MOLYBDATE METABOLISM OP AZOTOBACTER

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By RICHARD FAIRBANKS KEELER, B. S., M. S.

The Ohio State University 1957

Approved by:

Adviser Department of Agricultural Biochemistry ACKNOWLEDGMENTS

The author wishes to express his appreciation to his adviser, Dr,

Biochemistry for their help and guidance during the course of his graduate program.

Thanks go to L. B. Carr for the micrographs shown herein, and to Dr. H. J. Hausman and Paul Weiler for the preparation of the N*^.

Financial support for this investigation was very generously provided by the Research Corporation.

i± TABLE OF CONTENTS Page

INTROHJCTION 1

REVIEW OF IRE LITERATURE 4

EXPERIMENTAL METHODS 16 RESULTS AND DISCUSSION 23 SUMMARY 61 CONCLUSIONS AND OUTLOOK 65

BIBLIOGRAPHY 106

AUTOBIOGRAPHY 111

iii LIST OP ILLUSTRATIONS

Figure Page

1. Serial (NH4 )qS04 Fractionation of the Proteins of the Azotobacter vinelandil 25,000 Times Gravity Supernatant Fraction " 68 2. Paper Electrophoresis of Radioactive Tungsto- and Molybdoproteins from Partially Purified Homogenates of Azotobacter vinelandii Grown on N2 69

3. Serial (NH4 )gS04 Fractionation of the Molybdo­ proteins of the Azotobacter chroococcum 25,000 Times Gravity Supernatant Fraction 70 4. 1,500 Times Magnified Azotobacter vinelandii Preparations Photographed under tight Microscopy 71 5* 15,000 Times Magnified Electron Micrograph of the Membrane Preparation of Azotobacter vinelandii. 72

iv LIST OF TABLES Table Page 99 1. Uptake and Distribution of Mo in Centri­ fugal Fractions of Azotobacter vinelandii 73 2* Uptake and Distribution of M o " in Centri­ fugal Fractions of Azotobacter vinelandii 74

QQ 3. Mo Uptake by Azotobacter vinelandii as a Function of Growth 75 4. M o " Uptake by Azotobacter vinelandii as a Function of Aeration 76 99 5. Mo Uptake by Azotobacter vinelandii as a Function of the Iron Concentration 77 59 6 . Iron Uptake by Azotobacter vinelandii growing on TTg as a Function of Aeration 78

7. M o " Uptake and Distribution In Azotobacter vinelandii as a Function of Culture Con­ ditions » 79

8 . Effects of Tungsten, Vanadium, and Molybde­ num on the Growth of Azotobacter vinelandii 80 9. Tungsten as a Competitive Inhibitor of Molybde­ num in the Growth of Azotobacter vinelandii 81 10* The Effect of Tungsten on the Uptake of M o " by Azotobacter vinelandii 82

QQ 11. Uptake of Mo by Azotobacter vinelandii as a Function of the tungsten Level in the Medium 83 12. Uptake of w185 by Azotobacter vinelandii as a Function of the Molybdenum Level of the Medium 84 13* Inability of Vanadium to Substitute for Molybdenum In the Growth of Azotobacter vinelandii 85

v vi

Indirect Demonstration of a Molybdenum Re­ quirement fqr Azotobacter vinelandii Grown oti NH^ “ 86 Comparison of the Intracellular Distribution and Uptake of and Mo" in Azotobacter vinelandii 87 Dialysis of the Tungsten and Molybdenum of the Azotobacter vinelandii 25,000 Times Gravity Supernatant Praction 88 M o " Dialysis Loss from the 25,000 Times Gravity Supernatant Fraction of Azotobacter vinelandii as a Result of Various Treatments 89 yyl85 j5ialysis Loss from the 25,000 Times Gravity Supernatant Fraction of Azotobacter vinelandii as a Result of Various^ Treatments 90 Distribution of Fe59, W185, and M o " by Direct Lysozyme Lysis of Azotobacter vinelandii 91 Chromium as a Non-competitive Inhibitor of Molybdenum in Azotobacter vinelandii 92 Chromium as a Non-competitive Inhibitor of Iron in Azotobacter vinelandii 93 Comparison of the Molybdenum Requirements of Azotobacter vinelandii strain 0 and Azotobacter chroococcum C44 94 Competitive Inhibition of Molybdenum by Tungsten in the Growth of Azotobacter chroococcum 95 Relative Uptake of M o " by Azotobacter chroococcum as a Function of Various Treatments and as Compared to Azotobacter vinelandii 96 99 Distribution of Mo by Direct Lysozyme Lysis of Azotobacter chroococcum 97 Effect of Various Ge/Si Ratios on M o " Up­ take in Azotobacter vinelandii 98 vii

31 27. Uptake of Si as a Function of Germanium and Molybdenum Levels in Azotobacter vinelandii 99

28. Uptake of Si31 by Azotobacter vinelandii as a Function of Germanium, Molybdenum, and Phosphorus Levels 100 31 29. Distribution of Si in Cell-Free Fractions of Azotobacter vinelandii 101 31 30. Distribution of Si by Direct Lysozyme Lysis of Azotobacter vinelandii 102 31. Germanium Inhibition of Azotobacter vine­ landii as Influenced by"Pbosphate Level 103

32. Half-Life of Cyclotron Produced Nitrogen-*-5 104

33. N -*-3 Incorporation by Various Systems 105 MOLYBDATE METABOLISM OF AZOTOBACTER

INTRODUCTION

Molybdenum has received attention in recent years as a metal component in various enzyme systems* It has been implicated with some certainty in xanthine oxidase, aldehyde oxidase, nitrate reductase, hydrogenase, and as an absolute requirement for various organisms fixing elemental nitrogen. Whether or not it is an actual in vivo component of these systems has not been established unequivocally. The strength of the evidence varies with the enzyme in question. It appears rather certain that nitrate reductase and xanthine oxidase are molybdoproteins, while the evidence for the others is less certain than could be desired.

A few organisms are known which have the ability of fixing elemental nitrogen, that is, of using N2 gas as the sole nitrogen source. Azotobacter, an aerobic hetero- troph, is one of these organisms. It is known further that Azotobacter has a hydrogenase, can also reduce nitrate, and perhaps contains xanthine oxidase and aldehyde oxidase as well. It seemed, therefore, that Azotobacter might be an excellent organism in which to study the metabolism of molybdenum. 2 The study of the actual in vivo metabolism of a suspected enzymatic cofactor might be expected to reveal information which could not be obtained by the direct study of isolated enzyme systems. With information obtained by both approaches, one might be more likely to establish the means by which molybdenum facilitates various functions in the living cell. The requirement of molybdenum for nitrogen fixation offers an especially interesting challenge because the entire pathway of fixation has not yet been established, and thus the exact step(s) in which molybdenum is required and its exact function(s) are not known. Labeled molybdenum, under the right conditions, might possibly be utilized to localize the nitrogen fixing system as well as the other molybdenum containing enzymes. In this way one might identify at least a part of the nitrogenase system. Therefore the objective of this study was to examine the metabolism of molybdate, the form in which the molybdenum is supplied to the Azotobacter cell, and to de­ termine the nature of its disposition in the cell. It was hoped to be able to develop methods for determining the number and nature of the functional sites of molybdate utili­ zation. If this were possible, and If more than one site existed, and if one could assign a role to each of these sites by appropriate techniques, then one might be better able to ascribe unequivocal importance to molybdenum in individual in vivo enzyme systems. Furthermore, one might subsequently be able to purify toward the systems using

QQ the molybdenum1757 label. This would be especially useful in the case of nitrogenase, an enzyme system which has thus far escaped purification. REVIEW OF THE LITERATURE

Molybdenum, Tungsten, and Vanadium In Biological Systems

Interest in the role of molybdenum has Increased

greatly since the observation In 1950 by Bortels (8 ) that nitrogen fixation was enhanced by this element. A number of groups throughout the world are now actively engaged in Investigations of various systems in which molybdenum is a component.

The early work from the time of Bortels centered

around the role of molybdenum In nitrogen fixation. For

example, In 1932 Birch-HIrshfeld (7) reported no response by Azotobacter to molybdenum when nitrate was the nitrogen

source rather than elemental nitrogen. However, Burk and Horner (13) in 1939 were able to show a requirement for molybdenum when the cells were utilizing nitrate. The con­

centration of molybdenum required was less for cells utilizing nitrate than for nitrogen-gas-grown cells accord­ ing to Bortels (10). Thus, a requirement for molybdenum in nitrate reduction as well as in nitrogen fixation also became evident since, of all nitrogen sources tried by all

Investigators, only the utilization of Ug and NO3 was molyb­

date dependent. The molybdenum requirement under various

nitrogen sources is still undergoing active Investigation 4 (11,16,30,32,49). An interesting variation of the molybdate requirement by different species of Azotobacter was reported by Horner et al (26). A strain of A. vinelandii tested required much lower molybdate concentrations for optimum growth than various Azotobacter chroococcum strains.

More recent work has been in the area of molybdenum as a cofactor in various partially purified enzyme systems.

A recent review by Evans (17) describes the importance of molybdenum in nutrition as well as the role of the element in various enzymatic reactions. Mahler and Green (41) in an earlier review discussed the metallo-flavoproteins among which are found molybdenum containing systems. More specifically, Nason and Evans (56), and Nicholas and Nason

(52,53) were responsible for the isolation of a molybdenum containing nitrate reductase capable of catalyzing the oxi­ dation of reduced TPN by nitrate. Intestinal xanthine oxidase was shown by Westerfeld and Richert (76) to be rather low in rats maintained on molybdenum free diets.

Confirmation experiments and proof that molybdenum was an actual constituent of xanthine oxidase followed (21,57,61,70).

Mahler, Mackler and Green (38,42) described an aldehyde oxidase enzyme dependent on molybdenum. Shug £t jal (65) also described a molybdo-enzyme, this time a hydrogenase from

Clostridium pasteurianum. However Nicholas (51) suggests 6 that because hydrogenase from Neurospora crassa is de­ creased much more markedly by Iron deficiency than by molybdenum deficiency, it may, in fact, have no molyb­ denum requirement.

Tungsten and vanadium have been reported to support growth in the absence of molybdenum in Azotobacter cultures (9,26). However, Horner, et al (26) demonstrated that the effect of tungsten was very likely due to molybdenum contamination of the added tungsten. At the same time they showed molybdenum could be replaced by vanadium in cultures of A. chroococcum. However, Allen (4) has shown that vana­ dium cannot replace the molybdenum requirement of the blue- green alga, Anabaena. Vanadium likewise will not replace molybdenum in the hydrogenase enzyme according to Shug, et al (65). Arnon (5) reports that the green alga, Scene- desmus, apparently requires both mouLybdenum and vanadium.

A number of other workers have reported on the ability or lack of ability of vanadium to substitute for molybdenum, a phenomenon dependent, apparently, on the strain in question (11,16,29,30,51).

In spite of the work by Horner, et al (26) demon­ strating the effect of tungsten to be due to molybdenum

contamination, the idea that tungsten can substitute for molybdenum still persists. Nicholas (51) reports that tungsten lilce vanadium can partially replace the molybdenum required, by strains but not by others. This must be taken as valid considering the purity of the presently available tungsten.

That tungsten not only does not act as a substi­ tute, but is an actual inhibitor in some systems, has been recently shown. Higgins in a number of papers (23,24) which are summarized in his dissertation (2 2 ) has described a competitive inhibition of molybdenum by tungsten. He re­ ported that chicks maintained on a diet low in molybdenum and high in tungsten developed typical molybdenum deficiency symptoms. The molybdenum concentration in tissues under these conditions was found to be less. These symptoms could be reversed by adding molybdenum to the diet to overcome the tungsten inhibition. He also reported that the level of xanthine oxidase, a molybdo-enzyme, was lower in tungsten fed rats than in rats on adequate molybdenum and no tungsten.

He was able to demonstrate further the existence of a competitive tungsten inhibition in Aspergillus niger growing _ ~4- on NOg, but not on HH4 as a nitrogen source. Chromium, another member of the same chemical group, gave no inhibi­ tion even at levels of 15,000 parts chromium to one part molybdenum.

Shortly after the completion of the work to be described in section B of the Results and Discussion on the competitive tungsten inhibition of molybdenum in Azotobacter, a report of similar experiments done independently and at the same time by workers in another laboratory was published (69). These workers demonstrated a competitive inhibition of molybdenum by tungsten for Azotobacter growing on Ng or NO3 , but not NH^. Vanadium would not substitute for molybdenum.

One report exists of the use of W'*'88, the tungsten radioisotope, in distribution studies conducted in the rat

(73). No intracellular distribution was determined, however. Huang in some unpublished experiments in this laboratory has examined the Intracellular distribution of W-1-88, and M o " in rat liver cells and finds that most of the label is in the mitochondrial fraction with the microsome fraction next highest. In another study involving the radioisotope 99 Mo , Magee (39) effected a 150 fold purification of a molybdo"-protein from Azotobacter vinelandii which contained a large percentage of the molybdenum taken up by the grow­ ing cells. The readdition of this molybdenum-rich fraction to cell-free preparations did not enhance nitrogen fixation rates, however. No intracellular distribution of the label was determined by this worker.

A recent report by Lenhoff, Nicholas, and Kaplan

(36) implicates molybdenum in an alternate terminal oxidase system in Pseudomonas fluorescens. These cells take up more molybdenum when cultured under high oxygen tension on a low iron medium, than when cultured under low oxygen tension on high concentrations of iron, and further, the iron requirement is apparently reduced at high oxygen tensions. On the basis of these and other data they suggested that under low oxygen tension P. fluorescens respires via an iron-cytochrome peroxidase pathway, while under high oxygen tension the cells respire via a molybdo- flavoprotein terminal oxidase. Nicholas (50) has shown that a molybdenum deficiency in Neurospora crassa, while causing a 40-50$ reduction in growth, resulted in a re­ duction of the catalase and peroxidase in the cell-free extracts of the felts to 1/10 and 1/4 respectively of that in controls. Addition of molybdenum caused an adaptive formation of both enzymes. He speculated on the basis of his work with Lenhoff (above) that since it was established that a molybdenum deficiency caused a reduction in flavo- protein enzymes producing H2O2 , a deficiency in molybdenum in this case with Neurospora may have resulted in a low production of H2O2 so that the adaptive catalase and peroxidase were depleted.

Some of the results reported in this dissertation on molybdenum, tungsten, and vanadium in Azotobacter were published in biochemical journals during the authors Ph. D. graduate program (33,34,35).

Studies of Intracellular Localization

of Components in Bacteria

The success of studies on the intracellular distribution of enzyme systems and functional units in 10 plant and animal cells has been astounding in recent years.

The possibility of describing the function(s) of each individual cellular sub-unit in the near future seems rather good. There has been a desire on the part of many workers to utilize differential centrifugation techniques also in studies on the intracellular distribution of various components in bacteria.

Azotobacter is an organism rather widely used in studies of intracellular distribution of enzymes and other cellular components. Repaske (58), for example, reported 50-75$ of the succinic dehydrogenase activity of cell-free extracts of A. vinelandii prepared by sonic oscillation to be "soluble” after centrifugation at 144,000 times gravity for 30 minutes. Wilson and Wilson (77) have shown that all of the succinoxidase activity remained in the super­ natant fraction after centrifugation of cell-free extracts at 144,000 times gravity for 5 minutes, but was 90$ sedimented when the centrifugation was continued for three hours. Alexander and Wilson (2,3) have described the localization of various enzymes in Azotobacter, including

Krebs cycle enzymes, by differential centrifugation. In an excellent review article, Alexander (1) examined the reports available on the intracellular localization of enzymes in various bacteria, and discussed the application and values of the differential centrifugation technique in bacterial studies. 11 Since the report by Weibull (74) on the formation of protoplaata in Bacillus megaterium, and descriptions of methods of osmotic rupture of protoplasts and whole cells by many workers, interest has increased in the use of such methods in the preparation of cell-free systems for study. It was thought that these more gentle methods of rupture, as compared with sonic oscillation or grinding, might be expected to produce fewer artifacts, and result in a better separation of fractions. Furthermore, by the utilization of such techniques it is possible to isolate a fraction by centrifugation at about 2000 times gravity which contains the bacterial membrane in a rather well preserved state (75,78). Sonic oscillation, on the other hand, destroys the integrity of the original membrane and causes its disintegration into small particles. These particles sediment at about 100,000 to 144,000 times gravity in the case of Azotobacter (15,31,43), and also represent the so called "small particle” fraction of sonic disrupted

Staphylococci (48).

Repaske (59,60) has developed methods both for the direct lysis of Azotobacter vinelandii using lysozyme and versene In tris buffer, as well as the formation of protoplasts which can subsequently be lysed osmotically.

By either method, one can obtain the membrane fraction of

A. vinelandii. Jose and Pengra (31) using methods of this 12 kind were able to demonstrate a small but significant

fixation of by protoplasts* They were also able to

show that the hydrogenase activity, which had been pre­

viously shown to be a component of small particles sedi- menting at about 144,000 times gravity, was actually

present in the protoplast membrane fraction after osmotic

lysis of the protoplasts. This further suggests the

degradation of the membrane by sonic oscillation. Robrish,

et al (62) showed that not only hydrogenase, but also the

cytochrcmes were components of the ’’envelope” of A. vine­

landii by a somewhat similar direct osmotic lysis of whole

cells using glycerol*

Other workers, primarily interested in the mem­

brane as an agent involved in the transport of materials

into the out-of the cell, have examined the membrane on

the basis of its physical, chemical, and enzymatic makeup.

A leader in this area has been Peter Mitchell. In a series

of papers (45,46,47,48) culminating in a description of a

general theory of membrane transport (44), he has charac­

terized extensively the membranes of a variety of micro­

organisms. Of Staphylococci cells he reports the membranes

to be 5oS thick composed of 20$ lipid and 40$ protein, and

containing 90$ of the total cytochromes of the cell, 90$

of the succinic dehydrogenase, 80-95$ of the malic enzyme,

90$ of the malic dehydrogenase, 90$ of the formic dehydro­ 13 genase, 90% of an acid phosphatase plus various levels of other enzymes* He postulates that these enzymes are functional in the transport of their respective substrates into and out-of the cell* He further reporta that the membranes constitute 10% of the dry weight of the cell.

Storch and Wachsman (66,67) have reported similar find­ ings for the protoplast membranes of Bacillus megaterium. For example all of the malic, succinic, and a-keto glutaric dehydrogenases are components of this membrane fraction, which in these cells makes up 15% of the total cellular protein.

That the so called membrane fraction may not represent pure membrane material is suggested by the work of Repaske (60) who has shown that the 260 mu absorbing material and the specific activity of certain enzymes decreases on washing the fraction. However, Storch, ejfc al

(67) report little change, due to washing, of the specific activity of some enzymes associated with the membrane fraction of B. megaterium.

Silicon Metabolism and the Possibility of a

Slllco-Molybdate Complex in Electron Transport

Much interest has been shown recently in the possible importance of a silieo-molybdate complex or other complexed molybdate in electron transport. This was initially shown in the simultaneous phosphate and molybdate 14 requirement in various systems (21,38,65). Jacobs and

Sanadi (28) reported that silico-molybdate would enhance the oxidation of ascorbate by rat liver mitochondria.

They were considering, at that time, the possibility that this silico-molybdate complex might be coupled with phos­ phorylation. Mahler and Glenn (40) have summarized work in this area to late 1955. Glenn and Crane (20) described the chain of events leading to the demonstration that a silico-molybdate complex of 10 parts molybdenum and one part silicon was most effective in restoring the activity of aldehyde oxidase. M 0 O3 had been shown to be effective but not sodium or ammonium molybdate. The reason became evident to them when it was noted that the M0O3 used had been prepared by neutralizing with very old alkali which had been stored in glass containers and had much silicon from the glass dissolved in it. Thus, the neutralization of the MoOg had formed a silico-molybdate complex which was the actual effective agent. However they concluded that the silico-molybdate was only an artificial group sub­ stituting after the original natural site on the enzyme had been altered since no 650-1000 mu absorbing material was found in the purified enzyme (this is the region in which silico-molybdate absorbs).

The role of complex formation with raolybdo- enzymes is still under active investigation as evidenced by reports by Nicholas (50,51). He showed that purified 15 nitrate reductase from Neurospora crassa had a phosphate requirement which could be completely replaced by arsenate, tellurate, or selenate, and could be partially replaced by silicate or sulfate. This phosphate requirement does not involve phosphorylation since uncoupling agents do not inhibit the enzyme. The role may be to bind molybdenum to the apoenzyme since the effective replacement anions all have atomic radii similar to that of phosphate (in the range 2.4-2.8 $).

Incorporation of silicon by organisms or the possible actual requirement for silicon are both interesting areas for study. Diatoms of course, incorporate and re­ quire large quantities of silicon. Lewin, who has done considerable work on the metabolism of silicon in diatoms, has shown (37) that silicon uptake is linked with aerobic respiration. Cyanide, fluoride, iodoacetate, arsenate, azide and fluoroacetate inhibit respiration and uptake, while dinitrophenol enhances respiration but inhibits up­ take. Furthermore, the addition of silicate to silicon deficient cells enhances respiration by 40$.

Uptake of silicon has been studied by the use of silicon®^, a short lived radioisotope of silicon, in the rat by Holt and Yates (25), and in wheat plants by Rothbuhr and Scott (63). The very short half-life of Si3-*- (2.8 hours) has prevented very involved tracer experiments or much popularity of the Si®-*- isotope in tracer studies. EXPERIMENTAL METHODS

Growth of Cells

Azotobacter vinelandii strain 0, generously supplied by R. H. Burris, and Azotobacter chroococcum C44, donated by P. W. Wilson, were the organisms used through­ out this experimentation. The cells were cultured routinely on a sterilized, modified BurkTs (2) medium containing four times the suggested level of phosphate to further buffer the medium against pH changes. The levels of phosphate used in experiments where phosphate was varied are given on the tables describing those experiments. The buffering In these cultures was accomplished by using

0.025 molar tris at pH 7.2. The medium contained sucrose as an energy source plus added molybdenum and other metals or additives as Indicated in the individual experiments.

When combined nitrogen was used, it was added at a level of 0.7 mg. of nitrogen per ml. of medium as KNOg or

(NH4 )2 S0 4 * All routine components of the medium including sucrose were reagent grade. Inocula low in a particular component were prepared by repeated transfer of the cells on media deficient in that component.

In experiments where a large quantity of cells were required, such as the centrifugal distribution, ammonium sulfate fractionation, and paper electrophoresis experiments, the cells were grown in large quantities

16 17

(3-8 liters) in forced aeration cultures. When it was

desirable to change the oxygen tension in these cultures,

the level of air supplied was altered by changing the

pressure of the air supplied to the culture flasks.

Suitable sterile techniques were employed to insure pure

cultures. In the Inhibition, uptake of label, lysozyme

lysing, and protoplast preparation studies, the cells were grown as shake cultures of 25 ml. In 125 ml. erlen- meyer flasks. Oxygen tension was controlled In these

flasks by the rate of shaking. The temperature at which

the organisms were grown was 34°C in all instances.

Radioisotope Methodology

The Mo", w185, p32, and F e " were obtained from

Oak Ridge National Laboratory. The M o " was supplied to

the cells as Na2Mo9904, the W 185 as K2W18504 , the p32 as p3204, and the F e " as Fe^^cig. All labels were supplied

at the levels indicated in the individual experiments. 31 The Si was prepared by the following reaction

S i 30 -/■ n ..-> SI31-/ £T

In the Battelle Memorial Reactor. Reagent grade silica gel (Mallinckrodt) was used as the target. The ttormal

distribution of silicon isotopes in nature Includes 3$ 30 SI . Due to the close proximity of the Battelle Reactor

It was not necessary to utilize Isotopically pure silicon3® In spite of the short half-life (2.8 hours) of 18

the silicon3-*- produced. A IS hour irradiation provided 31 near saturation level of Si and allowed 12-14 hours

for experimentation at the level of silicon incorporation by Azotobacter. The target material was Irradiated in

specially constructed polystyrene containers, and sub­

sequently fused with sodium carbonate In platinum

crucibles. The soluble SI31 as NagSI^Og, obtained by

this treatment, was then supplied to the cells at the

Indicated levels.

The N-L3 was prepared in the Ohio State Uni­

versity cyclotron using reactor grade graphite as the target by the following reaction.

C 12 -/• p ------» N 13 -/ Y Due to the very short half-life (10 rain), all procedures were completed as rapidly as possible. The bombarded portion containing N-*-3 trapped in the lattices was removed carefully and transferred to a Vycor combustion tube with excess powered CuO. The graphite was burned in the combustion tube in Og to release the N13. After repeated passes across heated Cu and CuO, the N-*®

(presumably as Ng-*-®) was allowed to enter the atmosphere above the cells or other treatments.

All radioactive assays were performed with a

Geiger-Muller tube and standard scaling circuits. Suit­

able corrections were made for background, and for decay 19

where appropriate. Where the activity level was very low,

the duration of counting time was of such a length to

insure less than 1 0 ^ counting error.

Cell Harvesting, Assay Techniques and Miscellaneous

Cell growth was determined turbidimetrieally during the log phase by optical density readings at 660 mu. These readings have been shown earlier (19,32) to parallel Kjeldahl nitrogen values rather closely

during the log phase growth.

Since the length of the lag phase in the

competitive inhibition experiments was not altered by the different treatments, log phase optical density readings were considered equivalent to growth rates.

All results of experiments using the shake culture method are averages of 4 replicates, or averages of 2 re­ plicates for which a confirming, double replicate experi­ ment was run. The distribution, ammonium sulfate frac­ tionation, and the paper electrophoresis experiments were generally run at least 2 or more times to insure their reproducibility. The short half lives of the and 31 Si isotopes prevented replicates or reruns in some cases.

For experiments requiring cell homogenates, cells were prepared for disruption by harvesting by centri­ fugation. They were then washed twice and resuspended in 0.25 M sucrose containing 0.1 M phosphate (pH 7.2). 20 In early distribution experiments cells were disrupted by grinding with washed sea sand or alumina in cold mortars and pestles, or with a 10 KC Raytheon Sonic Oscillator. The latter technique was found most satisfactory and used thereafter in all experiments requiring cell breakage except in the lysing and protoplast formation experiments. The differential centrifugation procedure was as follows. Unbroken cells were removed by centrifu­ gation at 10,000 times gravity for 1 hour. Sedimentable fractions were obtained by successive centrifugations on a Spinco preparative ultracentifuge (Model L) at 25.000 times gravity for 1 hour, 144,000 times gravity for l/2 hour, 144,000 times gravity for 1 hour, and

144.000 times gravity for 6 hours. These fractions are designated as Rg5 > ^144-1/2 * ^i44«]_» ^144-6 respectively.

The corresponding supernatant fractions are designated as ^2 5 , ^i44-l/2> ^144-1 * and Si44-6» The disruption and centrifugation steps were accomplished at a temperature range of 0-4°C. The centrifugal values represent the relative centrifugal force at the bottom of the tube. In uptake experiments, the uptake of a labeled material was determined by centrifuging the log phase cells from the labeled medium and measuring the loss of activity in the medium. Incorporation is defined as the 21 uptake minus the loss by washing, or as in the case of

Mo®® and that actually bound to proteins. Protein was determined by the optical density values at 260 and 280 mp according to the method of Warburg and Christian (72). The ratios of the optical density readings are reported as an indication of the nucleic acid content of the fractions. Specific activi­ ties are defined as counts per minute per unit of protein. Dialysis of fractions was accomplished using 0.25 M sucrose - 0.1 M phosphate (pH 7.2), or 10“® M cyanide (pH 7.2), with stirring until equilibrium had been reached.

In the ammonium sulfate fractionation experi­ ments, the 25,000 times gravity supernatant fraction after 90 minutes centrifugation was used as the source material. To 10 ml. of the extract was added 1 or l/2 ml. increments of 3.9 M ammonium sulfate with centrifugation of the precipitate after each step until all the activity had been fractionated. Protein was determined on each fraction by the methods described above. The radio activity assay of each fraction was performed as above. All manipulations were done at 4°C.

The paper electrophoresis experiments were also conducted on the 25,000 times gravity supernatant fraction. Repeated trials at various pH levels and ionic strengths of a number of buffer systems showed pH 9 Veronal at an ionic strength of 0.01 to be most adequate and this system was used throughout. Whatmann 3MM paper in sheets 9 by 22-1/2 inches proved most satisfactory. A current of 40 mamps at 400 volts was used routinely, with runs of about 10 hours at 4°C. Protein color was developed by bromphenol blue staining. The lysozyme lysing and formation of proto­ plasts of Azotobacter were performed according to the methods of Repaske (59,60). Muscle phosphorylase was prepared by the methods of Illingworth and Cori (27), and the production of reducing sugars was assayed in the coupled phosphory- lase/malt phosphatase system by the dlnitrosalicylic acid method (6 8 ). The activity of the phosphorylase enzyme preparation was first assayed using the customary Fiske and Subbarow inorganic phosphate assay (18). The following abreviations are used: GSH - glutathione, TPN - triphosphopyridine nucleotide, TCA - trichloroacetic acid. RESULTS AND DISCUSSION

M o " Uptake by Azotobacter vinelandii Celia

and M o " Distribution in Cell-Free Preparations as a function of Culture Conditions

The desirability of determining the intra­ cellular fraction involved in Ng fixation prompted ex­ periments to examine the intracellular distribution of molybdenum in cells grown on Ng as the nitrogen source. The author has previously (32) reported experiments designed to test the possibility that a molybdenum-rich particulate fraction of A. vinelandii growing on Ng might be isolated. The results indicated that most of the molybdenum incorporated into the cells could be found in the supernatant fraction after 1 hour centrifugation at 144,000 times gravity on the Spinco preparative ultra­ centrifuge. Since that time, experiments have been run in which the time of centrifugation has been increased to 6 hours at 144,000 times gravity. Using a number of breakage 99 techniques, levels of Mo*70 added, and percentages of cell rupture, it has been found that one can isolate a partic­ ulate fraction, R-144-6, which contains the highest

specific activity of molybdate.

The results of an experiment, in which the best conditions for rupture were used, are found in Table 1. 23 Here, not only was the highest specific activity of molybdenum found in the R-144-6 fraction, but also about 2/3 of the total molybdenum taken up by the cell was found in this fraction. Centrifugal sediments below the R-144-1/2 contained only insignificant amounts of molybdenum which was probably present at simple occlusion on the sediments. Results in Table 2 indicate what occurred when percent cell rupture was increased to 60$. While the highest specific activity still remained in the R-144-6 fraction, the highest total activity was now found in the S-144-6 fraction. These data suggested that the higher proportion of cell rupture resulted also in a higher proportion of particle rupture. Prom both these experiments and a number of other similar experiments it was found that the highest 260/280 mp ratio was in the R-144-6 fraction, indicating a rather high proportion of nucleic acid in this fraction. Runs in which the cells were supplied other 99 levels of Mo , including runs where molybdate deficient cultures were supplied very low levels of molybdenum

(0.1 ppm), failed to reveal any differences in the distri­ bution of the highest specific activity fraction. As had been previously reported (32) Azotobacter vinelandii is capable of taking up about 100 times as much molybdenum as is required for optimum growth. That is, rapidly aerated cultures grow at an optimum rate on levels 25 of molybdate below 0.01 ppm even when the medium has been carefully purified. However, the cultures would take up about 1 ppm by the end of the log phase when presented with molybdenum at this level or higher. Once again in these experiments the same thing has been observed. If cultures were allowed to reach an optical density of about

0.850, which is approximately the point at which these cultures begin going out of the log phase, they took up about 1 ppm Mo®®. Interestingly enough, this uptake rather closely paralleled log phase growth. Table 3 indicates these results. With the demonstration of a molybdenum-rich fraction in cells growing on Ng gas it became desirable to test the possibility that a different distribution, or a different level of incorporation of molybdenum could be demonstrated for A. vinelandii utilizing NOg or NH^ as a source of nitrogen. Since both nitrate reductase and nitrogenase, presumably molybdo-proteins, are adaptive enzymes, and since neither would likely be present in cells using it was thought possible that marked differences might be observed. The initial experiment to examine this possibility indicated a rather marked variation in molyb­ denum uptake and some possible differences in distribution. Attempts to verify these results proved futile. Each additional time the experiment was run both uptake and distribution proved very similar for Ng, HOg, or NH^ cells. 26 It was thus apparent that the uptake and distribution of molybdenum was not a unique function of nitrogen source. It was finally discovered that the variation in uptake initially observed was more closely related to the level of aeration than to nitrogen source. At about this same time Lenhoff, ejb al (36) published an interesting proposal of an alternate molybdo- flavoprotein terminal oxidase pathway in Pseudomonas fluorescens. These organisms exhibited a marked variation in molybdenum content dependent upon the level of iron and aeration in the medium. Our results indicated a 99 variation in Mo uptake as a function of aeration, and in the light of this interesting proposal of Lenhoff, led to the following work. Experiments designed to test the influence of aeration (Table 4) and iron level in the medium (Table 5) on the uptake of molybdenum by A. vinelandii indicated that nearly twice the M o " uptake per unit growth was observed for well aerated versus poorly aerated cultures. Decreasing the level of iron also roughly doubled M o " uptake. These results were consistent with those observed by Lenhoff, ert al. Another Interesting observation was that there was little difference in uptake as influenced by iron level and oxygen tension as a function of the three nitrogen sources, Ng, NOg, or NHj.4 2 7 To teat more fully this iron/molybdenum/aeration 59 relationship, the uptake of Pe was examined as a function of aeration and molybdenum level. For cells growing on 59 N2 , Pe uptake per unit growth was constant for molybdenum levels varying from 0.001-10 ppm. However, as is evident CQ in Table 6 , about four times the Pe uptake per unit growth was observed for cultures poorly aerated as compared with well aerated cultures. Because of the considerable Influence of aeration OQ and iron concentration on Mo uptake, it was desirable to know the effects of these factors on M o " distribution, and 99 also to measure the uptake effect as Mo incorporation per

unit of protein formed. Table 7 shows such data for cells grown on N0£, HH^, and ^ in six liter cultures where the

aeration was controlled by varying the pressure of the air

supplied to the culture bottles. Although the effect of 99 iron and aeration levels on Mo uptake is very large, the 99 effect on Mo distribution is not significant except in the case of Ng grown cells. A large proportion of the total molybdenum taken up is found in the R-144-6 fraction, except where cell breakage was unusually high, indicating a high proportion of particulate rupture. Further evidence concerning the disposition of 99 Mo taken up by A« vinelandii was obtained by the following experiments. Cells were grown on the three nitrogen sources 28 in 1 ppm of M o " until moat of the molybdenum was taken up. The cells were then harvested and washed at 0°C three times in molybdate-free medium. In all cases about 15% of the Mo®®99 taken up was lost from the cells. When the cells were washed at 0°C in a medium containing 100 ppm of molybdenum

(unlabeled), about 30% of the M o " was lost. Labeled cells allowed to incubate for 4 hours at 34°C in a new medium containing 100 ppm of molybdenum also lost about 30 per­ cent of their Mo". There were no differences in the loss of M o " as a result of these treatments that could be ascribed to the different nitrogen sources. In addition, the M o " in the R-144-6 fractions from the cells grown on the different'nitrogen sources was not removed appreciably in any case by dialysis against 10"3 M cyanide at pH 7.2.

It therefore seemed probable that most of the molybdenum taken up was Incorporated into molybdoprotein(s).

The Relationship of Tungsten and Vanadium to Molybdenum in Azotobacter vinelandii

Due to the widespread variability in the reported effect of tungsten and vanadium as substitutes for molyb­ denum (see Literature Review) in Azotobacter, It was felt desirable to test the effects of these metals on the strain of A. vinelandii being used in this laboratory,

(strain 0 ). Inasmuch as tungsten is in the same chemical 29 group In the periodic table as molybdenum, one might expect tungsten to serve either as a partial substitute for molybdenum or as a competitive inhibitor. The case for vanadium is, of course, not so strong on these grounds.

The results of a shake culture experiment designed to test the effect of high levels of molybdenum, vanadium, and tungsten supplied as molybdate, tungstate, and vanadate on Azotobacter vinelandii are reported In Table 8 . This table shows that vanadium, and molybdenum, supplied at high levels, had little effect on growth of this A. vine­ landii strain, regardless of the nitrogen source. Tungsten, however, very markedly inhibited the growth of cells on either Ng, or NO3 but not on

Data obtained by supplying tungsten and molybdenum in varying ratios show (Table 9) that tungsten acts as a competitive inhibitor of molybdenum, since the inhibition is a function of the ratio and not the absolute level of tungsten. Furthermore, cultures growing on Ng or NO3 were equally sensitive to the tungsten inhibition. Half maximal growth rates were obtained with a tungsten/molybdenum ratio of 170/1.

Because it was known that under normal culture conditions (where molybdenum Is supplied at 1 ppm) the uptake of molybdenum parallels growth until about 95$ of the molybdenum is taken up, (Table 5), it was of interest 30 to know whether growth and molybdenum uptake would be inhibited to the same extent by tungsten. Table 10 illustrates that 100 ppm tungsten almost completely in- 99 - hibits Mo uptake by cells growing on Mg or NO3 with­ out seriously inhibiting growth. Thus the excessive up­ take and incorporation into non-essential molybdoproteins is more sensitive to tungsten inhibition than the synthesis of the molybdoprotein(s) required for growth. Similar experiments testing the effect of 100 QQ ppm vanadium on the uptake of Mo^° indicated a rather close correlation between the slight inhibition of growth 99 and the inhibition of Mo° uptake per unit growth. This, of course, contrasts with the effect of tungsten. Since it has been shown that tungsten was a competitive inhibitor of molybdenum in the growth of Azoto­ bacter vinelandii (Table 9), and that tungsten more markedly inhibits M o ^ uptake than growth (Table 10), it was felt QQ desirable to test the uptake of Mo as a function of tungsten level in the medium. It is evident in Table 11 that increasing the tungsten level decreased the relative 99 uptake of Mo per unit growth. However comparing, for example, treatments 3 and 5 one sees that 50 ppm tungsten lowered the growth to about 90# of the check culture while at the same time the Mo^® uptake was lowered to 16#

of the original, which once again shows the greater effect

of tungsten on Mo^9 uptake than on growth. 31 185 In Table 12 the uptake of W as a function of molybdenum level Is reported. In contrast to Table 11,

W uptake was rather independent of the molybdenum level except perhaps where molybdenum level was extremely high in treatment 1 as compared to the tungsten level. It is doubtful that a very high degree of significance can be attached to the lower reading of treatment 6 due to the rather low relative growth of this treatment. This rather

uniform uptake of W per unit growth as a function of molybdenum level may be a reflection of the more marked

effect by tungsten on M o " uptake than on growth.

The reported ability of vanadium to replace molyb­ denum in Azotobacter does not hold true for this strain.

As seen in Table 13 the tungsten inhibition can be re­ versed by molybdenum, but not by vanadium even at very high concentrations. If vanadium could actually substitute for molybdenum, one would expect it to reverse the tungsten

inhibition as does molybdenum. This inability to sub­ stitute for molybdenum does not, of course, constitute evidence against the possibility that Azotobacter may re­ quire vanadium in addition to molybdenum for some other function(s)•

If these cells do have an alternate terminal oxidase

requiring molybdenum (a possibility which is by no means remote as evidenced by the molybdenum/iron/aeration re­ lationship), and one could force respiration to proceed in 52 thia fashion, then one might be able to show a molybdenum J. requirement for cells growing on NH4 . In Table 14 are reported results which indicate an actual tungsten in­ hibition partially reversed by molybdate for cells growing on UH^ when the iron of the medium is at a very low level. These results have been repeated but not consistently*

This lack of consistency may have been due to the past history of the inocula, levels of iron in the medium, degree of aeration or some other variable not completely understood. It would be assumed that a very high degree of aeration coupled with this low level of iron would prove the best conditions for the tungsten inhibition and the molybdenum reversal. Experiments varying the degree of aeration intentionally have not as yet been run, however.

A Comparison of the Mature of the Incorporation of and

M o " into Azotobacter vinelandii

Considering the very interesting tungsten/molyb­ denum competitive relationship, it was deemed desirable to compare rather more closely the nature of the incorporation 99 T oe of Mo with that of W . I t seemed possible that because the antagonism was demonstrated at the cellular level, the actual competition might be occurring at the site of entry 33 of the metals Into the cell rather than at the site of incorporation into intracellular constituents (likely proteins). Perhaps tungsten served to block the uptake of molybdenum while remaining outside the cell itself. That uptake occurred was known from the results of Table

1 2 , and this, of course, suggested that tungsten and molybdenum might compete at the intracellular site. In addition, more information was desired as to the nature of the agent to which intracellular molybdate was bound, and to which tungsten was bound (if indeed it proved to be bound). It was further deemed desirable to attempt to separate the incorporated molybdate into various protein fractions (assuming the binding to be on proteins). The possibility that one might separate molybdoproteins from variously cultured cells, and thus assign a function to each molybdoprotein, loomed bright on the horizon.

Initially a comparison of the intracellular distribution and uptake of M o " and w 185 was made. It is evident in Table 15 that the distribution of w ^88 was similar to that of Mo". Most of the label, as well as the highest specific activity of that label was found in the R-144-6 fraction. The incorporation of w**-88 was similar to that of M o " and at a level of 30$ that of 99 Moi's' in terms of mgs. of metal per mg. of protein.

Considered In the light of the tungsten/molybdenum ratio 34 which was 10/1 , this suggests that, were conditions right, and if the cells were not inhibited by tungsten, tungsten incorporation might take place mole for mole in place of molybdenum, although the lack of any effect of molyb- 185 denum level on W incorporation (Table 12) does not support this view. It must be remembered that this in­ corporation of molybdenum or tungsten represents uptake over and above that found not bound in the cell, since unbound label (about 30$) is lost during the washing of the cells prior to rupture. As has been previously indicated the M o " taken up is not appreciably lost by dialysis against phosphate or cyanide which was taken as partial evidence of the protein bound nature of the molybdenum. Table 16 compares the dialysis characteristics of W"1-8^ and M o " of the 25,000 times gravity supernatant fraction of cell- free preparations. It is evident that less than 20$ of 99 185 either the Mo or W was lost by phosphate or cyanide dialysis. These data and that on washing and exchange 99 of Mo in whole cells (Section A) suggest a protein bound state for the label.

Further evidence supporting the conclusion that both molybdenum and tungsten incorporation is into pro- tein(s) was obtained by subjecting the 25,000 times gravity supernatant fractions of W185 and M o " grown cells to the 35 action of proteinasea and ribo-and deoxy-nucleases. It is evident in Table 17 that proteinase treatment by papain or trypsin, both of which caused about 80% de­ gradation of TCA precipitable material, resulted in the loss of nearly all the label by subsequent dialysis. RNAase and DNAase together also caused a considerable loss of activity presumably by the liberation of some internal Azotobacter proteinase masked by nucleic acid since some (20%) of the TCA precipitable material is lost by this treatment. Interestingly enough the blank kept at the incubation temperature of 34°C lost 20% more label to subsequent dialysis than did the cold temperature blank though no degradation was observable. This suggests an attack by endogenous proteinases at the elevated temperature, or a heat labile nature of the molybdoprotein. 185 A similar experiment for Incorporated W was run, and these results are reported in Table 18. While the incubation time was shorter and the temperature lower, resulting in differences of lower magnitude, It is evident that the same general pattern exists. That is, proteinases release the W 185, nucleases release It to a lesser extent, and some of the label is also released by elevated temperature. 56 It was shown in earlier work (32) that the 99 fraction normally containing high Mo would not take up label by simple adsorption to any extent. In fact, less than l/20 of the normal incorporation during growth was found to be adsorbed by this fraction during Incubation of the fraction along with the label. Similar experi­ ments have now been run for W^*88. Very little mA88 was adsorbed by this fraction during incubation of the label with the fraction. It thus appears that very little, if any, of the in vivo incorporation can be accounted for by simple adsorption onto proteins during preparation of the fraction. Rather, it would appear that both w -1-85 and Mo98 are incorporated into protein(s) during growth, thus representing actual in vivo synthesis of metallo- proteins. Further evidence for their protein nature was obtained In the ammonium sulfate fractionation and electrophoresis experiments to be discussed next. Magee (39) has shown that a large proportion of 99 the Mo taken up by A. vinelandii strain 0 can be pre­ cipitated from cell-free preparations by ammonium sulfate between 1.4 and 1.9 molar. Since there existed a good possibility that there would be more than one molybdo- protein in Azotobacter, experiments were set up to examine the possibility of using the ammonium sulfate 37 fractionation as Magee had done, hut by serial fraction­ ation using very small increments of ammonium sulfate in an effort to determine if one could locate more than a single molybdoprotein in cell-free preparations. Figure 1 illustrates results representative of such experiments. The Mo88 separates in one very sharp peak only, at an ammonium sulfate molarity of about 1.65, regardless of the nitrogen source. The slight difference in the placement of the peak fraction for the NOjj cells is not significant as other runs show experimental varia- tions to be of this magnitude. grown cells which incorporated W-^85, show a similar fractionation pattern, that is, only one peak appears. Cells on nitrogen gas incorporating W 185 fractionated identically. The small peak at a molarity of 1.46 is concluded to represent simple adsorption of label since it can be seen that a very large proportion of the total protein is recovered in that fraction. The concentrations of tungsten and molybdenum in Figure 1 are not directly comparable as mg. of metal/mg. of protein since specific activities of both labels were not the same. For an absolute com­ parison of the uptake of molybdenum and tungsten see Table 15 and the foregoing discussion.

The experiment in Figure 1 was conducted on cells grown under rather high levels of aeration and only 38 moderate Iron levels in the medium which, as has been QQ indicated above, enhances the Mo uptake into a

’’luxury1' site perhaps possessing terminal oxidase capa­ bilities. Other experiments on low oxygen tension and high levels of iron, where M o " uptake was reduced, failed to reveal any other peaks which, it was thought, may have been masked in the Figure 1 experiment by this excessive uptake. Regardless of the nitrogen source under which the cells had been grown, only one single peak was evident. While Figure 1 represents a plot of the total activities of the fractions, a plot of specific activities of the fractions also failed to reveal more than one molybdoprotein peak. A further attempt was made to compare the dis­ position of the molybdoproteins under varying conditions and further to compare the molybdo- and tungstoproteins with one another. Partially purified preparations of A. vinelandii were prepared by differential centrifuga­ tion of the crude homogenates for 90 minutes at 25,000 times gravity. The supernatant preparations were then subjected to paper electrophoresis. Radioautographs were prepared from these strips. Densitometer scannings of all treatments were run on the radioautographs and on the developed electrophoretic strips, and were compared with one another* Figure 2 shows a plot of the densi- 39 toraeter readings on preparations from highly aerated cells grown on N2 gas with either W 185 or M o " as a label. While the protein separation is not complete, as one might expect from only semi-purified preparations containing a very large number of different proteins, it is evident that only one labeled fraction is obtained on the electro­ phoretic strip. The mobilities of both the tungsto- and 4 molybdoproteins are identical. Homogenates of or

NOg grown cells revealed the same pattern. There was but one single peak in either case and it had the same mobility as that of the peak from homogenates of cells grown on

1^2 . Electrophoretic runs on preparations of cells grown so as to limit the excessive molybdenum uptake still failed to produce more than a single peak. It was initially felt that one might obtain a 99 number of Mo labeled spots on a paper electrophoretic run, or obtain a number of labeled fractions from serial ammonium sulfate fractionation in view of the number of molybdoproteins possible. However, because these cells do Incorporate 90-100 times more molybdate than is re­ quired for optimum growth, and because all this excessive uptake goes into a “luxury" fraction possibly having terminal oxidase capabilities, then the Inability to separate other molybdoproteins by these rather crude methods is perhaps explainable. However, the inability 40

QQ to observe other fractions when Mo uptake had been decreased by appropriate culture conditions remains unexplained if Azotobacter does have more than one molyb- doprotein.

Mo", w 185, and Fe59 Localization

in Azotobacter vinelandii by the Direct Lyaozyme Lysis and Protoplast Formation Techniques

The techniques of osmotically rupturing proto­ plasts and the direct lysis of cells by lysozyme or other treatment are becoming rather widely used as means of obtaining cell-free fractions of bacteria. These tech­ niques are especially useful in obtaining a fraction which has been labeled the "membrane ghost” fraction, which is very difficult to obtain by any other cell breakage technique. Repaske (59,60) has published methods which allow the direct lysis of A. vinelandii using lysozyme and versene in tris buffer, or indirect lysis by the initial formation of protoplasts and sub­ sequent osmotic lysis of the protoplasts. Presumably if one takes cells lysed in either way and centrifuges at

2000 times gravity, the precipitate represents the ’’membrane ghosts".

Assuming the fraction to represent primarily "membrane ghosts", it was felt desirable to examine 41 the distribution of iron, molybdenum, and tungsten using these methods* The localization of iron is of interest in relation to its role as a component of cytochromes, especially since cytochromes have been reported to be components of the membrane of Azotobacter (62). The recognized requirement of molybdenum in nitrogen fixa­ tion and in nitrate reduction in various organisms lends interest to its localization. Furthermore, workers (51, 62) have reported hydrogenase, which may be a molybdo- protein, to be a membrane component in A. vinelandii. In addition, if the excessive molybdate uptake by A* vinelandii is associated with a terminal oxidase, one would wonder if it might not be a membrane component as the cytochromes are. The similarities of tungsten and molybdenum incorporation reported above lend interest to localizing tungsten by these methods as well. In Table 19 are reported results of representative experiments in which the localization of the above metals was determined in Azotobacter vinelandil fractions prepared by direct lysis by lysozyme and versene in tris buffer.

Fraction 3 shows that, of the total incorporation after

washing (fraction 2), less than 25$ of the M o " or w 185 was found in the "membrane ghost" fraction. When the "ghosts" were washed in distilled water and resuspended (fraction 5), most of this label was lost. This indicates 42 an absence, or at least only a very low level, of tightly bound molybdenum or tungsten in the membrane. Iron on the other hand can be found in rather large proportions in the membrane fraction. About 2/3 of the total iron incorporated was found in that fraction, and even after washing, nearly half remained in the washed "membrane ghosts". Because a high count in fraction 4, the membrane supernatant fraction, does not distinguish between original localization in cell wall from that of the intracellular components, it Is necessary to obtain this information by formation of protoplasts* When one forms protoplasts directly, the label found In the supernatant solution after protoplasts are removed may be ascribed to the original cell wall material. This, of course, assumes that no protoplast rupture occurred, that the protoplasts have the same permeability characteristics of the original cell, and that the cell wall components after the action of the protoplast-forming medium are of such a state of division so as not to sediment at 2000 times gravity during the sedimentation of the protoplasts. Using such methods it was shown that the Mo^^

an

These results are taken as evidence for the absence of any large quantity of molybdenum, iron, or tungsten in the cell walls of Azotobacter vinelandii. It has been suggested that the membrane fraction contains components other than the original membrane,

some of which can be washed out (60). This may represent simple adsorption of materials, or may be due to com­ ponents which were actually bound loosely to the membrane in vivo. It may also represent formation of artifacts

of preparation. Assuming a thickness of 5oS for the membrane, one would not likely find more than 10$ of the total dry weight of the cell in the membrane. The dry weights of various fractions prepared by the above methods were examined by oven drying at 110°c for 6 hours. It was found that the membrane fraction obtained by direct lysis contained about 18% of the total dry weight of the

cell, and upon washing dropped to 12%. Protoplasts con­ tained 60% of the dry weight of the cell and if one

assumes 10-15% protoplast rupture during formation, this would leave a value of about 30% for the cell wall dry weight. Osmotic lysis of the protoplasts revealed that the "membrane ghost” fraction obtained this way contained 44 about 15% of the cellular dry weight. This rather closely parallels the "membrane ghost" fraction prepared by direct lysis. Thus it is very unlikely that unwashed membrane preparations represent pure membrane. Even the washed preparations likely contain some foreign material. L. B. Carr of this laboratory has examined this fraction both by light and electron microscopy and finds that in addition to membrane structures in this fraction there exists a certain amount of granular material— on occasion spherical granules of rather large dimension. This granulation may represent agglomerated membranes or may be intracellular material. No unequivocal evidence exists at present as to the origin of this material. It is sufficient to say, however, that the membrane fraction is not homogeneous upon preparation, that the washing of

the preparation likely removes many contaminants, but that even the washed material is not likely to be pure membrane. Photographs of whole cells, and preparations made by these methods can be seen on Figures 4 and 5.

The microscopy was by L. B. Carr.

Chromium as an Inhibitor of Azotobacter vinelandli The competitive inhibition of Azotobacter vinelandli by tungsten, a member of the same periodic 45 group as molybdenum, suggested that other elements might act in a similar fashion. Experiments were therefore undertaken using chromium, another member of the same group. The initial experiments proved chromium, as chromate, to be an even more potent inhibitor of the growth of A. vlnelandii than tungsten had been. One part per million of chromium added to Burk's medium reduced the growth of cultures growing in this medium markedly. Experiments were next run to determine if growth was a function of the chromium/molybdenum ratio (competitive inhibitior^ or if the inhibition was a function of the absolute level of chromium in the medium regardless of the molybdenum level (non-competitive inhibition). The results reported in Table 20 clearly demonstrate the non­ competitive nature of this inhibition. The inhibition is a function of the absolute level of chromium in the medium and is not related to the chromium/molybdenum ratio.

A recent report by Walker and Grover (71) suggests a possible relationship between chromium absorp­ tion and utilization of iron in maize plants. On the possibility that chromium might be a competitive inhibitor of iron In Azotobacter experiments were run to test the chromium/iron relationship. The results reported in 46 Table 21 clearly establish the lack of any competition between iron and chromium. Once again the growth inhibition is a function only of the absolute level of chromium added. Thus chromium, while being a very potent in­ hibitor of the growth of Azotobacter vine la ndli even at very low levels, is not a competitive inhibitor of molyb­ denum or iron. To the author’s knowledge there are no other reports of such a marked chromium inhibition by so low a level of chromium in any other organism.

A Comparison of the Molybdate Metabolism of Azotobacter chroococoum C44 with that of

Azotobacter vinelandii strain 0

The demonstration that A. vinelandii had the capability of incorporating into protein(s) about 100 times more molybdenum than was required to be present in the medium for optimum growth prompted an examination of another species of Azotobacter. It was known from the work of Horner, et al (26) that with most species of Azotobacter one could simply leave molybdenum out of the medium and obtain a considerable decrease in growth rate, while for an Azotobacter vinelandii strain tested, optimum growth rate was obtained by the molybdenum present in the medium as an accidental contaminant. In earlier work (32) 47 the author had shown that very elaborate medium purifi­ cation techniques were required to demonstrate a molyb­ denum deficiency with A. vinelandii strain 0. In the light of these facts it seemed that other species of Azotobacter might not have the ability to incorporate this excessive molybdenum. A strain of A. chroococcum was obtained and tested to determine if it would show a molybdenum de­ ficiency by simply leaving molybdenum out of the medium. Table 22 shows that while decreasing the molybdenum level in cultures of A. vinelandii had no significant effect,,

decreasing the level of molybdenum in cultures of A. chro­ ococcum C44 progressively decreased growth. Presumably if one were to repeatedly transfer molybdenum deficient cultures on medium purified to remove any molybdenum contamination, one could stop the growth of A. chroococcum completely.

This strain of A. chroococcum is likewise com­ petitively inhibited by tungsten just as is A. vinelandii. Table 23 presents evidence of this antagonism. Experiments were next designed to determine the 99 relative uptake of Mo by A. chroococcum as a function

of various culturing treatments and as compared to A» vinelandii. In Table 24 are given the very striking results. There seems to be no relationship between uptake 48 99 of Mo and the aeration or iron level as was previously observed for A. vinelandii. Further, cells grown on

as a nitrogen source incorporated only about l/5 to / 99 1/10 the Mo of parallel NOg or Ng grown cultures.

Perhaps the most striking thing of all was that this uptake represents only from about 5 to less than 1%

cells) of what similarly cultured A. vinelandii will incorporate. It thus seemed likely that A. chroococcum 044 did not incorporate large quantities of "luxury” molyb­ denum. The possibility immediately presented Itself that these cells might prove excellent cell-free preparation source material for ammonium sulfate fractionation and electrophoretic runs in an effort to demonstrate multiple molybdoproteins. The possibility would be much greater here, if multiple molybdoproteins exist, because the masking effect of the excessive molybdenum uptake would not be present.

After carefully considering the level of uptake by these cells and the maximum specific activity of the 99 Mo available in routine molybdenum shipments, the paper electrophoresis idea was abandoned since there would not have been sufficient label to obtain a satisfactory radioautograph from the electrophoretic strips. However, serial ammonium sulfate fractionation was run and these 49 results can be seen on Figure 3. In contrast to the results which might have been expected on the basis of 99 previous data and postulations, the Mo fractionated

Into only one peak at about 1.85 molar ammonium sulfate regardless of the nitrogen source. The activity peak at 1.45 molar, where most of the protein separates, is thought to represent simple adsorption of label as is probably the case for A. vinelandii. 99 The incorporated Mo , regardless of nitrogen source and regardless of the level of iron or oxygen tension in the medium, peaked in all cases in one single 99 similar fraction. The relative Mo concentrations in

Figure 3 are directly comparable in terms of absolute 99 concentration of Mo • Furthermore, since the total protein of each fraction was approximately the same, the areas under the curves represent roughly units of metal/ unit of protein. However one can not directly compare the data of Figures 1 and 3 since the specific activities of the labels were not the same. Table 24 gives an absolute comparison of the uptake of molybdenum by A* vinelandii with that of A. chroococcum.

The distribution of Mo®^ in A. chroococcnTn was also examined by the protoplast and direct lysis methods used earlier for A. vinelandii. If these techniques can be assumed to be satisfactory for 4* chroococcum, the 50 results of direct lysis reported on Table 25 possess a certain amount of significance. Optical microscope observations indicated that not all cells of a culture of A. chroococcum are lysed by these methods. Neverthe­ less, however preliminary the results may be, it appears rather likely that at least some (perhaps l/4) of the molybdenum uptake by A. chroococcum resides in the mem­ brane fraction. It would be very desirable to develop a lysing technique especially for A. chroococcum which would be as adequate as is the technique for A. vine­ landii. If such a technique were available, one would expect to be able to assay membranes for molybdenum with more accuracy.

31 Si Metabolism of Azotobacter vinelandii and Its Relation to Molybdenum, Germanium, and Phosphorus

The evidence discussed in the Review of Litera­ ture on the possible importance of silicon in a silico- molybdate complex functioning in electron transport prompted silicon metabolism studies with A. vinelandii. With the successful use of tungsten as a competitive inhibitor of molybdenum, it was felt that perhaps a similar approach with silicon might prove profitable. Germanium is an element which might be expected to be 51 either a partial substitute or an inhibitor providing the cells did prove to utilize silicon. A number of experiments were run to test the effects of various levels of germanium on the growth of Azotobacter. With addition of 100 ppm germanium to cultures a small inhibition was demonstrated. Germanium was added as Ge02 neutralized with base. Occasionally silicon, as silicate, when added at the same level partially reversed this inhibition. However, these results could not be consistently obtained. If there existed in A. vinelandii a silico- molybdate complex involving the "excessive” molybdenum incorporation, and if germanium was actually acting as a competitive inhibitor, then one might possibly observe a decrease in the molybdenum uptake per unit growth as a function of high concentrations of germanium, assuming no germano-molybdate formed. Prom Table 26 it is evident that there are no significant differences in the relative molybdenum uptake per unit growth as a function of either germanium or silicon level. Furthermore an alteration in iron level, which does affect the absolute uptake of molybdenum as shown earlier, has no effect on the relative uptake levels as a function of germanium or silicon.

This suggests the possibility that neither the "excessive” nor the essential molybdate uptake is affected by germanium or silicon. 52 However, a more direct approach waa obviously required in order to draw meaningful conclusions concern­ ing any possible "functional" silicon utilization in a

silico-molybdate complex by cells.

The possibility of utilizing Si for a silicon tracer had been investigated by others (25,63), and found

fairly satisfactory in spite of the production problems

and extremely short half-life (2.8 hours). The avail­

ability of the Battelle Memorial Reactor in this area made possible the production of Si31 by the S i ^

n ----- ^ Si51 reaction from reagent grade silica gel.

The normal isotope ratio of silicon provides 3% Si30.

With a reactor in the vicinity, this amount is sufficient without resorting to processed Si3*”* which, while avail­

able, is quite expensive. Were a reactor not available

in the immediate area, decay problems would necessitate

■ZQ using the processed Si in order to obtain a satisfactory

yield after shipping delays.

The production and preparation methods of the 31 Si were essentially similar to those described by Holt

and Yates (25) with alterations noted in the methods

section.

If silicon were complexed with the large excess

of molybdenum incorporated into proteins by A. vinelandii, then one would expect to be able to alter the uptake of 55

Si31 by altering the uptake of molybdenum by the cells. Furthermore, if germanium were a competitive inhibitor of silicon, then one would expect some alteration of

Si uptake as a function of germanium level. The data in Table 27 indicate that neither the germanium level nor the molybdenum level even when no molybdate was added significantly influenced the silicon uptake per unit growth. However, there was a fair amount of total uptake

71 of Si^ , representing about 0.25 ppm for a cell growth of about 0.850 optical density units in all cases.

Rothbuhr, et^ al (63) had suggested a possible relationship between the level of phosphorus and the 31 uptake of Si and also between the level of silicon and ttn the uptake of P , perhaps one being a partial substitute for the other. This was taken to indicate the possibility that some of the Si^ uptake observed in Azotobacter might actually represent exchange with, or utilization in place of intracellular phosphorus since Azotobacter is known to contain large quantities of poly-meta-phosphate granules. It was judged advisable to test the uptake 31 of Si as a function of germanium and molybdenum levels at various phosphate concentrations in the medium of cells inoculated from previously transfered cells on low phos­ phorus to develop an internal phosphorus deficiency.

Table 28 shows that the relative uptake of Si3]" per unit 54 growth varys little with any of the treatments. The differences are judged to be insignificant. There appears to be no exchange with intracellular phosphate. The lack 31 of any relationship between the uptake of Si and the levels of molybdenum or germanium can be taken in both this table and Table 27 as evidence that the germanium and molybdenum levels do not effect Si3-1- uptake. This, of course, suggests no germanium/silicon competition, nor the presence of a silico-molybdate complex involving any large proportion of the incorporated molybdenum and

silicon.

Experiments were also run to test the influence 32 of silicon and germanium on P uptake per unit growth

in A. vinelandii. Levels of 100 ppm of either germanium

or silicon had little effect on the relative uptake of

added to the medium at levels of 20 ppm.

The centrifugal distribution of Si 3 1 incorporated by cells (subsequently sonically disrupted) showed (Table

29) that only very little of the S i ^ could be found in

the 144,000 times gravity, 3 hour residue fraction, and

that found was at very low specific activity. This is

the fraction in which is found a high proportion of the molybdenum incorporated, and which also shows a high

specific activity of M o " per unit of protein. Thus,

this evidence further indicates that most of the molyb- 55 denum incorporated by A. vinelandii is not associated with the incorporated silicon in any silico-molybdate complex.

It is interesting to note in Table 29 that 2/3 of the Si 0.12 ppm Si uptake was washed out by the double wash to which the cells were subjected prior to sonication, indicating the very loosely bound state of most of the silicon taken up by the cells.

Table 30 indicates the distribution of Si^ as obtained by lysing experiments. It is evident that little of the Si taken up is associated with the mem­ brane fraction especially after washing. Whether or not the bulk of the silicon is associated with the wall components or the intracellular material is not known.

Time and the very short Si®^ half-life prevented such determinations as were made in section D for molybdenum,

Iron, and tungsten.

Throughout the experiments with germanium where growth inhibitions were obtained, it had appeared that the greatest inhibitions occurred on low levels of phosphorus. Table 31 reports the results of experiments specifically run to test this possibility. It Is evident in both runs that the percent decrease in growth over control cultures at the same phosphorus levels was much greater on low phosphorus than on high phosphorus. This 56 shows that there la a much better germanium inhibition at low levela of phosphorus, suggesting a possible competition between phosphorus and germanium. J. E. Varner in this laboratory in unpublished experiments has demonstrated what appears to be a com­ petition between germanium and phosphorus in the transfer reaction catalyzed by the glutamine synthetase enzyme. D. H* Slocum also of this laboratory has shown in un­ published experiments that germanium will catalyze a reaction analogous to arsenolysis by arsenate in the muscle phosphorylase system. However, the author has been unable to demonstrate any observable effect of germanium on the reaction between glycogen and phos­ phorus catalyzed by muscle phosphorylase. The re­ action was coupled to malt phosphatase for assay of reducing sugars since silicon and germanium also give color reactions with the Fiske and Subbarow reagent for inorganic phosphate.

Capabilities of the Isotope

in the Study of Nitrogen Fixation and in Other Systems Requiring a Nitrogen Label

A rather significant limitation in studies involving metabolic pathways of inorganic nitrogen com­ pounds is the lack of a suitable radioisotope of nitrogen. 57 15 The stable N Isotope is generally used, but suffers the usual limitations of stable isotopes such as elaborate methodology, time required, and lack of usefulness in purification studies. With the exception of the report by Bach (6 ) implicating a hydrazine complex, dihydro- pyridazinone-5-carboxylic acid, as an intermediate in nitrogen fixation, very little has been added to the knowledge of the intermediates in recent years. Burris in 1956 (14) summarized studies on mechanisms and suggested possible pathways including the possibility of hydrazine as an intermediate.

There are reports, notably that of Ruben, e_t al

(64), of attempts to use the very short half-life

isotope as a tracer. It has not gained wide spread

popularity due to the necessity of having a cyclotron

within a few minutes of the laboratory in which the

experiments are to be conducted, and the fact that the

experiment must be complete within a very few hours after

initiation.

It was felt that an attempt to establish the

usefulness of N as a label in nitrogen fixation studies,

and other studies requiring a nitrogen label would be of some value. Hence a number of runs were made using the Ohio State University cyclotron to produce by the

C12 / p » fll3 reaction. Yield and half-life 58 experiments were run initially to determine the feas- ability of routinely preparing N by this reaction.

Table 32 shows the results of half-life determinations and estimated yield of a typical run. It is evident that the target carbon used was very pure since only insig­ nificant amounts of long lived contaminants exist, and no C11, another possible contaminant (half life 20 minutes), was produced. Table 33 shows the results of 2 runs, the first of which was made to test the incorporation of into whole cells. Roughly 4000 counts/minute/ml. was incor­ porated. It was estimated that one would need over

100,000 counts/minute/ml. incorporation in order to make the method more useful by one or two orders of magnitude

than the currently used technique. This meant that

4 half-lives more speed would be required. The handling methods were streamlined to gain the required 4 half- lives, and a more adequate shaking device was designed

for the incubation which would increase the fixation rate of the cells. Further, an atmosphere of 80$ helium— 20$ oxygen was substituted for ordinary atmosphere to prevent dilution of the label. The only run made with these

improved techniques is reported as Experiment 2 on Table 33. Unfortunately, for some reason, perhaps the age of cells, the total incorporation levels were extremely low, 59 thus nullifing all the improvements designed to enhance incorporation. However, one important conclusion can be drawn. & comparison of the NH^ with the Ng grown cells indicates a high level of N^*3 incorporation for both cultures. The NH!^ culture incorporated 2/3 that of the

Hg culture. Since cells must be adapted before they will fix nitrogen, these results indicate that at least

2/3 of the N13 label was in the form of NH-j. This indicates the conversion methods in the combustion tube were not satisfactory. No doubt the incorporation by lysed cells and also protoplasts represents N-*-3Hg utili­ zation and not Ng-1-3 fixation.

However the possibility of utilizing N^3 as a tracer in spite of the results of the last run is very good. It has been possible by improvement of methods to transfer as much as 1/2 or more of the total N^3 trapped in the target to the atmosphere above the cells. This was done by determining the exact location of the bom­ barded area on the target and transfering only the very small amount of the target actually bombarded into the combustion tube. This small amount of carbon, in which the N*3 was trapped, was very rapidly and quantitatively ignited releasing nearly all the N^3.*

With this much label available plus more adequate methods of insuring that the N13 is in the proper chemical SUMMARY

Because of the interest in molybdenum as a cofactor in various enzyme systems, the metabolism of molybdenum in Azotobacter has been examined. The follow­ ing things have been observed. 99 The excessive uptake of Mo , earlier described by the author for Azotobacter vinelandii strain 0 growing on Ng was found localized in a particulate fraction pre­ pared by centrifugation of cell-free preparations for

6 hours at 144,000 times gravity. This fraction had a

QQ Mo17® to protein ratio higher than any other fraction, and contained a large proportion of the molybdenum taken up by the cells. Experiments with cells on NH^ or NOg showed neither uptake nor distribution of molybdate to be

QQ a unique function of nitrogen source. However, Mo*17 up­ take varys considerably with the oxygen tension and iron level in the medium. Low iron and high oxygen tension greatly increase uptake.

A competitive inhibition of molybdenum by tungsten in A> vinelandii growth was demonstrated. How­ ever, tungsten was found to have a much greater effect on inhibition of M o ^ uptake than on inhibition of growth.

Cells growing on NEfjf were indirectly shown to require

61 62 molybdenum provided iron concentration of the medium was low. Vanadium does not substitute for the required molybdenum.

Comparison of W185 and M o " incorporation re­ vealed that both to occur in the same centrifugal fraction of cell homogenates. Incorporation of both metals re­ presented actual formation of molybdo- or tungstoproteins characterized as follows. Neither label is appreciably lost by dialysis against cyanide. After incorporation of either label by whole cells, little can be washed or ex­ changed out at 0°C or 34°C. Incubation of homogenates with papain or trypsin causes release of both M o " and

W in subsequent dialysis. Neither tungsto- nor molyb- doprotein formation results from simple adsorption of label. Under ammonium sulfate fractionation and paper electrophoresis, both tungsto- and molybdoproteins behave identically. Paper electrophoretic and ammonium sulfate runs revealed only one single molybdenum or tungsten contain­ ing fraction, regardless of nitrogen source, even when incorporation was decreased markedly by lowering oxygen tension and increasing iron concentration in the medium.

CQ With lysozyme lysis Fe°® is largely localized in the ’’membrane ghosts” while w185 and M o " are largely 63 intracellular constituents* Hence centrifugal fractiona­ tion showing molybdenum in a particulate fraction does not represent an artifact formed from membrane material* Chromium was demonstrated to be a powerful inhibitor of A* vinelandli but it is competitive with neither molybdenum nor iron. Comparison of molybdenum metabolism of A. vine­ landii with that of Azotobacter chroococcum C44 revealed that the latter had no ability to incorporate the large excesses of M o " as does the former. Further, only l/5 to 1 / 1 0 as much molybdenum is incorporated by A. ohroococ-

on as on N2 or NOg. When molybdenum is not added to the medium. A* chroococcum shows a marked molybdenum deficiency not shown by A. vinelandii. The molybdate function of A. chroococcum is also competitively inhibited by tungsten. The M o " incorporated by A. chroococcum is also found largely in one fraction after ammonium sulfate fractionation. At least some (perhaps 1/4) of the 99 Mo incorporated, however, appears to reside in the membrane*

Experiments with Si showed that most of the 99 excessive Mo incorporated by A. vinelandii is not associated with silicon as a silico-molybdate complex. Germanium, while an inhibitor of growth, is not a 60 form (more adequate conversion plus suitable traps to remove unwanted forms), one could expect this isotope to be very useful. In purified enzyme systems such as in NH3 exchange or participation as a reactant in nitrate reduction or nitrification studies, for example, the isotope would be much more useful than in whole cells where the organism may use any one of a number of the chemical forms present as contaminants. 64 competitive inhibitor of silicon. However, germanium inhibition is inversely proportional to the phosphate level of the medium. Furthermore, incorporated silicon was not in the membrane fraction. The isotope was examined for its usefulness as a nitrogen label and found promising despite its short half-life of 10 minutes. CONCLUSIONS AND OUTLOOK

There is little doubt that molybdenum could function in a number of sites in Azotobacter. The absolute requirement while the cells are utilizing either Ng or NO3 suggests two possible sites. Other possibilities are likewise good--xanthine oxidase, aldehyde oxidase, and hydrogenase, not to mention the

"luxury” or excessive uptake form In A. vinelandli which possibly possesses terminal oxidase capabilities. The inability to demonstrate more than one molybdoprotein by either ammonium sulfate fractionation or paper electro­ phoresis seems strange in view of these possibilities.

One might assume that in A« vinelandii this is due to the masking effect of the excessive M o " uptake. How­ ever, even when the uptake is reduced by the appropriate culture conditions that M o " incorporated still represents 80-90^ luxury incorporation and could still cause masking. This may very well be true since, in this case, still only one molybdenum containing fraction was demonstrated. However, in A. chroococcum where very little if any "luxury" uptake occurs, there was still but one molybdo- fraction demonstrated. This, of course, suggests that either the methods are not sensitive enough to show a possible light labeling in other sites, or that, in

65 66 fact, only one raolybdoprotein exists. While the latter is not entirely unlikely, it seems a rather remote possibility. It is more probable that a demon­ stration of multiple molybdoproteins in Azotobacter will await the use of more sensitive methods. It seems that Azotobacter chroococcum C44 would be an excellent organism to use for further studies. Since it apparently possesses no ability to incorporate excess molybdate, the masking effect of these excesses would be eliminated. Paper electrophoresis runs pro­ ceeded by an ammonium sulfate fractionation to increase the specific activity of the label to a level useful in radioautography might provide the answer. It is entirely possible that some of the molybdoprotein(a) of A. chroo­ coccum might be localized in the membrane while others migjht be found intracellularly. An adequate lysozyme lysing method for A. chroococcum so that each fraction could be examined separately by electrophoresis etc. would be very useful. Furthermore, the more marked effect of tungsten on M o " uptake than on growth in

A. vinelandii, suggesting a differential uptake of tungsten by various proteins normally containing molyb­ denum, might be exploited using double labeling experi­ ments for subsequent fractionation. If this effect was also observable in A. chroococcum, these would again 67 be the cells to use. It would be interesting to compare the free-flow electrophoretic mobilities of the purified molybdoproteins from cells grown on Ng, NO3, and

One might also compare the mobilities from one species to another. Since there are some differences in the ammon­ ium sulfate fractionation, one might find electrophoretic mobilities to be different. Possibly the most likely means to demonstrate multiple molybdoproteins would be as follows. One would first run differential centrifugation of gently lysed

A. chroococcum so as to separate artifact-free fractions, notably the membrane, the R-144-6, and any other fraction with high molybdenum label. These fractions would then be sonically disrupted for an extended period of time.

Preliminary ammonium sulfate fractionation, to boost

specific activity, followed by comparative paper electro­ phoresis and autoradiography should reveal any differences in the mobilities of labeled proteins. If differences

tare observed, then comparisons by the various culture treatments to alter the proportion of each molybdoprotein would be in order.

If one were able to assign functions to various

molybdoproteins from cell-free preparations of Azotobacter,

the goal of localizing the cellular fraction important in nitrogen fixation would be at hand. Proteins of the Azotobacter vinelandli 25,000 XG Supernatant Supernatant XG 25,000 vinelandli Azotobacter the of Proteins Fraction Relative Protein and Activity Concentrations Fig. 1 Serial Ammonium Sulfate Fractionation of the of Fractionation Sulfate Ammonium Serial 1 Fig. Mo^9 Protein Protein Mo^9 (NH 4 Cells) * moimSlae Molarity Sulfate Ammonium 1.0 68 1.5 2.0 Mo99 protein protein Mo99 (N 2 Cells) atal uiid ooeae ofonvinelandiigrownArotobacter Homogenates N2. Purified Partially Relative Protein and Activity Concentrations Fig. 2 Paper Electrophoresis ofRadioactive from andMolybdoproteins Tungsto- Electrophoresis Paper Fig.2 \r\ eaieDsac rmte fciginfrom theRelative Distance Protein Proteins of an AiPtPbactal chroococcum 25,000 XG Supernatant Supernatant XG 25,000 Fraction chroococcum AiPtPbactal an of Proteins Relative Protein and Mo™ Concentrations Fig. 3 Serial Ammonium Sulfate Fractionation of the Mo^9 Mo^9 the of Fractionation Sulfate Ammonium Serial 3 Fig. Low Aeration Aeration Low L Protein (N2 Cells) (N2 (NOj" Cells) (NOj" m o 0.5 Aeration Aeration Ammonium SulfateAmmonium Molarity 1.0 70 1.5 2.0 \(N2 Cells) \(N2 High Aeration Aeration High 2.5 71

Whole Cells Protoplasts

"Membrane Ghosts"

Pig* 4 1,500 X Magnified Azotobacter vineland!i Preparations Photographed under Phase Microscopy Pig. 5 15,000 X Magnified Electron Micrograph of the Membrane Preparation of Azotobacter vinelandii 75

TABLE 1

Uptake and Distribution of M o " in Centrifugal g b Fractions of Azotobacter vinelandii 9

Total Activity 260m/u/ Total Specific Fraction in Fraction 280rau Protein Activity

R144-l/2 3,100 c/m 1.24 105 30 c/m/mg. protein r 144-6 22,600 1.60 143 158

S144-6 7,900 1.59 98 81

aDisrupted by 10 kc Raytheon sonic oscillator (33$ cell rupture). ^0.85 ppm Mo®9 uptake by the cells from 10 ppm supplied. 74

TABLE 2

Uptake and Distribution of M o " in Centrifugal

Fractions of Azotobacter vinelandii a,b

Total Activity 260mja/ Total Specific Fraction in Fraction 280np Protein Activity s25 24,800 c/m 1.60 540 mg. 46 c/m/mg. protein

R144-l/2 180 1.40 50 3.6 R144-6 6,200 1.76 104 60

S144-6 13,900 1.46 306 45

aDisrupted by 10 kc Raytheon sonic oscillator (60$ cell rupture). ^1.0 ppm Mo9® uptake by the cells from the 10 ppm supplied 75

TABLE 3

M o " Uptake by Azotobaeter vlnelandii aa a Function of Growth

Arbitrary Growth Nitrogen Source Units NH^ *2: so

0 (beginning of log 0 % a o 0 % a phase)

2 15 16 16 4 31 33 37

6 51 56 57

8 70 78 78

9 80 88 87

10 (end log phase) — 94 96

aEach figure represents the percent of the total available M o " (1 ppm) taken up. 76

TABLE 4

99 Mo Uptake by Azotobacter vinelandli

as a Function of Aeration8

Aeration N2 N0§

Stagnant Culture 26b 32 30 Rapid shake culture 47 45 53

al ppm M o " added. ^All numbers represent uptake of M o " per unit growth. 77

TABLE 5

Mo 99 Uptake by Azotobacter vinelandli aa a

Function of the Iron Concentration9

Iron Level N2 no3 nh4

0.05 ppm 345b 406 253

100 ppm 186 340 110

al ppm M o " added. All numbers represent uptake of M o " per unit growth. 78

TABLE 6

59 Iron Uptake by Azotobacter vinelandli Growing

on Ng aa a Function of Aeration®

Degree of Aeration Uptake per Unit Growth

low 4410 medium 2990 high 910

8 59 Fe added at a level of 2 ppm. Mo®® Uptake and Distribution in Azotobacter vinelandli as a Function of Culture Conditions41 Total Total Specific Total Mo®® Activity Protein Activity Uptake in per of Per Unit Cell Conditions Fraction Fraction Fraction Fraction of Protein Breakage counts/min • mg ■% 0.1 ppm Fe, high R-144-1 2,060 199 11 53 43 aeration R-144-6 13,700 116 118 S-144-6 7,880 135 58 100 ppm Fe, low aeration R-144-s 266 277 1 3 86 R-144-6 168 11 16 7 TABLE S-144-6 462 53 9 NOg 0.1 ppm Fe, high R-144-§ 2,310 183 13 70 43 aeration R-144-6 22,600 147 154 S-144-6 16,700 267 63 100 ppm Fe, low R-144-& 1,320 210 6 36 43 aeration R-144-6 17,600 277 64 S-144-6 6,560 214 31 n2 0.1 ppm Fe, high R-144-% 1,650 91 18 68 75 aeration R-144-6 7,200 48 147 S-144-6 8,930 124 72 100 ppm Fe, low R-144-J 1,070 60 17 23 16 aeration R-144-6 1,120 62 18 S-144-6 3,000 102 30 al ppm Mo®® added 80 TABLE 8

Effects of Tungsten, Vanadium, and Molybdenum on the Growth of Azotobacter vinelandli®

Nitrogen Addition: 100 ppm 100 ppm 100 ppm None Source V W Mo

N2 0.590b 0.110 0.700 0.690 no3 0.545 0.090 0.550 0.625

NH4 0.645 0.655 0.640 0.720

^Molybdenum level before additions was less than 0.001 ppm. ^All numbers represent relative growth (optical density units). 81

TABLE 9

Tungsten as a Competitive Inhibitor of Molybdenum in the growth of Azotobacter vinelandii IO HI Nitrogen W ppm 100 100 o o 100

o 1 0 10. Source Mo ppm 01001 0.01 • 0.1 0.01 0.1

% % inhibi­- 91 86 81 59 73 13 tion of growth:

no3 91 86 86 71 68 19 82

TABLE 10

The Effect of Tungsten on the Uptake of

90 Q Mo by Azotobacter vinelandli

Nitrogen Addition Percent Growth Percent Inhibition Source Inhibition of M o " uptake per Unit Growth

% 100 ppm W 10 96 no3 100 ppm W 10 100

nh4 100 ppm W 10 77

a qq Mo added at the level of 1 ppm. 83

TABLE 11

99 Uptake of Mo by Azotobacter vinelandli as a Function of the Tungsten Level In the Medium®

Relative Relative Mo®® Uptake Treatments Growth per Unit Growth

1) 1 ppm Mo®9 250 ppm W 0.387 66

2) 1 ppm Mo99 100 ppm W 0.495 121

3} 1 ppm Mo®9 50 ppm W 0.600 200

4) 1 ppm Mo99 10 ppm W 0,700 700

5) 1 ppm Mo®9 no W added 0.680 1100

aCells grown on N2 as a nitrogen source. 84

TABLE 12

Uptake of w -*-88 by Azotobacter vinelandli as a Function

of the Molybdenum Level of the Medium0

Relative Uptake of W^-85 per Treatments Growth Unit Growth

1) 1 ppm W 185 1 ppm Mo 0.640 405

2) 1 ppm W185 0.1 ppm Mo 0.585 1220

3) 1 ppm W 185 0.05 ppm Mo 0.615 1250

4) 1 ppm W -1-88 0.01 ppm Mo 0.602 1260

5) 1 ppm W 185 0.005 ppm Mo 0.335 1270

6 ) 1 ppm W185 no Mo added 0.100 950 7) no W added 1 ppm Mo 0.630

aCells grown on Ng as a nitrogen source. 85

TABLE 13

Inability of Vanadium to Substitute for Molybdenum

in the Growth of Azotobacter vinelandli8

Additions Relative Growth

None 0.320 100 ppm W 0.015 100 ppm W -f 1 ppm V 0.015

100 ppm W -f 10 ppm V 0.020 100 ppm W ■/ 100 ppm V 0.025

100 ppm V 0.285 10 ppm V 0.330 100 ppm !•/ 1 ppm Mo 0.325 1 ppm Mo 0.340

8Cells grown on Ng as a nitrogen source* 86

TABLE 14

Indirect Demonstration of a Molybdenum Requirement for Azotobacter vinelandli Grown on NHlj

Iron Relative Additions® Tungsten Additions Growth

None 100 ppm 0.212

None 100 ppm plus 0.440 100 ppm Mo

None None 0.610

10 ppm 100 ppm 0.670

10 ppm 100 ppm plus 0.670 100 ppm Mo

10 ppm None 0.678

Accidental iron contamination in the me dium estimated to be less than 0,01 ppm. 87

TABLE 15

Comparison of the Intracellular Distribution and

Uptake of VlA85 and Mo99 in Azotobacter vinelandii

Total Activity Total Specific Total Uptake In Fraction Protein Activity (mg. metal/ Fraction (counts/min) (mgs.) mg. protein)

Mo99 Cells:

R-144-1/2 3,100 105 30 0.0122 R-144-6 22,600 143 158 S-144-6 7,900 98 81

W 186 Cells:a

R-144-1/2 118 0.712 166 0.0034

R-144-6 3,650 0.300 12,100 S-144-6 1,588 0.204 9,200

aTungsten/molybdenum ratio was 10/1. 88

TABLE 16

Dialysis of the Tungsten and Molybdenum of the Azotobacter vinelandii 25,000 Times Gravity Supernatant Fraction®

Original Activity Activity After Activity After (counts/Minute) Phosphate Dialysis Cyanide Dialysis QQ Mo1'53 preparations: 1750 1650 c/m 1650 c/m

W 185 preparations:

2400 2400 1900

a o 16 hour dialysis at 3 C with stirring. 89 TABLE 17

99 Mo Dialysis Loss from the 25,000 Times Gravity Supernatant Fraction of Azotobacter vinelandli as a Result of Various Treatments®

Percent Activity Approximate Percent Treatments Loss by Dialysis Degradation*5

1) RNAase and 79 20 DNAase 2) Papain plus 99 80 10 “3 M GSH

3) Trypsin 96 80

4) Blank (34°C) 55 0

5) Blank (5°C) 35 0

Incubated for 6 hours at 34 C and subsequently dialysed for 40 hours against 0.1 M phosphate, and then dialysed for 20 hours against 10”3 M cyanide. Both dialysis at pH 7.2 at 5°C. ^Measured by the volume of TCA precipitable material. TABLE 18

1 W Dialysis Loss from the 25,000 Times Gravity Supernatant

Fraction of Azotobacter vinelandli as a Result of Various Treatments®

Percent Activity Loss Treatments by Dialysis

1 ) RNAase and 49 DNAase

2 ) Papain plus 54 10“5 M GSH

3) Trypsin 55 4) Blank (30°C) 36

5) Blank (5°C) 30

®Incubated for 5 hours at 30°C and subsequently dialysed for 36 hours at 5°C against 10"5 M cyanide at pH 7.2. 91

TABLE 19

Distribution of Fe^®, W^®^, and M o ^ by Direct Lysozyme

Lysis of Azotobacter vinelandii8

Relative Activity (counts/minute/ml.) Fraction Feby Mo«y w 114*3

i) Whole Gell 2822 3890 --- Suspension

2 ) Twice Washed, 2650 1525 160 Resuspended Cells

3) Resuspended 2042 155 30 "Membrane Ghosts"

4) Membrane Supernatant 800 1200 96

5) Washed 1360 30 10 "Membrane Ghosts"

6 ) Wash Losses of 370 125 20 Fraction 5

aFe59 added at a level of 0.1 ppm, Mo9® added at 4 ppm, and W-1-85 added at 40 ppm (W/Mo ratio of 40/l). 92

TABLE 20

Chromium as a Non-Competitive Inhibitor of Molybdenum

in Azotobacter1 1 — —■vinelandii - ■ \

Treatments Relative Growth

Experiment one: No Cr, 1 ppm Mo 0.960 0.01 ppm Cr, 1 ppm Mo 0.940 0.1 ppm Cr, 1 ppm Mo 0.920 1 ppm Cr, 1 ppm Mo 0.170 1 ppm Cr, 10 ppm Mo 0.070 1 ppm Cr, 100 ppm Mo 0.070 10 ppm Cr, No Mo 0.056 Experiment two: No Cr, 1 ppm Mo 0.730 0.5 ppm Cr, 1 ppm Mo 0.570 1.0 ppm Cr, 1 ppm Mo 0.440 1.5 ppm Cr, 1 ppm Mo 0.345 10 ppm Cr, 1 ppm Mo 0.076 0.2 ppm Cr, 10 ppm Mo 0.685 0.2 ppm Cr, 1 ppm Mo 0.710 0.2 ppm Cr, 0.2 ppm Mo 0.685 95

TABIE 21

Chromium aa a Non-Competitive Inhibitor of Iron in Azotobacter vinelandii

Corrected Relative Treatments Growth8

0.1 ppm Cr, 0.1 ppm Fe 0.425 1 ppm Cr, 1 ppm Fe 0.250 5 ppm Cr, 5 ppm Fe 0.064

0.5 ppm Cr, 1 ppm Fe 0.335 0.5 ppm Cr, 5 ppm Fe 0.298

0.5 ppm Cr, 0.1 ppm Fe 0.275

0.1 ppm Cr, 1 ppm Fe 0.418 0.1 ppm Cr, 0.1 ppm Fe 0.425

Relative Growth values corrected for the slight differences In Chromium-free cultures where the iron level was varied from 0.1 to 5 ppm. 94

TABLE 22

Comparison of the Molybdenum Requirements of Azotobacter Q vinelandii strain 0 and Azotobacter chroococcum C44

Organism Molybdenum Additions Relative Growth

A. Chroococcum: No Mo 0.310

0.01 ppm 0.400

0.1 ppm 0.795

1.0 ppm 0.920

A. vinelandii: No Mo 0.970

0.01 ppm 0.920

0.1 ppm 0.960

1.0 ppm 0.970

aReagent grade components used in the media which was otherwise not purified in any way. 95

TABLE 23

Competitive Inhibition of Molybdenum by Tungsten

in the Growth of Azotobacter chroococcum0

Tungsten/Molybdenum Percent Inhibition ratio (ppm) of Growth

100/0.001 88

100/0.01 88

10/0.001 86

10/0.01 64

1/0.001 75

10/0.1 52

0 ,1/0*001 49 l/o.l 32

0 *1/0.01 28

aCells grown on as a nitrogen source* 9 6

TABLE 24

Relative Uptake of M o " by Azotobacter chroococcum as a

Function of Various Treatments and as Compared To Azotobacter vinelandii

Uptake/Unit Treatment Growth^

Forced Aeration Cultures:.b

1) N03, low aeration, 10 ppm Fe, 1 ppm M o " 207 2) Np, low aeration, 10 ppm Fe, 1 ppm M o " 172 3) NH^, high aeration, 0.1 ppm Fe, 1 ppm M o " 39 4) Ng, high aeration, 0.1 ppm Fe, 1 ppm M o " 132

Rapid Shake Cultures:13

1) Ng 907 2) NIL 210

aThe absolute uptake for cells of about 0.85 optical density was from 0.02 - 0.05 ppm in all cases for cells grown o u /Sq ov n03 > and about 1/5 to 1/10 this amount when NEf£ was the nitrogen source. This represents from 5$ to less than 1% (NI% cells) of what similarly cultured A. vinelandii will incor­ porate . ^The specific activity of the M o " (Mo"/Motota'1-) supplied in the rapid shake experiment was about 5 times as high as in the forced aeration experiment. 97

TABLE 25

99 Distribution of Mo by Direct Lysozyme Lysis of Azotobacter chroococcum®

Relative Activity Fraction (c ount s/min. /ml.)

1) Whole cell suspension 170,000 2) Twice washed, re suspended 1,880 cells 3) Resuspended "membrane 920 ghosts” 4) Membrane supernatant 1,030 5) Washed "membrane 530 ghosts”

6 ) Wash losses from 480 fraction 5

®Mo9^ added at a level of 3 ppm. 98

TABLE 26

Effect of Various Ge/Si Ratios on M o " Uptake in

Azotobacter vinelandii0

Relative Uptake of Mo®9 Treatment per Unit Growth

Experiment 1 (0.01 ppm Fe)

1) No Si, No Ge 240 2) No Si, 100 ppm Ge 206 3) 100 ppm Si, No Ge 190

4) 100 ppm Si, 100 ppm Ge 240

Experiment 2 (1 ppm Fe) 1) No Si, No Ge 115 2) No Si, 100 ppm Ge 106 3) 100 ppm Si, No Ge 149 4) 100 ppm Si, 100 ppm Ge 129

aM o " added at the level of 1 ppm. 99

TABLE 27

31 Uptake of SI as a Function of Germanium and Molybdenum Levels in Azotobacter vinelandii®

Relative Si®^ Uptake Treatment per Unit Growth

1) No Ge, 1 ppm Mo 679 2) 100 ppm Ge, 1 ppm Mo 640 3) No Ge, No Mo 700 4) 100 ppm Ge, No Mo 750

31 Si added at the level of 10 ppm. Absolute uptake was 0.25 ppm for a cell growth of 0.85 optical density units. Phosphate level was 800 ppm (4 times Burks Media concentration). 100

TABLE 28

31 Uptake of Si by Azotobacter vinelandii as a function of Germanium, Molybdenum, and Phosphorus Levels®

Concentration of Treatment (ppm) Relative Si5-*- Uptake ------per Unit Growth P Ge Mo

200 0 1 23

20 0 1 32

20 100 1 31

20 100 0 34

200 0 0 24

20 0 0 28

200 100 0 31

200 100 1 34

Si5-*- added at a level of 2 ppm. Absolute uptake was 0.15 ppm for growth of 0.85 optical density units. 101 TABLE 29

31 Distribution of Si in Cell-Free Fractions of Azotobacter vlnelandiia,k

Total Activity Total Protein Specific Activity Fraction (c/ min) (mgs.) (c/m/mg protein)

R-144-1/4 748 57 13 R-144-3 324 18 18

S-144-3 5800 85 68

aDisrupted by 10 KC Raytheon Sonic Oscillator (61% cell rupture). bCells took up 0.12 ppm Si3"*- (0.04 ppm remained after double wash). 102 TABLE 30

31 Distribution of Si by Direct Lysozyme Lysis of Azotobacter vinelandii®

Relative Activity Fraction (c/min./ml.)

1) Whole cell suspension 1470 2) Twice washed, resuspended cells 28

3) Resuspended "membrane ghosts" 10

4) Membrane supernatant 20

5) Washed "membrane ghosts" 2

6 ) Wash losses of fraction 5 10

aCel!s supplied 10 ppm Si3^. Absolute uptake was 0.2 ppm for a growth of 0.85 optical density units. 105

TABLE 51

Germanium Inhibition of Azotobacter vinelandii as Influenced

by Phosphate Level

Percent Decrease Treatment Relative Growth in Growth over (ppm) (optical density) Controls

Experiment 1

200 ppm P, 100 ppm Gei 0.740 200 ppm P, No Ge 0.770 4

20 ppm P, 100 ppm Ge 0.456 20 ppm P, No Ge 0.545 16

Experiment 2

200 ppm P, 100 ppm Ge> 0.680 200 ppm P, No Ge 0.740 8

20 ppm P, 100 ppm Ge 0.423 20 ppm P, No Ge 0.523 19 104

TABLE 32

Half-Life of Cyclotron Produced Nitrogen

Time Elapsed after Count Remaining9 Count Expected Removal from Cyclotron (mr) (mr)

1 hr. 55 min. 18.7 20

2 hr. 15 min. 5.1 5

3 hr. 5 min. 0.21 0.16

3 hr. 45 min. 0.01 0.01

8 hr. 35 min.b 0.02 X 10-3 0

10 hr. 35 min.*5 0.02 X 10"3 0

Estimated original yield of approximately 1 Curie.

bCount in these two fractions represents the level of long-lived contaminants. 105

TABLE 33

13 N Incorporation by Various Systems

Relative Uptake System (Counts/min/ml)

Experiment li

grown cells 4075

Experiment 2:

1) Ng grown cells 336

2) NEt^ grown cells 228

3) basic NH^Cl solution (pH 11) 16

4) lysed grown cells 76

5) protoplasts of N£ cells 168

6) TCA treated lysate 20 BIBLIOGRAPHY

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I, Richard Fairbanks Keeler, was born in Provo, Utah, on January 24, 1930. I lived in Colonia JUarez, Mexico, until I was eight years old, attending elementary school there until that time. The remainder

of my elementary education was obtained in Palo Alto, California, and in various schools in the state of Utah. I completed my secondary education in 1948 in the Jordan School District, in Salt Lake County, Utah* My under­ graduate training was obtained at the University of Utah, and at the Brigham Young University, where I received the Bachelor of Science degree in Agronomy In 1954. I enrolled in the Graduate School of the Ohio State University In September, 1954. I received an appointment as Research Fellow in the Department of Agricultural Biochemistry In July, 1955, and received the degree of Master of Science In that department in December, 1955* I have held a position as Research Fellow while completing the requirements for the degree Doctor of Philosophy*

111