interactions with molybdate outside the cell (4, 12). Similar results were obtained with UN2454 (nifQ::Mu) and UN5075 (mol::Mu) (data not shown). Cystine and cysteine eliminated the inhibitory effect of very high concentrations of molybdate on acetylene reduction activity (Fig. 1). The combined addition of sulfate plus cystine to the derepressing medium had the same effect as the addition of cystine alone, i.e., almost identical molybdate requirement for maximum acetylene-reduction activity in the wild type and both mutants, as well as no inhibitory effect of very high concentrations of molybdate (data not shown). Effect of cystine on molybdenum accumulation during nitrogenase derepression. The accumulation of 9Mo label in cells derepressed in the presence of Na299Mo4 was studied for UN, UN2458 (nifQ::Mu), and UN5076 (mol::Mu) in medium containing sulfate (0.8 mM) or cystine (0.4 mM S) as the only sulfur source (Table 1). At the low levels of molybdate added for the assay, nonexchangeable 99Mo accumulation by UN2458 (nifQ::Mu) and UN5076 (mol::Mu) in the presence of sulfate was defective, in accordance with previous reports (9, 10). However, the addition of cystine restored the ability of both mutants to accumulate 99Mo. The amount of 99Mo in the ethanol-insoluble fraction has been shown to parallel nitrogenase activity under these conditions (25). Low amounts of 99Mo were present in this fraction when both mutants were derepressed in the presence of sulfate. 99Mo in the ethanol-insoluble fraction increased when the mutants were derepressed in the presence of cystine. The accumulation of 99Mo in nonexchangeable pools and in the ethanol-insoluble fraction of UN5076 (m0i::Mu) cells derepressed in the presence of cystine was lower than those in Un2458 (nifQ::Mu).

Fe-Mo AND Mo COFACTORS OF K. PNEUMONIAE

VOL. 164, 1985 TABLE 1. Effect of cystine on Mo accumulation during nitrogenase derepression % 99Mo % Ethanol-insoluble accumulateda Strain 99Mob Cystine

Sulfate

UN

100

UN2458

100

Cystine

Sulfate

100

100

12

150

6

122

7

80

8

55

(nifQ5S03: :Mu) UN5076 (mol4019: :Mu)

Nonexchangeable radioactivity as described previously (9); 100o represents 2.8 x 106 cpm/ml for sulfate cultures and 2.0 x 106 cpm/ml for cystine cultures. b Ethanol-insoluble 99Mo in cell extracts as discussed in Materials and Methods; 100o represents 1.7 x 106 cpm/ml for sulfate cultures and 1.0 x 106 cpm/ml for cystine cultures. a

Effect of cystine on molybdenum transport. An interaction between molybdate and sulfate at the level of transport into the cell could be the reason for the observations reported above. Transport of molybdate and molybdate requirement for maximum nitrogenase activity in UN5105 (cysA) and UN5099 (mol::Mu cysA), mutants lacking an active sulfate permease, were not affected with respect to their parent strains, UN and UN5076 (mol::Mu), respectively (data not shown). Derepression of NifQ- and Mol- mutants in the presence of cystine lowered the requirement for molybdate (Fig. 1). Cystine repressed the sulfate transport system (Fig. 2). Thus, the sulfate permease was not required for molybdate transport in the wild type or the NifQ- or Mol- mutants. The low requirement of molybdate in the wild type or NifQ- or Mol- mutants derepressed in the presence of cystine was not affected by the presence of high (0.8 mM) concentrations of

E 30[ 0.

201 -)

a.

@ 0

*a

10F

a A

n %A 0

1

2

3

4

5

Time (min) FIG. 2. Sulfate transport by K. pneumoniae (wild-type) cells grown with different sulfur sources. Assays were started by the addition of 20 j±Ci of Na235SO4 (specific activity, 167 mCi mmol-t) per ml. Samples of 50 ,ul were withdrawn at the indicated times and assayed. Cells were grown with 0.4 mM sulfur equivalents of sulfate (@) or cystine (A).

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TABLE 2. Effect of sulfate and cystine on sulfur metabolism in K. pneumoniae UN S source (mM)

Sulfate (0.8) Cystine (0.2)

o% Sulfate accumulation

ooa 8.7

%

% APS + PAPS formation

Cysteine sulfhydrylase

lb

23 100C

10

a 100o represents 3.3 x 106 cpm of 35S accumulated per ml of culture. b 100% represents an adenosine-5'-phosphosulfate (APS) and 3'phosphoadenosine-5'-phosphosulfate (PAPS) formation corresponding to 29%o of the Na2 35S04 added under the conditions described in Materials and Methods. c 100o represents 61.1 ,umol of S2- produced per min per ml of extract.

sulfate during derepression (see above). Thus, sulfate did not compete for the entry of molybdate into the cell. Regulation of sulfate metabolism by cystine in K. pneumoniae. Sulfate transport was very active in sulfate-grown UN cells, whereas it was repressed in cells grown on cystine (Fig. 2). Assimilation of sulfate by whole cells was equally inhibited in cystine-grown cells (Table 2). The ability of crude extracts to form adenosine-5'-phosphosulfate and 3'phosphoadenosine-5'-phosphosulfate was inhibited similarly. Cysteine sulfhydrylase, an enzyme whose activity depends upon intracellular cysteine levels (13), was very active in extracts from cystine-grown cells (Table 2). Effect of cystine on the molybdate requirement for nitrate reductase activity in K. pneumoniae Mol- and E. coli ChlDmutants. mol mutations pleiotropically affect the synthesis of both FeMo-co and Mo-co (10). Since cystine lowered the molybdate requirement for nitrogenase activity in Molmutants (Fig. 1), we tested this sulfur source for similar effects on nitrate reductase activity, a Mo-co enzyme. Figure 3A shows the dependence of nitrate reductase activity on molybdate concentration in K. pneumoniae UN5076 (mol::Mu) grown and derepressed for nitrate reductase in the presence of sulfate (0.8 mM) or cystine (0.4 mM S equivalents). Molybdate dependence was studied in the range of 40 nM to 40 mM. However, 4 and 40 mM inhibited growth of the wild-type strain UN900 and UN5076 (mol::Mu) by 50 and 100%, respectively (data not shown). This effect was not observed when cystine, alone or in combination with sulfate, was used as the sulfur source. When UN5076 (mol::Mu) was derepressed for nitrate reductase in the presence of sulfate, the levels of molybdate required to suppress the effect of the mutation on the enzyme activity were low when compared with the levels required to suppress the effects on nitrogenase (Fig. 1), in accord with a previous report (10). The addition of cystine did not affect the molybdate requirement in UN900 (data not shown), but increased it 100 to 1,000 times in UN5076 (mol::Mu). Mol- mutants have been shown to be equivalent to E. coli ChlD- mutants (10). The same effect of sulfur sources on the requirement of molybdate for nitrate reductase activity was observed in E. coli RK5202 (chlD::Mu) (Fig. 3B). Cystine did not suppress the effect of the mol mutation on nitrate reductase, whereas it suppressed the effect of the same mutation on nitrogenase. In addition, cystine dramatically increased the requirement of molybdate for nitrate reductase activity in both K. pneumoniae Mol- and E. coli ChID- mutants. Inhibition of nitrogenase activity by high concentrations of molybdate. Millimolar concentrations of molybdate inhibited nitrogenase activity, diazotrophic growth, and growth on nitrate when sulfate was the only sulfur source (see above). No inhibitory effects were observed with cystine (see

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FIG. 3. Nitrate reduction activity. Effect of the sulfur source on the molybdate requirement for maximum nitrate reduction activity of (A) K. pneumoniae UN5076 (mol::Mu) and (B) E. coli RK5202 (chlD::Mu). Symbols: *, sulfate (0.8 mM); A, cystine (0.2 mM).

above). The inhibitory effect on nitrogenase was further investigated. Nitrogenase activity was reduced to 21% when cells were derepressed in the presence of 40 mM molybdate (Table 3). The addition of the same amount of molybdate after derepression was not inhibitory. The same results were obtained with the wild type, UN5076 (mol::Mu), and UN2458 (nifQ::Mu) (data not shown). Thus, molybdate inhibited derepression, but not the activity of nitrogenase. High concentrations of molybdate caused parallel inhibition of acetylene-reduction activity and 35S incorporation into proteins from 35S042- added to the medium during derepression (Table 4). Increasing the sulfate content of the medium during derepression partially relieved the inhibitory effect (Table 5). A 60-fold excess of molybdate did not inhibit the transport of 35so42- (data not shown). The inhibition was not observed when sulfate, thiosulfate, or sulfide, intermediates in the assimilation of inorganic sulfur, was substituted for sulfite (Table 6). The combined evidence shows that the inhibitory effect of high levels of molybdate on the cells was due to a specific interference of molybdate with the intracellular utilization of sulfate as the sulfur source. DISCUSSION Low levels of molybdate can satisfy the requirements of K. pneumoniae for the synthesis of active nitrogenase and nitrate reductase (9, 10, 21). In the wild type, the utilization of molybdate for the synthesis of FeMo-co and Mo-co is an efficient process. This high efficiency requires the product of a nif-specific gene, nifQ (9), and a product of a non-nif gene, mol (10). NifQ- and Mol- mutants require higher (10-2 to 10-3 times) levels of molybdate in the medium to form active nitrogenase (9, 10). Mol- mutants pleiotropically affect nitrate reductase (and presumably other Mo-co enzymes) in the same way (10). All of these studies had been performed in a regular

J. BACTERIOL.

mineral salts medium containing sulfate as the only sulfur source. FeMo-co is an iron-molybdenum-sulfur cluster. In E. coli, cysteine has been suggested as the sulfur donor for the synthesis of iron-sulfur clusters (29). In K. pneumoniae, substitution of cystine for sulfate in the medium for nitrogenase derepression of NifQ- or Mol- mutants restored their ability to efficiently utilize molybdate, eliminating the effects of the mutation. In the wild-type strain, cystine did not modify the requirement of molybdate for nitrogenase activity. Cystine was used instead of cysteine to avoid the formation of Mo-cysteine complexes (4) in the media, but the same results were obtained with either form. These results could be explained in two ways: (i) cystine in these mutants allows the formation of FeMo-co in the presence of low levels of molybdate, or (ii) sulfate interferes with the utilization of molybdate, which becomes apparent only when the nifQ or mol products are not active. A competition between sulfate and molybdate was found in C. pasteurianum (7) and A. niger (24) at the level of transport into the cell. Several lines of evidence show that sulfate and molybdate do not interact at the transport level in K. pneumoniae. (i) The sulfate transport system was not required for molybdate transport since cysA mutations did not affect transport of 99MoO4-2 or utilization of molybdate for nitrogenase. In addition, cystine repressed the sulfate transport system in the same way as in E. coli (23) and S. typhimurium (6), but it lowered the amount of molybdate required for nitrogenase. Therefore, molybdate can be transported independently of sulfate in K. pneumoniae. (ii) It is unlikely that significant levels of molybdate can be transported into the cell through the sulfate transport system because transport of sulfate was unaffected by a 60-fold excess of molybdate. (iii) Sulfate did not interfere with molybdate transport. When 99MoO42- transport was studied in the presence of 0.8 mM s042- (40,000-fold excess with respect to molybdate), no inhibition was observed (9). In addition, the presence of sulfate did not affect the nitrogenase activity obtained with cystine at low levels of molybdate. These data prove that the systems for transport of sulfate and molybdate in K. pneumoniae are independent and specific, and that the sulfur source effect on molybdate requirement is intracellular. An interaction between the sulfur source and molybdate was also observed when very high concentrations of molybdate were added to the media. When sulfate was the sulfur source, millimolar concentrations of molybdate inhibited derepression of nitrogenase. Substitution of cystine or cysteine for sulfate abolished the effect. The inhibitory effect

TABLE 3. Effect of 40 mM molybdate on nitrogenase activity in K. pneumoniae UN derepressed in the presence of sulfate M0042- added (mM) % Ethylene formed After derepressiona During derepression 4 x 10-3 84 0 4 x 10-3 4 x 101 104 4 x 10-2 0 loob 4 x 10-2 4 x 101 120 4 x 10-0 0 80 4 x 10-1 0 21 a Cells were incubated for 30 min after the addition and assayed for nitrogenase. b 100%o represents 28.7 nmol/min per unit of absorbancy at 660 nm (10-ml culture).

Fe-Mo AND Mo COFACTORS OF K. PNEUMONIAE

VOL. 164, 1985 TABLE 4. Inhibition of 35S incorporation into proteins by high levels of molybdate present during nitrogenase derepression of K. pneumoniae UN' Molybdate added (nM)

0.4 4 12 40 120 400

% Ethylene

formed

lOOb 90 45 Mo-Y > >~ ~ pteridine

30

MO 04-

3- Mo042-

mo/(chID)

\ so2-_

J. BACTERIOL.

t)O so

t APS 4

:

30 MO-co

, Mo-X /

nifO

,)>cysteine

-o nitrate reductose

(other molybdoenzymes)

nifB,AI,E

FeMo-co

>nitrogenase

Fe,S

A

cysteine-

FIG. 4. Interaction of molybdenum and sulfur metabolism in the biosynthesis of FeMo-co. Symbols: ---- ->, repression; --->, inhibition; i>, reaction can proceed nonenzymatically in the presence of high substrate levels.

could be transformed either by the mol (chlD) gene product or spontaneously if the intracellular MoO42- concentrations are high into an intermediate (Mo-X) common to both the Mo-co and FeMo-co pathways. Along the Mo-co pathway, Mo-X is incorporated into a pteridine moiety (8) to yield active cofactor for nitrate reductase and other molybdoenzymes. Mo-X could also react with cysteine (or an immediate S-containing derivative) to yield the first intermediate (Mo-Y) specific for the FeMo-co pathway. This reaction would be facilitated by either a wild-type nifQ gene product or by a high intracellular concentration of either Mo-X or cysteine. Mo-Y would then be transformed into FeMo-co by the action of other nif genes (21). High intracellular levels of cysteine, caused by its addition to the medium (27), would allow synthesis of Mo-Y to proceed at wild-type rates in the absence of an active nifQ gene product without the need for elevated MoO42- additions. This could cause a scavenging of Mo-X when the absence of an active mol (chiD) gene product does not allow an optimal synthesis and, thus, a lower rate of synthesis of Mo-co. The addition of cysteine also repressed the sulfate metabolism pathway, thus overcoming the sulfur starvation that high levels of molybdate caused by competitive inhibition of ATP

sulfurylase. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by Public Health Service grant GM22310 from the National Institutes of Health. R.A.U. is a fellow of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina. J.I. was the recipient of a postdoctoral fellowship from the Ministry of Education of Spain. We thank Valley Stewart for the E. coli strains used in this study. LITERATURE CITED 1. Akagi, J. M., and L. L. Campbell. 1962. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphatesulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194-1201. 2. Banat, I. M., D. B. Nedwell, and M. T. Balba. 1983. Stimulation of methanogenesis by slurries of saltmarsh sediment after the addition of molybdate to inhibit sulphate-reducing bacteria. J. Gen. Microbiol. 129:123-129. 3. Brill, W. J., A. L. Steiner, and V. K. Shah. 1974. Effect of molybdenum starvation and tungsten on the synthesis of nitrogenase compounds in Klebsiella pneumoniae. J. Bacteriol.

118:986-989. 4. Callis, G. E., and R. A. D. Wentworth. 1977. Tungsten vs. molybdenum in models for biological systems. Bioinorganic

Chem. 7:57-70. 5. Cooper, A. J. L. 1983. Biochemistry of sulfur-containing amino acids. Annu. Rev. Biochem. 52:187-222. 6. Dreyfuss, J. 1964. Characterization of a sulfate- and thiosulfatetransporting system in Salmonella typhimurium. J. Biol. Chem. 239:2292-2297. 7. Elliott, B. B., and L. E. Mortenson. 1975. Transport of molybdate by Clostridium pasteurianum. J. Bacteriol. 124:1295-1301. 8. Hageman, R. V., and K. V. Rajagopalan. 1985. Characterization of molybdopterin, the organic portion of the molybdenum cofactor, p. 133-141. In P. W. Ludden and J. E. Burris (ed.), Nitrogen fixation and CO2 metabolism. Elsevier/North-Holland Publishing Co., New York. 9. Imperial, J., R. A. Ugalde, V. K. Shah, and W. J. Brill. 1984. Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae. J. Bacteriol. 158:187-194. 10. Imperial, J., R. A. Ugalde, V. K. Shah, and W. J. Brill. 1985. Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase activity. J. Bacteriol. 163:12851287. 11. Johnson, J. 1980. The molybdenum cofactor common to nitrate reductase, xanthine dehydrogenase and sulphite oxydase, p. 345-384. In M. Coughlan (ed.), Molybdenum and molybdenumcontaining enzymes. Pergamon Press, Oxford. 12. Kay, A., and P. C. H. Mitchell. 1968. Molybdenum-cysteine complex. Nature (London) 219:267-268. 13. Kredich, N. M. 1971. Regulation of L-cysteine biosynthesis in Salmonella typhimurium. I. Effect of growth on varying sulfur sources and O-acetyl-L-serine on gene expression. J. Biol. Chem. 246:3474-3484. 14. Lester, R. L., and J. A. DeMoss. 1971. Effects of molybdate and selenite on formate and nitrate metabolism in Escherichia coli. J. Bacteriol. 105:1006-1014. 15. Marquis, R. E. 1981. Permeability and transport, p. 393-404. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 16. Neilands, J. B. (ed.). 1974. Microbial iron metabolism. Academic Press, Inc., New York. 17. Pardee, A. B., L. S. Prestidge, M. B. Whipple, and J. Dreyfuss. 1966. A binding site for sulfate and its relation to sulfate transport into Salmonella typhimurium. J. Biol. Chem. 241:3962-3969. 18. Pasternak, C. A., R. J. Ellis, M. C. Jones-Mortimer, and C. E. Crichton. 1965. The control of sulphate reduction in bacteria. Biochem. J. 96:270-275. 19. Roberts, G. P., T. MacNeil, D. MacNeil, and W. J. Brill. 1978. Regulation and characterization of protein products coded by nif (nitrogen fixation) genes of Klebsiella pneumoniae. J. Bacteriol. 136:267-279. 20. Shah, V. K., and W. J. Brill. 1977. Isolation of an ironmolybdenum cofactor from nitrogenase. Proc. Natl. Acad. Sci. USA 74:3249-3253.

VOL. 164, 1985 21. Shah, V. K., R. A. Ugalde, J. Imperial, and W. J. Brill. 1984. Molybdenum in nitrogenase. Annu. Rev. Biochem. 53:231-257. 22. Stewart, V., and C. H. MacGregor. 1981. Nitrate reductase in Escherichia coli K-12: involvement of chiC, chlE, and chIG loci. J. Bacteriol. 151:788-799. 23. Trudinger, P. A., and R. E. Loughlin. 1981. Metabolism of simple sulfur .compounds, p. 165-256. In A. Neuberger and L. L. M. van Deenen (ed.), Amino acid metabolism and sulfur metabolism, vol. 19A of Comprehensive biochemistry. Elsevier Science Publishing Co., New York. 24. Tweedie, J. W., and I. H. Segel. 1970. Specificity of transport processes for sulfur, selenium, and molybdenum anions by filamentous fungi. Biochim. Biophys. Acta 196:95-106. 25. Ugalde, R. A., J. Imperial, V. K. Shah, and W. J. Brill. 1984. Biosynthesis of iron-molybdenum cofactor in the absence of

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