Azotobacter Vinelandii Cysts Approved

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THE PHYSIOLOGY OF

AZOTOBACTER VINELANDII CYSTS

APPROVED:

GRADUATE COMMITTEE:

Ma'Yor Professor

Minor jlrof/ssor

onmmftee Member

Comm itee Membe

Crr tee Member

7,.- V / ~ 41 ~- / N ~ -~ - K- L Director ~6\f the Oepartment of Biological'~: ~ Sciences

4-4e Dean o 'the Graduate School ,7cI

A'I

THE PHYSIOLOGY OF

AZOTOBACTER VINELANDII CYSTS

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

Solomon L. Aladegbami, B.S.

Denton, Texas

December, 1980 Aladegbami, Solomon L., The Physiology of Azotobacter vinelandii cysts. Doctor of Philosophy (Biology). December,

1980, 149 pp., 18 tables, 28 figures, bibliography, 147 titles.

The value of the adenylate energy charge [(ATP) +

1/2 (ADP)/(ATP) + (ADP) +(AMP)] in Azotobacter vinelandii cells was monitored during growth and germination in flask cultures. The miximal value of 0.88 was attained during mid-log phase; this declined gradually to 0.50 by late stationary phase. When these cultures were transferred to encystment media, the adenylate energy charge decreased to an average value of 0.40 as the vegetative cells encysted and remained unchanged during the next 20 days. Encysting cultures were composed of vegetative cells, encysting cells and nature cysts but the proportionate value of the energy charge could be assigned. Viability of the total population remained 95% or higher during the entire period studied.

Azotobacter vinelandii cysts cultivated on phosphate- deficient media were compared to cysts grown in phosphate- sufficient media. Although cell protein and nucleic acids were unaffected by phosphate deficiency, cell wall structures, oxygen uptake and encystment were significantly affected. Phosphate-limited cysts contained much larger amounts of poly-beta-hydroxybutyric acid but had a lower adenylate energy charge than did control cysts. The ATP/ADP ratio was much lower in phosphate-deficient cysts than in the control cysts. The data indicate a "substrate saving" choice of three metabolic pathways available to cells of Azotobacter under different growth conditions. TABLE OF CONTENTS

LIST OF TABLES...... ii

LIST OF ILLUSTRATIONS...... iv

Chapter

I. INTRODUCTION...... 1

II. MATERIALS AND METHODS...... 34 III. RESULTS...... 50

IV. DISCUSSI0N...... 124

BIBLI0GRAPHY...... 138

i LIST OF TABLES

Table Page

I. Composition of Burk's nitrogen- free medium...... 34

II. Tryptic Soy Broth (TSB)...... 35

III. Method of preparation of A. vinelandii for isoelectrofocusing on polyacrylamide gels...... 47

IV. Distribution of adenine nucleotides (ATP, ADP, and AMP) and energy charge in A. vinelandii during growth in Burk's medium supplemented with ammonium chloride...... 65

V. Distribution of adenine nucleotides and energy charge in A. vinelandii during growth in Tryptic Soy Broth...... 66 VI. Effect of growth medium on oxygen uptake and ratio of ATP to ADP in A. vinelandii...... 67

VII. Oxygen uptake and adenylate energy charge of dry cysts of A. vinelandii held on filter membranes...... 68

VIII. Oxygen consumption and content of poly- beta-hydroxybutyric acid in cells and cysts of A. vinelandii...... 69

IX. Distribution of adenine nucleotides, adenylate charge, and the ratio of ATP to ADP in vegetative cells of A. vinelandii grown in phosphate-sufficient and phosphate-deficient media...... 70

X. Effect of phosphate-deficiency on cyst formation in A. vinelandii...... 71

XI. Effect of phosphate-deficiency on oxygen uptake and ratio of ATP to ADP in A. vinelandii...... 72

ii Table Page

XII. Effect of phosphate-deficiency of poly-beta-hydroxybutyric acid storage and protein content in cysts of A. vinelandii...... 73

XIII. Comparative rates of cyst formation in A. vinelandii on substrates chemically similar to n-butanol...... 74

XIV. Comparative ratio of energy charge and ATP to ADP ratio of n-butanol-related compounds inA. vinelandii cysts...... 75 XV. Distribution of adenine nucleotides and energy charge in germinating cysts of A. vinelandii...... 76

XVI. Relative oxygen consumption and ATP/ADP ratio in germinating cysts of A. vinelandii...... 77

XVII. Relative protein content of A. vinelandii grown in Burk's nitrogen-free medium supplemented with glucose as compared to cells in different growthmedia...... 78

XVIII. Relative protein content of A. vinelandii grown in Burk's nitrogen free medium supplemented with glucose as compared to cysts grown in different substrates...... 79

iii TABLE OF ILLUSTRATIONS

Figure Page

1. Degradation of glucose...... 20

2. Reactions involved in adenine nucleotide formation, utilization and interconversion...... 27

3. Detection of flourescence signal...... 45

4. Adenine nucleotide concentrations as a function of culture age and adenylate energy charge in A. vinelandii. Cells were grown in Burk's nitrogen-free liquid medium to which glucose was added to a final concentration of 2% (w/v)...... 81

5. Adenine nucleotide concentration in mature cysts of A. vinelandii as a function of time. Cysts were grown in Burk's nitrogen- free media to which n-butanol was added to a final concentration of 0.3% (w/v)...... 83

6. Effect of phosphate limitation on adenine nucleotides inA. vinelandii cysts...... 85

7. Oxygen - uptake of A. vinelandii grown in phosphate-sufficient media; and phosphate-deficient media...... 87

8. Effect of n-butanol-related substrates on oxygen consumption by cysts of A. vinelandii...... 89

9. Level of NAD as a function of culture age in A. vinelandii grown in Burk's nitrogen-free media, media supplemented with ammonium chloride and in Tryptic Soy Broth...... 91

10. Soluble proteins from vegetative cells of A. vinelandii grown on different media...... 93

11. Densitometer scanning patterns of the isoelectrofocusing separations of soluble proteins shown in Figure10...... 95

iv Fi gure Pag9e

12. Isoelectrofocusing patterns of proteins from cells of A. vinelandii grown in Burk's nitrogen-free media supplemented with glucose and in phosphate-limited Burk's nitrogen- free medium...... 97

13. Densitometer scanning patterns of soluble protein from cells of A. vinelandii grown in Burk's nitrogen-free medium supplemented with glucose and in phosphate-limited Burk's medium supplemented with glucose...... 99

14. Soluble proteins of A. vinelandii cysts grown in Burk's medium supllemented with n-butanol instead of glucose...... 101

15. Densitometer scanning patterns of the isoelectrofocusing separation of the soluble proteins shown in Figure 14...... 103

16. The effect of growth medium on cell protein as determined by flow microfluorometry. The substrates employed were Burk's medium supplemented with glucose, Burk's medium supplemented with ammonium chloride, phosphate- limited Burk's nitrogen-free medium and Tryptic SoyBroth...... 105

17. Cyst proteins of A. vinelandii determined by flow microfluorometry...... 107

18. The effect of substrates on protein of A. vinelandii cyst as determined by fTow microfluorometry...... 109

19. Relative DNA content of A. vinelandii cysts grown on Burk's nitrogen-free medium with n-butanol...... 111

20. Effect of phosphate-deficiency on the morphology of A. vinelandii...... 113

21. Scanning electron micrograph of cells of A. vinelandii grown in Tryptic Soy Brot~hI and fixed in glutaraldehyde...... 115

V Figure Pag e

22. Scanning electron micrograph of cells of A. vinelandii grown in Burk' s medium supplemented with ammonium chloride and fixed with glutaraldehyde...... 115 23. Early precystic cells visualized by scanning electron microscopy...... 117

24. Precystic forms observed on day three by scanning electron microscopy...... 117

25. Inner core of matured cysts grown on n-butanol and observed by scanning electron microscopy after glutaldehyde treatment...... 119

26. Inner core of matured cysts grown on n-butanol and observed by scanning electron microscopy after glutaraldehyde treatment...... 119

27. Alternate pathway for substrate energy utilization in A. vinelandii...... 121

28. Distribution of energy charge during morphogenetic stages in cells of A. vinelandii ...... 123

vi Chapter I

INTRODUCTION

Members of the family Azotobacteraceae are said to form large gram-negative cells which are capable of fixing nitrogen in a nitrogen-free medium with an organic carbon source. They produce rod-shaped cells which become coccoid when mature. The Azotobacter are characterized physiologi- cally by an aerobic mode of life and metabolically by growth in media free of combined nitrogen. The other heterotrophic bacteria that utilize atmospheric nitrogen non-symbiotically, belong to the genus Clostridium and these are anaerobic gram- positive, spore-forming, rod-shaped bacteria. Several other groups of microorganisms show the ability to utilize small amounts of atmospheric nitrogen, including certain species of

Aerobacter, Bacillus, Pseudomonas and Serratia.

While the Azotobacter do not form spores, they do form cysts which are special structures that encase the entire vegetative cell. These spherical structures are morphologi- cally distinct from the vegetative cell and have been described in all the species of the genus Azotobacter (142).

They were named cysts in 1938 by Winogradsky (142) and shown by him to have three anatomical areas which he called the exine, intine and central body (142). The area consisting

1 2 of a contracted, highly vaculated, inner mass of cytoplasm which stains black to brown with violamine stains is the central body; this area is considered analogous to the vegetative cell. It is now well established (128, 143) that the appearance of vocoulated cytoplasm is caused by

large deposits of poly-beta-hydroxybutyric acid. The central

body is surrounded by a thick layer, the intine, which does

not stain, and a thinner outer layer called the exine (78).

In contrast, the vegetative cells of these organisms appear

as long rods or cocco-bacillary cells with fairly homogenous

cytoplasm and relatively thin cell walls. Young cells are

peritrichously flagellated and actively motile, but as they

age, the flagella are lost and the cells become larger and

rounded. The typical vegetative cells arise from cysts

which germinate when transferred to appropriate culture

media. The formation of cysts from vegetative cells probably

occurs in the soil and can be induced in the laboratory by

growing the organism in Burk's nitrogen-free media containing 0.3% butanol as the carbon source (142, 143).

It has long been known that phosphorous (phosphate) is

a vital element in all living cells but little is known of

the effect of phosphate deprivation on the physiology of

the Azotobacter. Although the role of phosphate in the

structure and function of other microorganisms has been

studied in great detail (41, 92,), only a few reports regard-

ing specific effects of phosphate limitation on Azotobacter

appear in the literature (25, 26, 72). Of these, the 3 work which describes the loss of cell viability in phosphate- deficient cultures of A. chroococcum (25, 26, 72) raises the most important questions.

Dalton and Postgate (25, 26) reported growth inhibition

and decreased viability in phosphate-limited cultures and a

correlation between cellular oxygen consumption and nitro-

genase function. This lead them to the erroneous conclusion

that phosphate-limited cultures stopped growing in the pre-

sence of oxygen because adenosine diphosphate was converted

to adenosine triphosphate and the resulting increased ratio

of adenosine triphosphate to adenosine diphosphate "shut off"

respiration. They further postulated that this allows

oxygen to accumulate in the cell, causing the inactivation

of the nitrogenase system and death of the cell.

Lees and Postgate (72) reiterated this explanation

and offered supporting evidence by showing that phosphate-

sufficient cells removed dissolved oxygen from the cell

interior, whereas phosphate-deficient cells failed to

do so under the same conditions. Tsai, Aladegbami, and

Vela (129) have now proved that phosphate limitation affects

growing cells of Azotobacter vinelandii by limiting the

synthesis of adenosine triphosphate. It was postulated that

the reaction leading to adenosine triphosphate production

required phosphate and would not proceed under conditions

of deficiency. Laboratory experiments described in the

results section of this dissertation prove that this

hypothesis was correct. As a consequence of these studies, 4 it appears that decreased viability of cells grown in phosphate-deficient media is due to cell wall weakening and lowered ATP/ADP ratios.

The adenine nucleotides participate directly or in- directly in all the metabolic sequences and this participa- tion is different from that of the pyridine nucleotides

(22). Energy transduction and energy storage, involving

adenosine triphosphate, adenosine diphosphate, adenosine

monophosphate are at the heart of metabolism as it is

understood at this tine. Uncontrolled changes in the

relative concentrations on these nucleotides probably affect

the rates of all metabolic reactions and are highly disrup-

tive to the cell. Reports concerning the concentrations of

the adenine nucleotides and ATP/ADP ratios under various

environmental conditions such as depletion of nitrogen (33),

phosphate (56), substrate and oxygen (22), and also during

morphogenic changes in the cell (1, 129), have not been con-

sistent for most microorganisms studied.

Chapman, Fall, and Atkinson (22) reported the sharp

drop in adenosine triphosphate and the total adenylate pool

during the exhaustion of substrate in cultures of E._ coli,

but the value of the energy charge was observed to change

less drastically. Both these changes are rapidly reversible

if the energy source is restored. Although this is true for

E. coli, it does not hold for all microorganisms. Transi-

tion of cells from aerobic to anaerobic metabolism has been

reported to cause a decrease in total adenylate pool and 5 energy charge in E. coli (22) and Klebsiella aerogenes

(49). Aeration of washed, anaerobically-grown cells causes the energy charge to increase from 0.1 to 0.74 in Proteus mirabilis, but the total adenylate pool remained constant

(130).

Hutchinson and Hanson (56) showed that phosphate depletion in B._ subtilis causes a decline in energy charge to

0.67, which is well below the range normally observed in

growing cells, while the concentration of adenosine triphos-

phate decreases to approximately 56% of normal. Unusually

low values for the energy charge and adenosine triphosphate

were also reported on phosphate-limited cells by Tsai, Aladegbami and Vela (129).

Viable endospores of many bacteria have been shown to

have an adenylate charge close to zero, but the adenylate

charge of cysts has not been measured. Since cysts are

capable of some biological activity (19, 20, 81, 104, 131,

132, 133), it is assumed that they have an energy charge

approximating that of inactive, viable vegetative cells but

probably greater than that of viable endospores i.e., between 0.50 and zero. In this study, it has been shown that Azoto-

bacter cells survive in the soil for periods greater than

fifteen years (131). This implies an active adenosine tri-

phosphate regeneration system and storage of an energy-rich

substrate sufficient to last for a long time. This is an

astonishing situation in that the storage of sufficient

energy to last for more than 15 years would require much 6 more space inside the cell than that devoted to poly-beta- hydroxybutyric acid and intine combined (assuming intine carbohydrate can be utilized as has been suggested). The explanation for this paradox appears later in this dissertation.

The value of the adenylate charge in E. coli during

growth is approximately 0.90 but varies from 0.80 to 0.98

(22). During the stationary phase, after cessation of

growth, or during starvation in carbon-limited cultures, the

energy charge declines slowly to about 0.5. During the slow

decline seen in the value of the energy charge in a given

population of cells, all cells are capable of forming colo-

nies. The number of viable cells falls off rapidly in res-

ponse to the rapid decline of adenylate charge during the

death phase of the culture. This modern view of cell via-

bility serves to explain the difference between viable and

non-viable cells and spores. It has been worked out for

many organisms but not for those that form cysts instead of

spores such as the Azotobacter.

Although there have been reports on induction of cysts

and on their survival, little is known about the relationship

between these events, respiration and energy management.

The major objectives of this research are:

(1) determine values of the adenylate energy charge and the

oxygen uptake rate in A. vinelandii 12837 growing in

different media. Since a relation could exist between 7

the adenylate energy charge and respiration, the energy

charge states may be useful to bacterial physiologists

in understanding the effect of growth medium on physio-

logical state.

(2) measure the adentylate energy charge and the ratios of

adenine nucleotides of Azotobacter vinelandii during

encystment and relate them to the conservation of energy

during cyst survival.

(3) determine the energy requried to maintain viability of

dry cysts of A. vinelandii.

(4) determine the role of poly-beta-hydroxybutyric acid during encystment.

(5) measure the physiological effect of phosphate-limitation

on cells and cysts of A. vinelandii. Review of the Literature

Winogradsky (141) first isolated the spore-forming oligonitrophiles of the genus Clostridium from garden soil using anaerobic culture conditions. He used media devoid of fixed nitrogen and air while nitrogen gas was bubbled through the culture. In 1901 (8), Beijerinck first isolated aerobic oligonitrophilic bacteria which he placed in a genus given the name of Azotobacter. He named two species;

Azotobacter chroococcum, which is very common in garden soil, and Azotobacter agile, which is wide-spread in water.

The latter he isolated from canal water in the city of Delft,

Holland. Beijerincks's experiments were a modified form of Winogradksy's in that he suppressed the butyric acid anaerobes by supplying air to his cultures. Winogradsky also used aerobic bacteria to remove oxygen from cultures in order to establish anaerobiosis. In this way, he was also able to isolate nitrogen-fixing bacteria without adding nitrogenous substances present as contaminants in nitrogen gas. Earlier suggestions by Frank (42), that blue-green algae could fix nitrogen gas were not proven until the late 1920's (2, 35). In 1949 Gest and Kamen (47) demonstrated that the photosynthetic bacterium Rhodospirillum rubrum was a nitrogen fixer. This idea was tested and proved using

15 isotopic nitrogen gas ( N2 ). The two important groups of autotrophic, nitrogen fixing organisms are the blue-green algae and a number of photosynthetic bacteria (138, 139).

8 9

The heterotrophic bacteria capable of independent nitrogen fixation and commonly known as free-living are now divided into two broad groups: a family of aerobic bacteria, the

Azotobacteraceae and numerous anaerobic sporeformers of the genus Clostridium (10).

Morphology

There have probably been more reports on pleomorphism in Azotobacter than on any other bacterium. Jensen (57) noted that it is impossible to obtain a pure culture of

Azotobacter containing only one cell form. The morphology of Azotobacter in reliably pure cultures or even in those said to be clonal is remarkably variable (57). Some of the reported cell morphologies include (59):

1. Bluntly rod-shaped or oval cells measuring roughly

2 - 4 micrometers in diameter, which are subject to

a large degree of variation in size;

2. Spherical cells 2 - 3 micrometers in diameter;

usually in short chains or clumps; 3. Smaller rod-shaped or spherical cells, sometimes

less than 1 micrometer in diameter, arising in

aging cultures or under special conditions of nutri-

tion and described by Winogradsky (141) as "nannocy-

tosi s";

4. Resting cells (cysts), of roughly spherical shape,

with contracted cytoplasm and a double-contoured

cell wall; according to Winogradsky (141), their 10

formation is induced when simple organic compounds

such as n-hutanol are employed as the oxidizable substrate.

5. The large, often irregularly swollen or filamentous

cells which Lohnis and Smith (81) described as

"gonidangia", their nature is not known, but they

seem to arise under conditions of nutrition dif-

ferent from those in simple nitrogen-free media,

or under the influence of unfavorable environments

(31, 142), they are usually termed involution forms,

and appear in medi a unfavorable to the growth of

the organisri.

6. The other morphological form reported was the filterable forms (68, 70).

Lewis (75) and Stapp (121) have shown granular inclusions, partly fat and volutin, in cells of Azotobacter. Jones (59) has also identified and differentiated two granules within Azotobacter using differential stains. He considered the stainable granules as jonidia and called the nonstainable structures glycogen.

The extensive pleomorphism in this group has prompted some investigators, including Lohnis and Smith (81) and Bi set (13), to claim that the Azotobacter possess a complex life-cycle. This conclusion was based on the observation that .any di f fe rent ce 1 type s occurred as stabili zed staes in the growth cycle of this organism. Although much work has bcen done in thi: area, little progress has been 11 made in understanding pleomorphi sm in these bacteria. The first breakthrough in this area may be that recently reported by Gonzalez and Vela (submitted for publication in Nature).

Mutants

The genetic nature of Azotobacter is unique in that no stable biochemical mutants have been obtained by means normally employed with other bacteria (63, 88). Most of the mutants reported are unstable and are characterized by a high rate of reversion to the wild type. It has been reported (106), that this high rate of reversion may be related to the presence of large amounts of intracellular deoxyribonucleic acid

(DNA) which may contain gene duplications. Recently shop and Brill (12) and others have obtained mutants of Azotobac- ter deficient in the nitrogenase system. Using these mutants, it has been shown that two enzymes are required for nitrogen fixation and that one of these is oxygen-labile while the other is stable.

Classification

While Azotobacter is easily recognizable as a genus, the differentiation into species is riot clear cut. Beijerinck

(8 ) found simiI ari ties etween )1ue-green al gae and two species of Azotobacter he had isolated. He named one of the species A. chroococcun, a fter the Cyanophyton of the family Chroococcaceae in order to emphasize the similarity he perceivcd. 12

In the 7th edition of Bergey's Manual of Determinative

Bacteriology (10), only three species were described: A. chroococcum, A. agilis, and A. indicus. Three genera Azoto- bacter, Beijerinckia and Derxia were listed in this edition based on morphological characteristics and the ability to utilize certain substrates. The 8th edition of Bergey's

Manual of Determinative Bacteriology (11) divides the family

Azotobacteraceae into four genera (Azotobacter, Beijerinckia,

Derxia and Azomonas) and twelve species. On the other hand, the Index bergyana lists the following species: A. agilis,

A. beijerinckii, A. chroococcum, A. hilgardii, A. indicum,

A. insigne, A. lacticogenes, A. paspali, A. protens, A. macrocytogenes, A. nigricans, A. smyrnii, A. spirillum, A. vinelandii, A. vitreum and A. woodstownii.

Several attempts have been made to better distinguish the species of Azotobacter but without complete success.

More recently, De Ley (29) reported DNA base composition expressed as molar averages of guanine, and cytosine (% GC) in members of this family and confirmed the generic status of those species listed in the 8th edition of Bergey's Manual of Determinative Bacteriology. In addition, along with phenotypic characters, it is suggested that the family Azoto- bacteraceae consist of three groups (Beijerinckia, Derxia and Azotobacter) and three subgroups in Azotobacter (chroococcum-beijerinckii-vinelandii, macrocytogenes and agilis).

The above conclusions have received criticism because of the difference in the % CC of Derxia (70.4 % GC) and Beijerinckia 13

(54.7 - 59.1% GC). From this and the previous reports, it is obvious that there is some controversy regarding the classification of the family Azotobacteraceae.

Nitrogen Fixation

The Azotobacteraceae are heterotropic microbes with the main uniting criterion that they are capable of aerobic, non-symbiotic, nitrogen fixation. The genus Azotobacter is the most ubiquitous of the family, being found in most soils and waters. Because of their ability to fix atmospheric nitrogen, they have been considered a potential source of nitrogen for human and animal nutrition. Nitrogen is con- tinually being lost from the soil to the atmosphere by the metabolic activities of the denitrifying bacteria but, it is assumed, that the balance is restored by the nitrogen-fixing organisms such as the Azotobacteraceae.

Many factors influence growth and nitrogen-fixation by the Azotobacteraceae. Requirements for phosphorous, molybdenum, magnesiumi, iron and calcium have all been demonstrated for optimal nitrogen fixation. The optimal pH value lies between 7 and 8 for fixation and the temperature between 25 and 3C C.

The major distinguishing characteristic of Azotobacter is its ability to utilize atmospheric nitrogen. In addition to free nitrogen, only a limited number of relatively simple nitrogen compounds are available to these bacteria (14).

Horner and Allison (52) showed that when free nitrogen is 14 not available, only ammonia and compounds that can be converted to ammonia are utilized by A. chroococcum. These bacteria are also capable of utilizing adenine, asparagine, aspartic acid, glutamic acid, nitrates and urea while al- lantoin, nitrites, cytosine, guanine and uracil are used to a lesser extent. Ammonia, which can be directly utilized by

Azotobacter, has but recently been shown to play a key role in the nitrogen-fixation cycle as it reacts with alphaketo- glutaric acid to form glutamic acid. This compound has an

inhibitory effect on the nitrogen-fixation process by feed- back inhibition. Studies by Wilson (138) on nitrate-adapted cells of A. vinelandii reveal that the cells use nitrate practically to the exclusion of free nitrogen, with adapta- tion being a rapid, strain-specific process.

Bortels (14) first showed that molybdenum is essential for nitrogen-fixation in A. chroococcum. This finding was subsequently confirmed and it was also shown that other species

of Azotobacter had the same requirement (52). Further studies

by Horner et al (55) showed molybdenum and nitrogen to be es-

sential for growth of A. chroococcum and stimulatory for the

growth of A. vinelandii. Although molybdenum promotes growth

with nitrogen, it is not uniquely specific for nitrogen-fixa-

tion. Vanadium has been shown capable of replacing molybdenum

in nitrogen-fixation (14) although not in nitrate assimilation.

Growth Requirements

Phosphorous, sulfur, potassium, calcium, iron and 15 molybdenum are essential nutrients for the growth of Azoto- bacter. Phosphorous is required in the largest quantities and these bacteria like all others, will not grow unless phosphorous is present in the medium in certain quantities.

Graves and Anderson (46) showed that these bacteria can use sulfur in the form of sulfate. Burk, Lineweaver and Horner

(18) found calcium to be required in amounts of 20 to 50 parts per million (ppm) for optimal growth with free nitrogen.

Several authors maintain that calcium is not needed for the assimilation of nitrate or ammonia (54, 108, 122), but Burk and Horner (17) reiterated this explanation and concluded that identical concentrations of calcium are necessary for growth with nitrogen gas, nitrates, ammonia, and asparagine.

The specific physiological function of calcium remains unknown.

Horner and Burk (54) showed that 3 to 5 ppm of magnesium were required for optimal growth of A. vinelandii while a value between 28 to 40 ppm of magnesium was required for optimal growth of A. chroococcum. This is an unusually high demand for a gram-negative organism. This may be related to the extraordinarly high respiratory activity of the Azotobac- ter and may rel ate to the high concentration of phosphory- lating enzymes. Magnesium functions as an activator of phosphorylation in the Azotobacter as in other organisms.

It has been shown that manganese can replace magnesium in many enzymatic processes in Azotobacter, but growth with manganese shows a longer latency period. Bortels (14), 16

Krzemieniewski and Kovats (67) and Rippel (101) found that

20 to 40 ppm of iron either as ferrous or ferric sulfate were necessary for optimal fixation of nitrogen in media with adequate molybdenum content. It was reported (38, 39, 126) that when P. subtilis and B. licheniformis were grown under conditions of phosphate-limitation in the chemostat, their cell wall composition changed. When B. licheniformis is subjected to growth limitation by decreasing the supply of inorganic phosphate, the cells cease to make teichoic acid arid their walls are found to contain an increased amount of mucopeptide. Teichuronic acids which are components of cell walls, sometimes replace teichoic acids. It is important to note that changes in morphology and wall chemistry during phosphate-limitations are fully reversible however long a particular steady state has been imposed. Under these conditions, the cells appear as spheres which change back to rods when inorganic phosphate is supplied. Both cell walls and protein synthesis are necessary for change in morphology.

Tsai, Aladegbami and Vela (129) reported that cells of

A. vinelandii grown under phosphate limitation contain approximately the same amount of nucleic acid and protein as the control cells but more cell debris and more spheroplasts were observed in phosphate-limited cultures. Electron microscopy studies by the same group confirmed the effects of phosphate-limitation on cell wall integrity. They also showed that phosphate-limited stained cells accumulate much more 17 poly-beta-hydroxybutyric acid than do cells grown in media containing sufficient phosphate.

Cyst Formation

While members of the genus Azotobacter in the family

Azotobacteraceae do not sporulate, they are characterized by the ability to form specialized resting cells called cysts.

Each cyst arises from an individual vegetative cell. In

1938, Winogradsky (142) showed that cyst formation could be

induced in Azotobacter by incorporation of organic compounds

such as ethanol or butanol into the growth medium. Socolofsky

and Wyss (119) found that cyst formation could be induced with 0.3% n-butanol as the sole carbon source. However, they were unable to induce cyst formation in liquid Burk's medium with either sucrose or n-butanol as the carbon source.

Layne and Johnson (71) reported cyst formation in liquid media containing sucrose provided that iron and magnesium were omitted from the medium. Further studies by the same

group showed that deletion of these two minerals (iron and

magnesium) from the medium increased the number of cysts

formed more than did omission of only one of the minerals.

A well defined life cycle in A. vinelandii includes

rounded, nonmotile pre-cysts; and cysts (143). Cysts are

oblate spheriods 1.5 by 2.0 micrometers in diameter and

occupy approximately half the volume of vegetative cells.

Cytological studies by Wyss, Neumann and Socolofky (143)

arid electron micrographic studies of mature cysts (78) This page has been inserted during digitization.

Either the original page was missing or the original pagination was incorrect. 19 possess readily available energy for metabolic activity.

Metabolism

Azotobacter show a great versatility in their ability to metabolize organic compounds as sources of energy. It has been reported that alcohols, organic acids, and mono-, di- and trisaccharides are utilized by most strains of Azoto- bacter (57). In addition, the Azotobacter utilize some cyclic compounds: benzoic acid (142), salicylic acid and even phenol (4b). Some of the non-utilizable compounds

include methanol, formic acid, oxalic acid, erythritol, xylose, rhamnose and mannose (122). 20

Glucose or other carbon source

Pentose Phosphate

Fructose 6 - phosphate

Entner-Doudoroff pathway Embden-M eyerhoff-Parnas pathway

Glyceraldehyde- Fructose 1,6- 3-phosphate diphosphate

Glyceraldehyde 2-Triose phosphate 2-phosphate

Pyruvate

Figure 1. Substrate catabolism in A. vinelandii. Adapted from Still, G.G., and H. Wang. 1964. Archives of Biochemistry and Biophysics. 105: p. 131. 21

Metabolism in Azotobacter is unique in that the cells are capable of oxidizing a wide variety of carbon sources with the initial stages of carbohydrate dissimilation probably passing through the hexose monophosphate pathway (88,

89, 90). Isotopic studies have shown that the Entner-

Doudoroff pathway is the most important path of carbohydrate metabolism in these bacteria (89) although evidence for two key enzymes in this pathway, 6-phosphogluconic dehydrase and

2-keto-3-deoxy-6-phosphogluconic aldolase are lacking in

Azotobacter. Glucose utilization involves phosphorylation to glucose 6-phosphate and its oxidation to 6-phosphogluconic acid. The latter compound is then split to give rise to pyruvic acid and glyceraldehyde (Fig. 1). Further oxidation yields carbon dioxide. Azotobacter grow well on acetate and

intermediates of the tricarboxylic acid cycle (TCA) (57) indicating that this pathway is functional in substrate oxidation. These substrates, as well as glucose, induce specific transmembrane carriers and the energy for their active trans-

port is presumably derived from the oxidation of malate.

The presence of TCA enzymes in growing cells of A. vinelandii

provides further evidence for the operation of the cycle.

Respiration

The most interesting aspect of the physiology of

Azotobacter involves its respiratory activity. Jensen (57)

noted that the Q02 values may be as high as 2,000 in A.

chroococcum (57), and 4,000 in A. vinelandii. These are 22 the highest values observed in any kind of living matter.

The respiration results in a complete oxidation of the sub-

strate to carbon dioxide and water (80).

Resistant Properties

Bacteria possess the ability of forming several struc-

tural devices which enable them to resist the detrimental

effects of the physical environment. The genus Azotobacter is distinguished from the other genera of the family, Azonomas,

Beijerinckia, and Derxia, by the ability to form cysts.

Cysts are considerably more resistant to deleterious chemical

agents or physical conditions than the parent vegetative

cells (43, 120). Desiccation resistance is used to distin-

guish between cells and cysts in a mixture of the morphotypes

of Azotobacter in a manner analogous to use of heat resis-

tance in ennumerating bacterial endospores. Of all the

resistant properties reported, perhaps resistance to heat

has yielded the most confusing results. It has been shown

that cysts have the same degree of susceptibility to heat as

vegetative cells. However, Garbosky and Giambiagi (43)

reported that cysts are quite resistant to heat because of

the heat resistant corpuscles present within the cysts. Socolofsky and Wyss (119) showed that resistance to desic-

cation was the most pronounced difference between cysts and vegetative cells. They showed that encysted cells could

withstand conditions of moderate desiccation for two weeks

without a detectable drop in viability. Only 1% of the vege- 23 tative cells remained viable after one day of similar treatment. Further studies have shown that dried cysts survive at least three years at room temperature (119), and fifteen or more years in dry soil (131).

Resistance to ultraviolet radiation by vegetative cells and cysts of Azotobacter further revealed the differences between these morphological forms. It takes twice as much ultraviolet light to inactivate cysts as it does to inactivate vegetative cells. In addition, Vela and Peterson (133) proved that cysts inactivated in this manner were capable of being photoreactivated.

Studies with gamma rays showed that cysts were seven times more resistant to this form of ionizing radiation than the nonencysted cells (120). Vela and Wyss (134) showed that the soil Azotobacter were much more resistant than laboratory cultures. The effect of soniccation on the viability of vegetative and encysted Azotobacter was also reported (120). The results indicate that only three minutes were required to bring about 90% inactivation of vegetative cells, while sixty minutes were required to effect the same degree of inactivation in encysted cells. Parker and Socolof- sky (94) reported that the resistant properties of cyst were due to the exine. They further showed that the degree of heat resistance of encysted cells was proportional to the amount of exine. 24

The Adenylate Energy Charge All living cells have been shown to have three funda- mental requirements: energy in the form of adenosine triphosphate (ATP), reducing power in the form of nicotinamide adenine dinucleotide (NADPH) and an oxidizable substrate

(4). The metabolic distinction between autotrophs and hetero- trophs has been related to the ways in which these needs are satisfied. Autotrophs obtain these requirements from the environment without recourse to compounds producd by other organisms. In a typical heterotrophic cell, foods are oxidized to carbon dioxide during catabolism. Most of the electrons liberated in the oxidative reactions are transferred to oxygen, with concominate production of adenosine triphosphate (electron transport phyosphorylation). Other electrons were used in the regeneration of NADPH, the reducing agent for biosynthesis. The materials produced by glycolysis and the citrate cycle serve as the starting materials for the elaboration of cell component during the biosynthesis phase. The NADPH serves as a reducing agent and adenosine triphosphate as the energy transfering compound.

Metabolic sequences, such as glycolysis and the citrate cycle that lead to the regeneration of adenosine triphosphate are controlled by the energy level of the cell (the balance between ATP, ADP, and AMP). It was assumed that the availability of metabolic energy in the form of adenosine triphosphate is an important factor in the control of bio-

synthesis. 25

Since adenosine triphosphate is not a primary biosynthetic material, it may be that these metabolic sequences can be regulated simultaneously at other steps by other intermediates. One must also assume that adenosine triphosphate, as the major determinant of the energy level of the cell, must be relevant in one way or another to all metabolic reactions. Some enzymes respond to the individual concentrations of adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate; whereas, others may respond to either the (ATP)/(AMP) or the (ATP)/(ADP) ratio.

In 1971 Atkinson (5) devised a widely used model for describing the physiological state of living cells. The model is based on the relationship among the adenine nucleotides (ATP, ADP and AMP). In this model, Atkinson assumed that all the metabolic sequences of living cells could be described in terms of the relative concentration of the

adenine nucleotides. He classified the roles of the adenine

nucleotides under three headings based on the following data:

1. The adenine nucleotides are intermediates in the

biosynthesis of nucleic acids and histidine, and

nucleotide co-factors such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleo-

tide phosphate (NADP), flavin adenine dinucleotide (FAD), and co-enzyme A.

2. The adenine nucleotides constitute an energy-trans-

ducing system that stoichiometrically couples all 26

metabolic process.

3. The adenine nucleotides regulate the activities of

a large number of enzyme reactions and probably of

all metabolic sequences.

The phosphorylated adenylates are similar in general character or properties to other biologically important compounds, but the second and third functional phosphate groups are unique in that they contain large amounts of energy which can be released on hydrolysis. The process in which the adenine nucleotides participate stoichiometrically

(categories 1 and 2 in the above classification) are sum- marized in Figure 2. 27

NADP NAD FAD CoASH HISTIDINE, PROTEIN

dADP A <---> ADP DN A

CAMP mR NA AMP

Figure 2. Reactions involved in adenine nucleotide formation, utilization and interconversion. Adapted from an unpublished figure by Jean S. Swedes, University of California, Los Angeles, California. 28

Interconversions among the different adenine nucleotide species that occur in the course of energy transduction and stoichiometric coupling between metabolic sequences are indicated within the circle; while the reactions in category

1, in which the adenylates serve as biosynthetic intermediates, are indicated by arrows extending from or entering the adenine nucleotide circle.

According to Atkinson (4), the adenylate system, (ATP +

ADP + AMP), resembles an electrochemical storage cell in its ability to accept, store, and supply energy. Most chemical energy-storage systems function stiochiometrically; that

is, a given amount of energy put into the system causes a

proportional chemical change in the system. The adenylate

energy-storage system is represented by the equations show below.

AMP + 2Pi <======>-ATP + 2H20 (1)

2ADP + 2Pi <======>-2ATP + 2H 20 (2)

ATP + AMP (======>-2ADP (3)

Equation 2 describes adenosine triphosphate regeneration, by

electron transport phosphorylation or the utilization of

adenosine triphosphate in metabolism, and equation 3 describes

the reaction catalyzed by adenylate kinase. In equation

1, the number of anhydride-bound phosphates per adenosine moiety varies from 0 (only AMP present) to 2 at full charge

(only ATP present). Division by 2 gives a parameter varying between 0 and 1, which has been named the energy charge of 29 the adenylate system (5). Since the number of anhydride- bound phosphate was 2(ATP) + (ADP), the energy charge was defined in terms of actual concentrations:

[(ATP) + 1/2(ADP)] energy charge (E.C.)= [FATP) + (ADP) + (AMP)] (4)

When only adenosine triphosphate and adenosine monophosphate are present, the expression for energy charge is reduced to a simple ratio or fraction of ATP: that is

(ATP) E.C. = (ATP + (AMP) (5)

The energy charge has been shown to be a linear measure of the amount of metabolic energy stored in the adenine nucleotide pool. The basic premise is that an organism attempts to maintain a particular cellular ratio of adenosine triphosphate to adenosine diphosphate or adenosine monophosphate and that this ratio depends on the state of its metabolism.

The ratio is high when substrate is oxidized and also when

inorganic phosphate is added to adenosine diphosphate but

low when adenosine triphosphate is used at a high rate without the concomitant oxidative phosphorylation. These varying

rates are discernable in terms of culture growth phases,

such as a logarithmic growth phase, stationary phase, death phase, etc.

There have been conflicting reports regarding the

balances of the adenylate nucleotide pool during sporulation.

Hanson and Dworkin (48), Hutchinson and Hanson (56) and 30

Stelow and Kornberg (112) have reported either a constant or an increasing adenine nucleotide concentration during sporulation and germination but Chow and Takaliski (23) and Leitz- mann and Bernlohr (73) have shown a low value of 0.05 during sporulation. Aladegbami, Tsai and Vela (1) recently showed the adenylate energy charge in A. vinelandii during encystment drops to a value of approximately 0.35. Cyst viability was maintained at 95 to 100% for prolonged periods of time at this low value. This low energy charge value is not consistant with Atkinson's descriptions but it is in keeping with the nature of cysts as they are understood today. Further studies by the same group showed that cysts exhibit some oxidative metabolism during the long dehydrated state associated with cyst survival.

Comparative Aspects of Cysts, Endospores, and Myxospores

The endospores of Bacillus, myxospores of Myxococcus and cysts of Azotobacter are structurally different from their progenitor vegetative cells. Under suitable conditions, these spores and cysts germinate to yield the cell type from which they arose, thus completing the morphogenetic cycle.

Sporulation in Bacillus and Myxococcus and encystment in Azotobacter all occur when cells are in a state of nitrogen deficiency (105). These organisms metabolize glucose via the Embden-Meyerhof pathway and accumulate acetate, pyruvate and lactate. Metabolism of acetate and other short-chain 31 fatty acids uncouples amino acid carrier proteins from their cytochrome-linked electron transport systems thus blocking amino acid uptake (113, 114). It is expected that RNA turnover occurs to provide nucleotides for the synthesis of sporulation-specific messenger RNA (6, 7). The ribonucleic acid fractions from both vegetative and sporulating cells of different species of Bacillus (3, 32, 34, 144, 145) have shown that both systems transcribe those genes associated with the vegetative cell. Sadoff (104) reported acetate metabolism as a common characteristic of morphogenesis in

Bacillus, Myxococcus and Azotobacter, and also stated that it occured via the tricarboxylic acid cycle and the glyoxy- late shunt.

Germination

The terms activation, germination and out-growth have been described as the process by which resting cells break dormancy and regenerate their vegetative (reproductive) forms. Like bacterial endospores, cysts germinate under suitable conditions of pH and temperature utilizing many of the substrates upon which the cell normally grows (104).

The sequence of macromolecular events, like extensive turnover of RNA and protein synthesis in this transition process, is similar to that which occurs during the germination and outgrowth of bacterial endospores except that more time is usually required. Cyst germination occurs over a 4 hr period but DNA synthesis and normal nitrogen-fixation do not begin 32 for another 4 hr after the cysts rupture. It is fairly well established that cyst to cell conversion occurs 8 hr after the initiation of germination (81).

The hydrolysis of poly-beta-hyroxybutyric acid and

beta-hydroxybutyric acid (BHB) and their rapid metabolism in

the outgrowing cyst was found coincident with the onset of

both nitrogen-fixation and DNA synthesis. Aladegbami, Tsai

and Vela (1) showed a quick jump in the energy charge within

4 hr after initiation of germination. However, little is

known of the physical or biological properties of these newly emerged cells.

Utilization of poly-beta-hydroxybutyric acid

One of the most interesting chemicals found in both

gram positive and gram negative bacteria is poly-beta-

hydroxybutyric acid. The physiological role of this poly-

merized form of beta-hydroxybutyric acid remained unknown

for a long period of time after its discovery. The polyester

was first identified in 1927 by Lemoigne (74). There have

been several reports on the properties of poly-beta-

hydroxybutyric acid (136), its physiological role (36, 107,

147) and its synthesis (107, 110, 115) among different classes

of bacteria. The polymer (PHB) has been identified in the

following genera: Azotobacter, Bacillus, Caryophanon, Chromatium,

Chromobacterium, Ferrobacillus, Hydrogenomonas, Micrococcus, Pseudomonas, Rhizobium, Sphaerotilus, Spirillum, Vibrio and

also in fungi and blue-green algae (50, 57, 60, 83, 87, 96, 33

103, 115, 116, 119). It was first described in Azotobacter by Lewis (75). The content of poly-beta-hydroxybutyric acid in Azotobacter varies from 9.1% to 70.4% of the cell dry weight. This value decreases with the onset of stationary phase but the level can be increased by adding glucose to the medium. There have been reports suggesting that poly- beta-hydroxybutyric acid serves as endogenous substrate reserve for A. agilis during endogenous metabolism (50).

Viability of the cell during starvation may then be depen- dent on the presence of poly-beta-hydroxybutyric acid.

The role of poly-beta-hydroxybutyric acid in bacterial metabolism has been described as analogous to that of starch and glycogen in the metabolism of higher organisms but the evidence presented is not as convincing. CHAPTER II

MATERIALS AND METHODS

The organism used for this study was Azotobacter vinelandii ATCC 12837. The basic culture medium employed was Burk's nitrogen-free salts solution (136), supplemented

in most cases with 1% (w/v) glucose or 0.1% (w/v) ammonium chloride in order to obtain cell growth. The composition of

Burk's nitrogen-free medium is given in Table I. The other medium employed in this study was Tryptic Soy Broth. The composition of Tryptic Soy Broth (a commercial preparation)

is given in Table II.

TABLE I

COMPOSITION OF BURK'S NITROGEN FREE MEDIUM

Component Amount

KH 2 PO 4 0.16 g

K2 HP0 4 ...... 0.64 g

ee'''me'me 0.20 g MgS0 4 .7H 2 0 *e* em- ee-00 eeeee' ee''* ''''. Fe SO4 ...... ees*0 00 .. 0* 0** ..0.003... g

N a2 MoO4 ...... 0.001 g 0.20 g

CaSO4 e...... ee...... e...e e...... 0.005 g

Distilled water...... e 1 liter

34 35

TABLE II

TRYPTIC SOY BROTH (Baltimore Biological Laboratories, Baltimore, MD)

Component Amount Trypticase peptone 17.0 g

Phytone peptone ...... 3.0 g

NaC1 ...... 6.0 g

KH 2 PO4 ------e--- ....------' ----- '''O.O ...... CO 2.5 g

Glucose .... O...... 2.5 g

Distilled water ...... 1 liter The complete medium was sterilized by autocl avi ng

All cultures were grown at 30 C in an incubator-shaker to insure adequate aeration, namely, a residual oxygen level of more than 1 mg dissolved oxygen per liter of medium.

Cysts were produced on Burk's nitrogen-free agar medium supplemented with various substrates as carbon sources instead of glucose. The three groups of substrates employed were the alcohols, n-butanol (0.3%; v/v) and n-pentanol (0.1%; v/v), an acid, beta-hydroxybutyric acid (0.1%; v/v) and an ester, ethylacetoacetate (0.1%; v/v). Solid cultures were prepared by adding 20 g of agar (Difco Labora- tories, Detroit, Michigan) per liter of the desired liquid medium. Media were sterilized in an autoclave at 121 C, for

15 min.

Phosphate-deficient encystment media were prepared 36 essentially as the n-butanol medium except for a change in the phosphate concentration. Only 6.4 mg K2 HPO4 and 1.6 mg

KH 2 PO4 per liter (which is 1/100 the amount of phosphate present in the control medium) were used.

Cells were harvested at late stationary phase in order to allow poly-beta-hydroxybutyric acid accumulation and cyst

formation. These cells were collected after centrifugation for 15 min in a refrigerated (4 C) centrifuge (Ivan Sorvall

Inc. Norwalk, CT) at 12,000 g. The samples were washed three times in distilled water to remove excess glucose. Large quantities of cysts were obtained by heavy inoculation

of Burk's nitrogen-free agar in Petri plates with vegetative cells.

Twenty encysting plate cultures were selected randomly

every day for seven consecutive days. Cysts were harvested

by placing 5 ml of distilled water on each plate and carefully

scraping the agar surface. The suspended cysts were collected

with sterile Pasteur pipettes, washed three times, resuspended in 20 ml of water, and frozen immediately.

Determination of Encystment

The degree of encystment was determined microscopically

by using a stain specific for Azotobacter cysts (134) and by

counting approximately 300 cells in a minimum of three fields. The other method was by the desiccation technique described

by Socolofsky and Wyss (119) using the criteria proposed by

Stevenson and Socolofsky (123). 37

Viable counts were determined by the spread plate technique, using sterile distilled water dilution blanks. Each dilution was plated in triplicate on Burk's nitrogen-free salts supplemented with 2% glucose. The plates were incubated for 2-3 days at 30 C before colonies were counted.

Assay of Poly-beta-hydroxybutyric Acid

The spectrometric method of Law and Slepecky (69) was used for the quantitative assay of poly-beta-hydroxybutyric acid. However, one significant modification was made in that the cells were digested for 24 hr at 37 C with shaking.

For the assay of polymer, the organisms were centrifuged in poly-propylene centrifuge tubes which had been previously washed thoroughly with ethanol and hot chloroform to remove plasticizers. The cell paste was resuspended in a volume of commercial sodium hypochlorite solution (Clorox) equal to the original volume of medium. After 1 hr at 37 C the lipid granules were centrifuged, washed with water, and then washed with acetone and alcohol. Finally, the polymer was dissolved by extraction with three small portions of boiling chloroform, the chloroform solution was filtered, and the filtrate was used for poly-beta-hydroxybutyrate assay. The filterate containing the polymer was transferred to a clean test tube and 10 ml of concentrated sulfuric acid were added. The tube was capped with a glass marble and heated for 10 min at 100

C in the water bath. The solution was cooled, and the absorbance was measured at 235 nm against a sulfuric acid blank. 38

The amount of crotonic acid was calculated by its molar extinction coefficient, which is 1.55 x 105 (117).

Protein Determination

The method used was that described by Bradford (15) using a standard solution of bovine serum albumin as a reference. Protein reagent was prepared by dissolving 100 mg of

Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol. To this solution 100 ml of 85% (w/v) phosphoric acid were added.

The resulting solution was diluted to a final volume of one liter. Protein solution containing 10 to 100 mg protein in a volume of 0.1 ml was pipetted into a test tube. Five milliliters of protein reagent were added to the test tube and the contents mixed with the vortex mixer. The absorbance at 595 nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of the appropriate buffer and 5 ml of protein reagent.

Extraction of the Adenine Nucleotides

Adenine nucleotides were extracted from cells, cysts and mixed populations of cells and cysts by the methods described by Johnson, Gentile and Cheer (58). The most important modification was that 2 nM ethylenediaminetetraace- tic acid (EDTA) was included in all extraction reagents.

According to this procedure, a 1 ml sample of the bacterial culture was rapidly injected into 1 ml hot ethanol . After incubation in a hot-water bath (100 C) for 10 min, the sample 39 was cooled in ice, the volume readjusted to 2 ml with cold water, and the denatured protein was removed by centrifugation. The pH of the extracts was adjusted with 0.1N sulfuric acid to pH 7.75, which was found to be optimal for bio- luminescence in the assay buffer used.

Determination of Adenine Nucleotides by the Luciferin-

Luciferase Assay

The procedure suggested by Chapman, Fall and Atkinson

(22) was used for measuring adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and also for calculating the energy charge of the adenylate pool. The adenosine triphosphate was determined by means of the luciferin-luciferase reaction (58) with a Luminescene

Biometer model 2000 integrating photometer (SAI Scientific

Co., San Diego, CA). The contents (50 mg) of a vial of the luciferin-luciferase mixture were dissolved in 5 ml of distilled water. Commercial firefly lantern extract was recon- stituted with cold water, gently mixed by inversion and centrifuged at 12,000 g for 15 min (4 C). The supernatant was refrigerated until used. During the assays, the preparation was kept at room temperature. Both adenosine diphosphate and adenosine monophosphate in cell extract were determined after enzymatic conversion to adenosine triphosphate (98) by

pyruvate kinase and myokinase (Sigma Chemical Co., St. Louis,

MO). For ATP determination, 200 bPl of the cell extract were

added to a mixture containing 50 pl of 75 mM potassium phos- 40 phate buffer, pH 7.3, and 15 mM magnesium chloride. For adenosine triphosphate plus adenosine diphosphate determinations, the above mixture was made and 0.5 mM phosphoenolpyruvate, and 20 -Pg of pryuvate kinase were added. For total adenylate determinations, 200 yil of the cell extract were added to 50 pl of a solution containing 75 mM potassium phosphate (pH 7.3), 15 mM magnesium chloride, 0.5 mM phosphoenolpryuvate,

20 pl of pyruvate kinase, and 25 pg of myokinase. The adenosine diphosphate and adenosine monophosphate were determined by difference (98). The three mixtures were incubated at 30 C for 15 min and then held at 0 C until assayed. The assay sample was 10 pl and the enzyme solution was 100pl. As a reference standard, adenosine triphosphate solutions of known concentrations (10 pM to 100 pM/10 pl) were always included in the assay (98). Blanks were assayed (with 10 pl potassium phosphate buffer and 100 p1 enzyme) after every 16 assays to determine the background enzyme count which was caused by traces of adenosine triphosphate in the crude enzyme. These background counts were subtracted from the corresponding assays to attain a corrected count for calculating adenosine triphosphate concentration. The adenosine triphosphate values given in the experimental results each represent mean values of 3 parallel measurements.

Extraction of Pyridine Nucleotides

Three culture media were employed in this study: chemically defined media, modified Burk's nitrogen-free 41 media supplemented with 1% glucose and 1% ammonium chloride; and Tryptic Soy Broth. Each supplemented with 10 ici of

[14C] nicotinamide/100 ml culture. Viable cell counts were determined by serial diluation and plating on Burk's nitrogen- free agar. Uptake of radiolabled nicotinamide was determined by pipeting 100 y1 of the radiolabeled culture directly onto a saline-soaked 0.45-um membrane filter. The cells on the filter were dried, and radioactivity was determined. The above procedure was repeated after each 24 hr interval for five consecutive days.

Respiratory Activities

Oxygen uptake by whole cells and cysts was measured

polarographically with a Gilson oxygraph Model KIC with

Clark type electrode (Gilson Medical Electronics, Inc.,

Middleton, WI). The electrode was covered with Teflon mem-

brane and maintained at a potential of +0.8 volts. The elec-

trode was inserted into a 2 ml water-jacketed sample compartment maintained at 30 C with a constant temperature water

circulator. The compartment was placed on a magnetic stirrer

and constantly mixed with a magnetic bar to prevent the

formation of oxygen concentration gradients. The instrument

was standardized with deionized water and the base line of

the instrument was readjusted between samples.

Calculations of the rates of oxygen consumption were

accomplished by the methods proposed by Hogman (51). The

solubility of oxygen in water at 30 C and 760 mM pressure is: 42

8mg 02 per 1000 ml of water (a)

Since the concentration of oxygen in air dissolved in water

at 30 C and 760 mM pressure is 33.6%, then there are:

5.26 ml 02/1000 ml H2 0 (b)

Since 1 mole of oxygen occupies a volume of 24.9 1 at 30 C

and 760 mM pressure, then under the same conditions:

5.26 ml 02 = 211.6 nmoles 02 (c)

Therefore, the solubility of oxygen in water at 30 C and

760 mM pressure is 211.6 nmoles 02 /ml. Using these calculations, each 2.0 ml of water in the sample compartment con-

tain approximately 423.2 nmoles of oxygen. The total deflec-

tion of the recorder represents the difference betwen 0 and

100% oxygen concentration; therefore, oxygen concentration

per recorder division gives 4.232 nmoles 02 /min. Therefore, the results of the respiration measurements represent the

rate of change of oxygen concentration as a function of time

d(dx/dt)dt or (02 /min/min) since the individual points are themselves plots of rates (dx/dt or 02/min) (102).

Butanol Metabolism

Cysts were grown on Burk's nitrogen-free medium supplemented with n-butanol as previously described and then washed

and dried on filter membranes (119). At predetermined times,

dehydrated cysts were removed from the filter membranes by 43 shaking vigorously in distilled water and immediately adding one portion to hot ethanol. Another portion of the same sample was placed in the oxygraph, and a third portion was used for determining the number of viable cysts in the preparations examined. The time elapsed between wetting the cysts and adding hot ethanol (approximately 10 min) was thought to be not sufficient to allow physiological changes from dehydrated to the wet condition.

Assay of Protein

The cells and the cysts were harvested by washing three times with 0.9% saline and fixing in 70% ethanol for 9 hr at

4 C. The fixed cells were stained for 1 hr in flourescein isothiocynate (FITC, specific for protein), and centrifuged in the Beckman SW 27 rotor at 10,000 rpm for 5 min at 4 C.

The precipitate was resuspended in normal saline before assay. Protein was measured at a wavelength of 540 nm (124) by flow microfluorometry (FMF) Figure 3. The laser flow microfluorometry (62), which has been applied exclusively to mammalian cells in the past (124), provides a convenient means of rapidly measuring the protein and nucleic acid content of individual cells. In this instrument (Figure 3) cells stained with specific flourescent dyes flow at rate of 500 to 3000 cells per second through a 0.5 watt continuous argon laser beam of wavelength 488 nm. The resulting flourescence which is proportional to cell content of the stained components, is detected by photomultiplier tubes for 0 0.

cm z

0 z..J

LA. C), 0 wO LL

0 z 0 a) ).- N>

4-0 C.,. 0

E 0 o- . 0 ., Ew wj -- 0 oo .j - E'4. 0.

I I I I I I I I I = c3

2 45 storage in a computer or multichannel pulse height analyzer.

Assay of DNA

Cells and cysts were fixed as described above for protein. The fixed cells were washed with saline and incubated in ribonuclease (100 mg RNAase/100 ml in 0.2 M Tris buffer pH 6.8) (Sigma Chemical Co., St. Louis, MO) at 37 C for 1 hr. The cell suspension was centrifuged and resuspended with

1000 units of pepsin (0.5 g pepsin in 94.5 ml of water and

4.5 ml 1N hydrochloric acid). An aliquot of the above mixture was incubated for 5 min at 25 C. The precipitate was suspended in propidium iodine (0.03 mg/ml propidium iodine in 1.12% sodium citrate) and incubated at 25 C for 1 hr. The supernatant was resuspended in 10 ml of saline and assayed at wavelength 340 nm. The architecture and operational principles for the flow microfluorometry were described by Steinkamp et al. (124).

The fluorescent signals emitted from the cells were recorded electronically and displayed on a 256-channel pulse-height analyzer according to relative intensity. The intensity of the fluorescence emitted is proportional to

DNA content (95). The displayed histograms were photographed, and the graphic reproduction of the distribution was reported.

Sample Application and Isoelectric Focusing Ampholine polyacrylamide gel plates for thin layer gel electrofocusing were obtained from LKB Instruments, Rockville, 46

MD with pH range 3.5 to 9.5. The sample holders, 5 x 10 mm

piece of Whatman number 3 paper, were soaked in purified protein extract and placed near the anode. Electrolyte solutions were 1 M sodium hydroxide at the cathode and 1 M phosphoric

acid at the anode. The sample application pieces were removed after 0.5 hr run. Since the proteins may absorb to

the paper and produce tailing; the power was turned off to

remove papers with forceps and the power again turned on.

When the experiment was finished, the power was switched off

and the gel was removed. The pH gradient was determined by

placing a surface pH electrode along one of several lines

between the anode (+) and the cathode (-).

Staining and Destaining of Polyacrylamide Gel Plate

The plates were fixed in the fixing solution (57.5 g

trichlorlacetic acid and 17.25 g sulfosalicyclic acid in

500 ml distilled water) for 0.5 - 1 hr as described in Table

III. This solution precipitated the proteins and allowed

ampholines to diffuse out. The plates were washed in de-

staining solution (500 ml ethanol and 160 ml acetic acid

diluted to 2 liters with distilled water for 5 min), and later stained in 0.46 g Coomassie Brilliant Blue R-250 in

400 ml destaining solution for 10 min at 60 C.

The plates were preserved in glycerol (40 ml glycerol

to 400 ml of the destaining solution) for 1 hr. The gel was

removed, placed on a glass plate, allowed to dry at room

temperature until sticky, and then stored. 47

TABLE III

METHOD FOR PREPARATION OF AZOTOBACTER FOR ISOELECTROFOCUSING ON POLYACRYLAMIDE GELS

Cells and cysts were harvested

Samples were centrifuged and washed 6 times in saline. To the pellets were added 2.5 ml of saline. 11 Samples were sonicated for 30 sec and cooled for 30 sec and the cycle repeated for a total sonication time of 5 min.

To the sonicated samples, were added solubulizing solution containing (50 mM Tris-HC1 + 20 mM mercapthanol + 5 mM MgC1 at pH 7.5 plus 1% glycine at a ratio of 1:1. 4V Centrifuge at 80,000 g for 1 hr.

Collected the supernatants. Protein concentration was determined by Bradford's (15) method.

Samples were concentrated or diluted to the desired concentration (1-5 mg protein/ml).

Samples were assayed immediately or stored for periods no longer than two weeks at 4 C until used. 48

Electron Microscopy

Cells for electron microscopy were cultivated on Burk's nitrogen-free medium supplemented with 2% glucose, and 0.1% ammonium chloride. Cells were also grown in phosphate- deficient Burk's medium and in Tryptic Soy Broth as previously described. Cysts were obtained after 7 days incubation on the same basal medium supplemented with n-butanol instead of glucose.

Cells and cysts of A. vinelandii were harvested by centrifugation, washed 3 times in distilled water, and suspended in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate.

They were fixed for 2-4 hr as described by Vela, Cagle and

Holmgren (132), washed 2 times, and then suspended in the buffer. Cells grown in phosphate-deficient media were placed in 1% (vol/vol) osmium tetroxide in cacodylate buffer at 4 C for 1 hr. This preparation was washed in buffer and then dehydrated by passing through 30, 50, 75, 85, 95 and 100% ethanol solutions before embedding in Epon 812. Thin sections were cut with a Porter-Blum ultramicrotome (Ivan

Sorvall Inc., Norwalk, CT) equipped with a glass knife.

All sections were stained with uranyl acetate for 25 min and then with tartrate for 30 min and examined with an RCA-EMU-3G electron microscope at initial magnification of 11,500 to

16,000.

Studies of the surface of cells and cysts were done with the scanning electron microscope (SEM). For these studies, both cells and cysts were harvested, washed and 49 fixed as described above.

All the samples were kept in 100% ethanol overnight before mounting on stubs. The stubs were then placed in the metal vapor coater, where they were coated with gold to an 0 approximate thickness of 230-250 A. The stubs were then removed from the sporter coater and stored in dust-free containers until they were examined. Scanning electron microscopy of the cells was performed with a ISI mini scanning electron microscope (Tokyo, Japan) at a 20 Kv beam accelerating voltage. Photographic negatives were taken at total magnification of 8,000 to 20,000. CHAPTER III

RESULTS

The results of the experiments conducted in the pre- ceding chapter are reported in the following discussion.

The energy charge of many cultures of Azotobacter vinelandii

12837 was followed throughout the physiological transforma- tion associated with growth, encystment and germination. The data in Fig. 4 represent the concentrations of the adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). From these values one can calculate the energy charge ratio during growth.

The concentrations of adenosine triphosphate, and to a lesser extent the pool of total adenylates (ATP + ADP + AMP), were higher in exponentially growing cells than in stationary phase cells. The energy charge values increased to 0.90 during exponential growth before decreasing to 0.77 in stationary phase cultures in which the concentrations of adenosine monophosphate increase with culture growth. A decrease of 20% in the level of the adenosine diphosphate pool was accompanied by a fall of about 40% in the adenosine triphosphate pool on day three of culture. In contrast, the adenosine triphosphate and total adenylates rose for the first two days of culture growth before a decline. The

50 51 energy charge remained constant at 0.77 during the stationary phase and the total adenine nucleotide concentration decreased to less than 25% of its normal value. These data support the findings of Chapman, Fall and Atkinson (22) in that bacteria in the reproductive stage should have an energy charge between 0.85 and 0.95.

The next series of experiments was designed to investi-

gate the effect of the growth medium on the energy balance of cells of A. vinelandii. The growth media were Burk's medium with 0.1% (w/v) ammonium chloride and Tryptic Soy

Broth. Azotobacter vinelandii was grown in these media, and the results of energy charge measurements are shown in

Tables IV and V. The adenosine triphosphate concentration was low throughout the study with Burk's medium supplemented with ammonium chloride and the concentrations of adenosine triphosphate was 20% that of cells grown in Burk's nitroge-

free medium supplemented with glucose. The energy charge

reached a maximum of 0.76 on the first day of growth and it

is assumed that this value was well below the active physio-

logical level expected of normally growing cells of other

organisms studied. The energy charge values were consistently

lower in cells grown in Burk's medium supplemented with

ammonium chloride than the cells grown in Burk's nitrogen-

free medium supplemented with glucose. The concentration

of adenosine triphosphate in the cells grown in Tryptic Soy Broth (Table V) followed closely that of the total adenine

nucleotides, and constituted approximately 70% of the total 52

adenine nucleotides. The energy charge values of these cells were similar to the ones observed in cells grown in

Burk's nitrogen-free medium supplemented with glucose and also to the values reported by Chapman, Fall and Atkinson (22) for E. coli growing at optimal condition. The level of adenosine diphosphate remained fairly constant in cells grown in Tryptic Soy Broth and the adenosine monophosphate concentration increased by as much as 35% of the total adenylate nucleotide pool on the fourth day of culture growth.

Since respiration has been shown to be a distinctive characteristic of Azotobacter, the ratio of adenosine triphosphate to adenosine diphosphate was related to consumption of oxygen in different growth media (Table VI). Respiration rates of cells grown in Burk's nitrogen-free medium supplemented with glucose and in the Tryptic Soy Broth were essentially the same, in contrast, cells grown in Burk's medium supplemented with ammonium chloride had much lower values.

Minimal values of the energy charge occured at the same time that the ATP/ADP ratio was at its lowest point. There was a two-fold decrease in the respiratory rate in cells grown in

Burk's medium supplemented with ammonium chloride. The low respiration rates in these cells appear to be correlated to low ATP/ADP ratios.

The data in Figure 6 are representative of the results obtained in measurements of the adenine nucleotides and the energy charge for Azotobacter plated on agar media containing 53

0.3% n-butanol. When these results are compared to cells grown in Burk's nitrogen-free medium supplemented with glucose, the measurements showed that the energy charge had dropped from values of 0.90 - 0.87 during logarithmic growth

(Fig. 4) to 0.35 during dormancy (Fig. 5). Cyst viability was maintained at 95 - 100% during the entire course of these measurements. It should be noted that although the energy charge was much lower for cysts than for vegetative cells, the total adenylate nucleotide pool was not. This points to some condition in the cell which limits conversion of adenosine diphosphate to adenosine triphosphate. Since this conversion depends on oxygen-consuming reactions in Azotobacter, it can be assumed that the low energy charge in cysts resulted from decreased oxidative metabolism.

The data in Table VIII indicate the fact that cysts of

Azotobacter respire at much lower rates than do the same cells in the vegetative state. From these data and from other (unpublished) studies, it appears that cysts consume only about one-tenth the amount of oxygen that vegetative cells consume. It seems that this amount of oxygen is required to maintain the energy charge at the level of 0.35.

The encystment medium contains n-butanol, an oxidizable substrate for these bacteria, and it could be reasonably assumed that cysts maintain their adenylate energy charge by metabolizing it. We interpret the data in Table VII as a strong indication of the fact that cysts of Azotobacter retain a high (e.g., 0.30) energy charge and some oxidative 54 metabolic activity even in the dehydrated state.

Cysts that were washed, placed on filter membranes and dried had no exogenous substrate available, and consequently, any metabolic activity evident was probably due to endogenous respiration. It is assumed that dehydrated cysts were found to consume oxygen within 1 or 2 min of the time they were placed in the oxygraph because they retain this capacity during the period of dehydration and not because they undergo some physiological reorganization from an inactive, dormant form to one capable of oxidative metabolism.

Obviously, the maintenance of a high energy charge by active oxidative metabolism would depend on the amount of oxidizable substrate stored in the cysts (105). The data shown in Table VIII were derived from experiments designed to show the correlation between the amount of poly-beta-hydroxybutyrate and oxygen uptake in cells and cysts. These measurements were used as a basis for speculation on cyst survival in nature. The arrow in Table VIII indicates transfer of cells from growth media to encystment media. Day seven on the table corresponds to the first day of encystment. There was an early accumulation of poly-beta-hydroxybutyric acid in the cells when transferred to the encystment media. The amount of poly-beta-hydroxybutyric acid, which increased during the early days of encystment reached a maximum on the second day before declining. It is important to note that a concurrent decrease in the amount of poly-beta-hydroxybutyric acid after day nine leads to a steady state in res- 55 piration. At the end of day six of encystment, the poly- beta-hydroxybutyric acid content was reduced by 30% of the maximum previously attained. The level of respiration rates was higher in the vegetative cells than in the cysts. Res- piration rates decrease as cells transform into mature cysts.

It is interesting to note that respiration rates reached the lowest rate as poly-beta-hydroxybutyric acid attained its maximum values. It was also assumed that these cells may be facultatively anaerobic since oxygen is not required for deposition of poly-beta-hydroxybutyric acid. To test this hypothesis, we streaked Burk's nitrogen-free agar supplemented with glucose, and n-butanol with washed and harvested cells.

The plates were incubated under anaerobic conditions at 30 C.

Cells on plates of Burk's nitrogen-free medium supplemented with glucose produced poor growth and there was no encystment observed on plates with Burk's nitrogen-free medium supplemented with n-butanol under these conditions. Further incubation at room temperature produced better growth in the former and cyst formation in the latter medium. From these results, it is evident that further research is needed to determine if Azotobacter is really aerobic as previously assumed.

The data in Table IX represents a series of studies designed to show the level of phosphate required to support cell formation in A. vinelandii under the conditions of these experiments. This limiting level of phosphate (1/100 the amount in Burk's medium) was chosen from preliminary studies because it yielded approximately 60% of the number 56 of cells found in control cultures. Table IX shows that energy charge of cells grown in phosphate-deficient media was slightly lower than that of the control cells. In addition, the data in the same table show regeneration of adenosine triphosphate and ratio of adenosine triphosphate to adenosine diphosphate in cells grown in phosphate-deficient media was con- sidereably lower than that in phosphate-sufficient cultures.

The growth rate was essentially the same as in the control cultures but encystment in phosphate-deficient media was greatly reduced (Table X). The maximum level of encystment reached was less than 10% of that observed for phosphate- sufficient media during the time studied. The nucleotides concentration of cysts grown in phosphate-limited medium are shown in Fig. 6. The rate of respiration was determined in phosphate-sufficient and phosphate-deficient media, and the results are shown in Table XI and Fig. 7. The phosphate- sufficient cells consume more oxygen than the phosphate- deficient cells. Further studies show that cysts grown in phosphate-deficient media contain approximately the same amount of protein as cysts grown in phosphate-sufficient media (Table XII). On the other hand, phosphate-limited cysts contain greater amounts of poly-beta-hydroxybutyric acid than the control cysts.

The data in Table XIII show the effectiveness of n- butanol related compounds in inducing cyst formation under the conditions of these experiments. Cultures of A. vinelandii were grown to the stationary phase before harvesting and 57 the cells transfered to Burk's nitrogen-free medium supplemented with 0.1% of beta-hydroxybutyric acid (BHB) or ethylacetoacetate (EAA) or n-pentanol (nP). At predetermined times, aliquants were removed for adenylate nucleotide determination, viable counts and scoring for per cent encystment.

The comparative rates of encystment for each substrate employed, given in Table XIII, show that n-pentanol and ethylacetoacetate induce cyst formation than in Burk's nitrogen-free medium supplemented with beta-hydroxybutyric acid.

Correlation between the energy charge and ATP/ADP ratio of cysts recorded from cultures of A. vinelandii grown on n-butanol-related compounds is shown in Table XIV. Minimal values of the adenylate energy charge occur at similar times as the minimal ATP/ADP ratios in these cysts. There was correlation between the results obtained for cysts grown in

Burk's nitrogen-free media supplemented with n-pentanol and n-butanol (Table X). The maximum values of energy charge and

ATP/ADP ratios was fairly constant in the different growth medium. The lowest value of energy charge (0.25) was found in cysts obtained from Burk's nitrogen-free medium supplemented with beta-hyrdroxybutyrate. The values obtained for oxygen uptake by the cysts, were obtained by means of a conventional oxygen electrode (Fig. 8) and found to be similar to the ones reported by Sadoff (104). Respiration reached maximal level on day three in all cultures (Fig. 9) and the ATP/ADP ratio decreased with an increase in encystment. 58

Table XV summarizes the data on the energetics of A. vinelandii cysts during germination which occured in Burk's nitrogen-free medium supplemented with glucose under aerobic conditions. Samples of germinating cysts were taken at hourly intervals. The total adenylate pool, as well as the proportions of the three adenine nucleotides within the

pool, increased to normal values within four hours of placing cysts in the germination medium. The energy charge rose to values typical of growing cells within the same period. The results of relative oxygen consumption and ATP/ADP ratio

in the germinating cysts are shown in Table XVI. Respiratory

activities started immediately upon induction, and there were pronounced changes in the ATP/ADP ratios.

Typical growth curves and nicotinamide adenine dinculeotide

(NAD) levels of A. vinelandii grown in Burk's medium supplemented with glucose or ammonium chloride and in Tryptic Soy Broth

at room temperature are shown in Fig. 9. The nicotinamide adenine dinucleotide content remained approximately the same in each culture on the first day before increasing concomitant with cell growth. The nicotinamide adenine dinucleotide content had increased three-fold by day three in cells grown in the the three media; cell numbers increased over 100-fold in the same time. Cells in the Burk's nitrogen-free medium with glucose showed the highest nicotinamide adenine dinucleotide level. It is interesting that the nicotinamide adenine dinucleotide level increased with culture growth and that the reverse was observed for adenine nucleotides. 59

The techniques for separation of proteins by polyacrylamide gel electrophoresis have been developed to a point where relatively small amounts can be quantitated with good reproducibility. This method has been used to separate proteins in cells and cysts of A. vinelandii grown on different media. Protein patterns can be used to visualize physiological changes which occur during growth and resting stages in different nutritional environments. In preliminary studies, most of the proteins were found to migrate to both acidic and basic regions; a pH range between 3.5 to 9.5 was therefore determined to be adequate for this study. Since the number of protein bands was difficult to count, scanning with the densitometer was used to interpret these results. The reproducibility of the protein patterns from any one isolate was generally good. Figues 10 - 13 show the bands and densitometer scanning of soluble proteins from A. vinelandii grown on Burk's nitrogen-free medium supplemented with glucose, or with limited-phosphate; also in Burk's medium with ammonium chloride and in Tryptic Soy Broth. The frequency of occurrence of the protein bands and the intensity of their staining (dark or light) varied somewhat; intensity of color is related to concentration. There were 33 different protein bands found in the cells studied. A tally of the protein bands revealed a mean value of 30 bands in cells grown in Burk's nitrogen-free medium supplemented with glucose,

24 in Burk's medium supplemented with ammonium chloride and

27 in Tryptic Soy Broth. The main bands varied with different 60 media. A comparison of densitometer scanning patterns of proteins separated by isoelectrofocusing in different growth media (Figs. 11 and 13) reveals a number of common protein bands. The nine common protein bands in the cells grown in the several media focused at the following pH values: 3.9,

4.1, 4.3, 4.6, 5.1, 5.6, 5.9, 6.1, 7.5. Cells grown in Burk's nitrogen-free medium supplemented with glucose lacked proteins focusing at pH 6.2 and 8.7; bands missing in cells grown in Burk's medium supplemented with ammonium chloride were those of pH 4.1, 7.9 and 8.1 and bands not found in cells grown in Tryptic Soy Broth were those of pH 6.5 and

6.8. It is reasonable to expect such qualitative and quantitative differences because other physiological parameters

(such as energy charge, ATP/ADP ratio and respiration) are severely affected by the growth substrates.

Comparison of protein from cells grown in Burk's nitrogen-free medium supplemented with glucose and proteins from encysting cells grown on Burk's nitrogen-free medium supplemented with n-butanol are shown in Figs. 14 and 15. There were 36 different bands observed in such cell and cyst extracts.

Thirteen protein bands were common to cells and cysts, namely, those banding at pH: 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.8,

5.1, 5.2, 5.4, 5.9 and 6.4. The number of bands was different and caused an "overall" protein profile difference. Cysts obtained on the second day of encystment contained the greatest number of soluble proteins (33 bands) while those obtained on the seventh day of encystment had the lowest number (22 61 bands). These results indicate that the types of protein shift daily during encystment and that the number of protein bands decreases as cells enter encystment.

The protein and nucleic acid concentrations of individual bacterial cells can be measured at the rate of several thousand individual cells per second by means of flow microfluorometry (FMF). Methods such as turbidimetry and conventional biochemical analyses provide data on relatively large-

sized samples but the flow microfluorometry provides a con-

venient means of measuring the protein and nucleic acid content of individual cells. Flow microfluorometry was used to study changes during growth as shown in Fig. 16. The

cell populations, which were mostly vegetative cells, had a

narrow protein distribution with a relatively small mean

value of 30. The peaks appeared between channels 30 and 50 on day one of incubation. After three days of incubation, the cells had a much larger mean protein content than did cysts and the peaks moved further right to channels 90 and

120, with a wider area under each peak, indicating an increase

in the relative protein content of the cell. The protein

values increased two-fold on day three and remained fairly constant at this value. These findings are consistant with those previously reported by conventional analyses (79).

The above experiment was repeated for Azotobacter cysts

(Fig. 17). The relative protein content on days one, two and three during cyst formation is similar to that observed in the vegetative cells with the peak appearing at channel 62

80. As the cyst population increased over 50%, the relative amount of protein decreased. On the seventh day, the cyst population showed a peak at channel 30. The relative mean

value of protein was 21, which means that there was almost a three-fold decrease when this is compared with the vegetative

cells. These findings strongly support the work of Lin and

Sadoff (79) in that actively growing cells contain three times

the amount of protein found in cysts. Further studies with flow microfluorometry showed that cysts obtained in Burk's

nitrogen-free media supplemented with n-butanol and n-pentanol

contain greater amount of protein than cysts obtained in

Burk's nitrogen-free media with ethylacetoacetate and beta-

hydroxybutyric acid (Fig. 18).

The data in Fig. 19 show the nucleic acid distribution

in cysts of A. vinelandii grown on Burk's nitrogen-free medium supplemented with n-butanol. The wavelength of the

laser beam was 488 nm. The intensity of the fluorescence

signal was proportional to the nucleic acid content (in this

case DNA) emitted from each of the ethidium bromide-stained

cells. Cyst populations were recorded electronically on the counter. The results of nucleic acid distribution were displaced on pulse-amplitude frequency distributions using a 256-channel pulse-height analyzer. The nucleic acid distribution showed two peaks clustered at channels 40 for deoxyribonucleic acid from the first day of encystment and

100 for vegetative cells. On day three, peak "a" which represents the nucleic acid distribution in the cysts was 63 observed on channel 50 and peak "b", which represents the nucleic acid content of the vegetative cells, was widespread and showed a broad plateau covering channels 60 to 125. As incubation time increased, peak "b" decreased and peak "a" increased. The peak representing the nucleic acid of the vegetative cells accumulated at a higher channel than that of the cysts. It is important to note that peak "b" on the right faded as encystment exceeded 90%.

Phosphate-limited cultures revealed more pleomorphism than the control cultures. There was also more cell debris and many more spheroplasts in phosphate-deficient cultures than in control cultures during the late stationary phase

(42 h). Electron microscopy revelaed many abnormal cell walls (Fig. 20). Scanning electron micrographs of vegetative cells treated with glutaraldehyde are shown in Figs. 21 and

22. The micrograph in Fig. 21 of cells grown in Tryptic

Soy Broth for 60 hr, shows some large and round cells with smooth regular surfaces. Figure 22 represents cells grown in Burk's medium supplemented with ammonium chloride. These cells were surrounded with a polysaccharide deposit and were about one-third the size of those found in Tryptic

Soy Broth. Azotobacter cysts were treated with glutaraldehyde and observed under scanning electron microscopy. The precyst forms are shown in Figs. 23 (36 hr) and 24 (60 hr); some of the precystic forms have rigid surfaces but these are air-dried samples. The inner core of a mature cyst is shown in Fig. 25. The smooth and regular surface, typical of 64

Azotobacter, is evident; the cysts inner core is distinct in

Fig. 26. The significance of the results of the experiments reported in this chapter will be discussed in the following chapter. 65

TABLE IV

DISTRIBUTION OF ADENINE NUCLEOTIDES AND

ENERGY CHARGE IN A. VINELANDII DURING GROWTH IN

BURK'S MEDIUM WITH AMMONIUM CHLORIDE

Total Time Adenine (Days) ATP ADP AMP Nucleotides E.C.a

1 7 *2 3b 5.56 0.37 13.16 0.76 2 5.78 4.92 1.07 11.77 0.70

3 4.47 3.84 1.68 10.01 0.64

4 2.74 3.36 2.42 8.79 0.52 a Adenylate energy charge b All values expressed as n moles ATP/10mg protein. All determinations were carried out four times, starting with a new sample of cells each time. The standard error of the mean was 0.02 to 0.05 energy charge value. 66

TABLE V

DISTRIBUTION OF ADENINE NUCLEOTIDES AND ENERGY CHARGE

IN A. VINELANDII DURING GROWTH IN TRYPTIC SOY BROTH

Total Time Adenine (Days) ATP ADP AMP Nucleotides E.G.

1 11.42 2.74 0.63 14.79 0.86

2 9.35 2.52 1.42 13.29 0.80

3 8.27 2.38 2.56 13.21 0.72

4 7.25 2.12 3.26 12.93 0.65 67

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00 ;:L-1C C C r-t H ~ C) CD

m:t o o S C) C) C) C C < CI:O CO LUJ * 0 0 0o LU:: ?-- r Lr-i q-t (LJ> 0 - 0 I >< e

0 - SZ 1>1 a)C :LU SE r LU 0-- 0 -H o- C E ;- CD D H D E r- 2 C <

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TABLE VIT

OXYGEN UPTAKE AND ADENYLATE ENERGY CHARGE IN DRY CYSTS

OF A. VINELANDII HELD ON FILTER MEMBRANES

n moles 02 consumed Total Age per min per ATP/ Nucleo- (days) 108 cysts ATP ADP AMP ADP tides E.C.

20 0.32 1.1 2.89 4.6 0.38 8.59 0.30

26 0.28 1.0 3.11 4.8 0.32 8.91 0.29 69

TABLE VIII

OXYGEN CONSUMPTION AND CONTENT OF POLY-BETA-

HYDROXYBUTYRIC ACID IN CELLS AND CYSTS OF A. VINELANDII

moles moles 02 con- Age PHB per 108 sumed per min (Days) cells or cyst per 108 cells

1 vegetative cells 0 2.32

2 0 2.20

3 0 1.68

4 3.00 1.64

5 3.50 0.98

6 4.00 0.86

7 mixture 4.32 0.06

8 " 18.05 0.08

9 " 10.41 0.18

10 cysts 8.31 0.16

11 " 8.30 0.17

12 " 6.42 0.16

13 " 6.53 0.16

16 5.23 0.16

Arrow indicates transfer of cells from growth media to encystment media. 70 4- 0 O

C\J r-4l C) C) .r-... C kO O -r- C.. O C) LO C) '-I 'I 0I 4-) :C<

E kO C)

'--:3 C\j LO 00 LC) C) 00 N LU 'I C) E C) kO C ) C . C LU H Co 'I e--l r-i - -4 2: C; . C-) LU< C) 4- C) Co CO) C) C) C) r4 LUJ F-- -C

-j LU

C. r*-J 00 C\J 00 C) Co C) C\J LUJ Co C-o 0 H H 4-) .4 >- CD LO V) oC o_i C) 0 0 .~ H

0L 0-0 CY) H.. 00 c4 C) c*) Co 1- Co LUJ C) CN CD <:Z C) I I>- O LUj LU C 4- 0 .) 04-) C) LU C:L(/H co LO C\J C) Co '-4 cc 2fLUL LCO) D LU C) r- o 1- C) r~- C) U 2: 0 C) : S.- V) 4-) H H LU /) cd LLCO 0 'I C) 040 C) C) LO C)i L. C; Co C) 00J Co C) CO 1-4 O 00 'I C) CD CO LO C) C)j C) : C)<:C 4- C) C) H 4- Co:1 LC) 4- '-4 2: c400 t. Co) LO r-O r--q '-4 U, C) C) 1o cc co03 C 110 C. C\J '- C\cc '- (0 .C o

Co.4 '- 0.. '-4 C) Co) C) LC) LO (\J 00 LO c~C C) . C) '-4 --4 '- -4

tO C\j cc i- C) O Ni c00 *"1 0 - - C\ Co Co ::%- z- 71

TABLE X

EFFECT OF PHOSPHATE-DEFICIENCY ON CYST FORMATION

IN A. VINELANDII

Phosphate-sufficient Phosphate-deficient control culture

Time Total viable Cysts Total viable Cysts (Days) cells and cysts (%) cells and cysts (%)

ia 1.4 x 108 5 1.1 x 108 0

2 2.5 x 108 15 6.2 x 108 0

3 3.8 x 108 45 1.1 x 10 8 3

4 5.7 x 108 68 1.2 x 108 7

5 5.6 x 108 86 1.3 x 108 8

6 3.4 x 108 96 3.2 x 108 9

7 5.2 x 108 98 2.3 x 108 9 a Age of culture on n-butanol media. 72

4- < 0 4-) 0 CI o- c c co00 m 0 ok %: C\J C~j C\J C\J -4 . HO * m 1 0 0 C0 0

4-0) EC

0 C +)E CL C\J-r-

V) W) 0.. C) CL V) 0 0 0 C- 4--0 U * 0 0 0 0-

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4- "

Z:LU - : o~ C\ O hO LO L-O C)C-)

.4- CLN

-J C) :C Q 0 cc U- * 0 0 n 0 0 N CLLU< 0 cyl N j - N -I r- LO UlO Ht Eo '

LUJ -r- roHF

U- 4-- >- CD 4- CC 0L. 4- U- 31O

- uU- 4JC L)01 (AU

C. CL U. -) : 0)C u_ 0 0) r- t.0 00 co ko - (.0 Q N r-O - 0 0 0 0 0 0 4-o -ve Em 8..- r

*1NO E 8-O

mC I0 LO 110 r-_

a)C 73

TABLE XII

EFFECT OF PHOSPHATE-DEFICIENCY ON PHB STORAGE AND

PROTEIN CONTENT IN CYST OF A. VINELANDII

phosphate-sufficient Phosphate-deficient control medium

Protein PHBb Protein PHB Time mg/108 -Pmol e/ mg/10 8 imol e/ (Days) cysts 108 cysts cysts 108 cysts

1 2 .2 3a 4.32 2.34 3.45 2 3.34 18.05 3.51 15.33

3 3.26 10.41 3.44 21.87

4 2.67 8.31 2.31 25.12

5 2.03 8.30 1.85 30.20

6 1.95 6.42 1.69 26.35

7 1.55 5.23 1.60 20.23 a All figures are the average of three separate determinations. b Poly-beta-hydroxybutyric acid. 74

TABLE XIII

COMPARATIVE RATES OF CYST FORMATION IN A. VINELANDII

ON SUBSTRATES CHEMICALLY SIMILAR TO n-BUTANOL

Time Total viable Cysts Substrate/Concentrationa (Days) cells and cysts (%) beta-Hydroxybutyric 1.6 X 107 10 acid (0.1%) 5.1 X 107 30 1.1 X 108 80

3.2 X 108 98

Ethyl acetoacetate 5.8 X 107 12 (0.1%) 1.4 X 108 62

3.2 X 108 83

4.7 X 108 90

n-Pentanol (0.1%) 4.3 X 107 15 1.6 X 108 55

3.5 X 108 82

4.3 X 108 97

a All substrates were filter-sterilized and added to Burk's nitrogen-free medium to a final concentration of 0.1% (w/v). 75

TABLE XIV

COMPARATIVE RATIO OF ENERGY CHARGE AND ATP

TO ADP RATIO OF n-BUTANOL-RELATED COMPOUNDS

IN CYSTS OF A. VINELANDII

Time Substrate (Days) ATP/ADP E.C. beta-Hydroxybutyric acid 1 2.21 0.32

(BHB) 3 0.74 0.29

5 0.52 0.25

7 0.37 0.20

Ethyl acetoacetate 1 1.95 0.35

(EAA) 3 0.92 0.31 5 0.91 0.30

7 0.53 0.30

n-Pentanol 1 2.19 0.43

(nP) 3 1.58 0.39

5 0.96 0.38

7 0.47 0.34 76

00 00 C C LO D co 00 00 00 (Y) 0 9 9 *

C)~

-- o Lr) (10 C\J (1 r- C) S0 r-- co LiJ S v-q C C\J Lr) - .--4 r- re r--

Ce 4- C)

LiJ H Z: Z: ~ 2: v--i C\J '~d- 0)~ C) cM) LO CNJ V) 1 0 e0 e cz C\J .- ,-4 CY)

Li d- C) C\J C) C) CF) (10 I o - Li I 0 LiJ I) C'J C\j CNJ r-) 2: C3 H0 F- NI

L. LO : LC) C\J o

Ii .0 o cc0 cc 0 Li- 2:

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Z:cr CDH (I) C:) R::- LC) LC ( C:) C) C:) C) C) A 4- I >< >< >< X ><

- O C C

EL'

CD:O C:tS:- N mO - qzCNC:

C-)

C 77

TABLE XVI

RELATIVE OXYGEN CONSUMPTION AND ATP/ADP RATIO IN GERMINATING CYSTS OF A. VINELANDII

Age mole of 02 uptake (Hours) per min per 108 cells ATP/ADP E.C.

0 - 0.35

1 0.54 2.2 0.68

2 1.02 3.6 0.88

3 1.36 3.4 0.80

4 1.82 3.5 0.83 78

TABLE XVII

RELATIVE PROTEIN CONTENT OF A. VINELANDII GROWN IN

BURK'S NITROGEN-FREE MEDIUM SUPPLEMENTED WITH GLUCOSE

AS COMPARED TO CELLS IN DIFFERENT GROWTH MEDIA

Growth medium and time of Relative Proteina incubation Channel protein content ratio

Glu 24 40 32 1.00

p- 24 38 30 0.94 NH+ 24 35 29 0.91

TSB 24 37 31 0.97

Glu 72 102 66 2.06

p- 72 105 69 2.16 NH- 72 117 79 2.47

TSB 72 90 60 1.98 a Compared to protein content of cells grown on Burk's nitrogen-free medium supplemented with glucose for 24 hrs;

P~0 def. 4 2 4 -. 094

Glu def- 24

The abbreviations represent Glu = glucose; p- = phosphate deficient; NH4+ = ammonium chloride; and TSB = Tryptic Soy Broth. 79

TABLE XVIII

RELATIVE PROTEIN CONTENT OF A. VINELANDII GROWN IN

BURK'S NITROGEN-FREE MEDIUM SUPPLEMENTED WITH GLUCOSE

AS COMPARED TO CYSTS GROWN IN DIFFERENT SUBSTRATES

Growth medium and time of Relative Proteina incubation Channel protein content ratio in days

nB 1 86 70 1.06

nB 2 90 72 1.20

nB 3 108 72 1.20

nB 5 73 59 0.89

nB 7 25 21 0.32

nB 10 15 13 0.20

nP 5 67 49 0.74

nP 7 26 22 0.33

BHB 5 60 48 0.73

BHB 7 17 15 0.23

EAA 5 50 40 0.61

p- 5 62 50 0.76 p- 7 18 16 0.24 a Compared to protein content of cells grown on Burk's nitrogen-free medium supplemented with glucose for 24 hrs. nB 24 = 1.06 Gl u2 4

The abbreviations represent nB = n-butanol; nP = n-pentanol; BHB = beta-hydroxybutyric acid; EAA = ethylacetoacetate; and p- = phosphate-deficient. The numbers represent days of incubation 80

Figure 4. Adenine nucleotide concentrations as a function of culture age and adenylate energy charge in A. vinelandii. Cells were grown in Burk's nitrogen-free liquid medium to which glucose was added to a final concentration of 1% (w/v). The symbols represent ATP 0; ADP 0 AMP A ; total adenylates{0 and EC 1$. egieq3 AJeu3 co co " c1 --D *i C6 C

7 I:)

*ME% C-

CV)J

(u!oJd Bw O/diV SOIOW iUJ Sep!IoSlOnN SU!UPV 82

Figure 5. Adenine nucleotide concentrations in mature cysts of A. vinelandii as a function of time. Cysts were grown in Burk's nitrogen-free media to which n-butanol was added to a final concentration of 0.3% (w/v). The symbols represent ATP 0; ADP 0 ; AMP A total adenylates } ; and ECI Adenine Nucleotides ( n Moles ATP/10 mg Protein) N)

CA3

OEM

*4 0 LfJI I

CDh N) Energy Charge 84

Figure 6. Effect of phosphate limitation on adenine nucleotides in A vinelandii cysts. The symbols indicate ATPQ; ADP50 ; and AMP A. Adenine Nucleotides (n Moles ATP/10mg. protein)

4016 C" CD

I I

Ec m

#INON 86

Figure 7. Oxygen-uptake of A. vinelandii cysts grown in phosphate- sufficient media (p+); and phosphate- deficient media (p-). yMMoles 02 Uptake/Min./108 Cysts

- - I .1 I I |

u'r

ONE# 88

Figure 8. Effect of n-butanol - related substrates on oxygen consumption by cysts of A. vinelandii. The symbols represent Ethylacetoacetate EAA ; n-Pentanol NP; and beta-hydroxybutyric acid BHB. 8 mMOLE of 02/MIN/10 CYSTS

I I I I I

C4 -

-I

-C

cJ1-

Orih- W m2z

Clo 90

Figure 9. Level of NAD as a function of culture age in A. vinelandii grown in Burk's nitrogen-free media, in Burk's medium supplemented with ammonium chloride and in Tryptic Soy Broth. The total NAD content was expressed as picomoles of NAD per 1 X 108 cells. The open symbols represent cell numbers; closed symbols represent NAD level. El, Burk's nitrogen-free medium supplemented with glucose; 0, Burk's medium supplemented with ammonium chloride; andA, Tryptic Soy Broth. S11O 801/OVN Blow d

I I I I

Co CO CO r Immmm o (iw/sileol BoI 92

Figure 10. Soluble proteins from vegetative cells of A. vinelandii grown on different media. ~~The numbers represent hours of incubation. The designations are G: Burk's nitrogen-free medium with glucose; T: Tryptic Soy Broth; A: cells grown in Burk's medium with ammonium chloride. v 94

Figure 11. Densitometer scanning patterns of the isoelectrofocusing separations of soluble proteins shown in Figure 10. The numbers represent hours of incubation. The designations G: Burk's nitrogen free medium with glucose; T: Tryptic Soy Broth; and A: Burk's medium with ammonium chloride. U, 7 -T' i-- I 1 __-- Co -4_ I I 3 4 co

4 1 - j I

CC! <11-7? - -~ - Cc cn

7+-- 4 _ C+

iit U, -i - -- T

C6 -co

I Ja,

I N

L _!

C-3

2I ui

T-4

CL 96

Figure 12. Isoelectrofocusing patterns of proteins from cells of A. vinelandii grown in Burk's nitrogen- free media supplemented with glucose (60G) and in phosphate-limited Burk's nitrogen-free medium (60P-). The numbers represent hours of incubation. Protein Profile of Azotobacter Cyst

2nB 4nB 5nB 6nB 7nB 98

Figure 13. Densitometer scanning patterns of soluble protein from cells of A. vinelandii grown in in phosphate- limited Burk's nitrogen-free medium with glucose (top) and Burk's nitrogen-free medium supplemented with glucose (bottom). The letters represent hours of incubation. LO cr; cc

2c Ic co

L. C4 cm C aa I- 'C o =L LL- LL. CL $ -

~--+------

cI-

J- 1 1C. 100

Figure 14. Soluble proteins of A. vinelandii cysts grown in Burk's nitrogen- free medium with n-butanol instead of glucose; extracted by deionized water and stained with Commassie Brilliant Blue R-250. The numbers represent the days of incubation and nB for n-butanol. Iw' 102

Figure 15. Densitometer scanning patterns of the isoelectrofocusing separation of the water soluble proteins shown in Figure 14. The numbers represent days of incubation and nB for Burk's nitrogen- free medium with n-butanol. ------C"

cz) -- --

7-A --F-. -~ - j

cO -j

I-

C) -- --

CL,

CD CL CL,

L. CD Lo a,

-- CO I- T LUJ - 4 cO I------

0A 1= - cm 7- 1~ - L 7 cc LO 77 7 7-j:

L U, -K- -F CL 1u -I U, - T -r -4- 71T a,uj 77

4L 104

Figure 16. The effect of growth medium on cell protein as determined by flow microfluorometry. The substrates employed were Burk's nitrogen-free medium supplemented with glucose (Glu), Burk's medium supplemented with ammonium chloride (NH+), phosphate limited Burk's nitrogen- free medium (p-) and Tryptic Soy Broth (TSB). Data are obtained as numbers of cells (ordinate) corresponding to relative protein content (channel numbers; abscisso). The numbers 24 and 72 represent hours of incubation. Relative protein contendt of A. vinelandii

3 T 24 Glu 72 Glu 2

I I I

3 -A 24 1TSB 72 TSB 2

CD, a ~ I *MN=x 3 CA 24 NH4 + 72 NH4+ 2

L- IA II I I I I I I I I I 3 I I 24P- 72 P~ 2

- A- I I j 50 100 150 200 50 100 150 200 Channel Number (Relat Protein Content) 106

Figure 17. Cysts proteins of A. vinelandii determined by flow micro.- fluorometry. The substrate was n-butanol (nB) and the numbers before the substrate represent days of incubation. Relative Protein Content of Azotobacter Cysts on n-Butanol

3 nB 1 nB I, 2

I I I -

II I CV) 3 2 nB 1 nB CD,

CD,

E 2c 3 3 nB 10 nB 2

50 100 150 200 50 100 150 200 Channel Number (Relative Protein Content) 108

Figure 18. The effect of substrates on protein of A. vinelandii cysts as determined by flow microfluorometry. The symbols indicate the substrate used: nB for n-butanol; nP for n-pentanol; BHB for beta- hydroxybutyric acid; and p- for phosphate limitation. The numbers indicate days of incubation. Relative Protein Content of Azotobacter Cysts

3 5P- 7P-

I I I I I I I I

3 5 nB 1 7 nB

CM, 1 a x, 7 S I I |7| CM 3 5 np 7 7 np

SI I I ~~ ~~ I |

I I I I | i I n 3 5 BHB -' gB

I I I I I I I i S i I 4 I I -I, 50 100 150 200 50 100 150 200 Channel Number (Relative Protein Content) 110

Figure 19. Relative DNA content of A. vinelandii cysts grown on Burk's nitrogen-free medium with n-butanol (nB). The peaks "b" represents vegetative cells and "a" for cysts. The numbers represent days of incubation. Relative DNA Content of Azotobacter Cyst

3 1 nB I 4 nB 2 _a

-a 1 b

I I I |I I I I I I I 2nB _ 5 nB x

0 -a L3

="2 - I I I I I | | I I I

S1 3 nB 7 nB -a a

I I I I I I I I l I 2 0 50 100 150 200 50 100 150 200 Channel Number (Relative DNA Content) 112

Figure 20. Effect of phosphate deficiency of the morphology of A. vinelandii. (A) Normal cells. (W~ Cells grown in phosphate-deficient media. Arrows indicate differences in cell wall appearance. X20,000. 642 TSAI, ALADEGBAMI, AND VELA J. BACTERIOT_ IIAMMALo, 47wl- 142M,awl

-moor

N Aff"41

V -'1 414 WV

WE,

FIG. 3. Effect of phosphate deficiency on the rnotpholog of A. -inel-ndA) Noral in phosphate-deficientmedia. Arrows cells. (B) Cells grown indicate differences in cell wall appearance. X20,00.( These three observations are in agreement with nucleotide phosphate the scheme suggested in Fig. goes to ATP. This is prob- 4. ably not so. It i' difficult to accPrit flc, i 114

Figure 21. Scanning electron micrograph of cells of A. vinelandii grown in Tryptic Soy Broth and fixed in glutaraldehyde (10,000 X).

Figure 22. Scanning electron micrograph of cells of A. vinelandii grown in Burk's medium supplemented with ammonium chloride and fixed with glutaraldehyde (10,000 X). 9V 116

Figure 23. Early precystic cells visualized by scanning electron microscopy. These were grown on n-butanol and incubated for 2 days. Some have rigid cell surface (10,000 X).

Figure 24. Precystic forms visualized on day three by scanning electron microscopy. Some smooth cysts with even surface are evident (10,000 X). slow-, 118

Figure 25. Inner core of matured cyst grown on n-butanol and observed by scanning electron microscopy after glutaraldehyde treatment. The inner layers are shown. The cyst surface appears even (20,000 X).

Figure 26. Inner core of matured cysts grown on n-butanol and observed by scanning electron microscopy after glutaraldehyde treatment. The layers are clearly distinct (20,000 X). *- AL 120

Figure 27. Alternate pathway for substrate energy utilization in A. vinelandii. Roman numerals indicate priority and X's indicate the inhibiting or "switching" condition. AC-S-CoA, acetyl coenzyme A. Glucose

Phosphoenol-pyruvate

Pyruvate

l - Ac-S-CoA

High PH Low Adenylate , -Ketoglutarate/Malate Adenylate Charge Charge

ADP-ATP BIOSYNTHESIS

Phosphate Deficiency 122

Figure 28. Distribtuion of energy charge during morphogenetic stages in cells of A. vinelandii. C4, LUI C0 RC's >Luj CM*

Co C02c CD, -ig I L" C-, i- CD 0 -m 0 LUJ C3 co- U ix= I-M

to ,.4 4 C.)

CDZ z LL.

2co z

C),

oil E

CD CHAPTER IV

DISCUSSION

The results reported in Chapter III show that the adenylate energy charge varies in the same organism with changes in the physiological condition. This report clearly shows that the nature of the growth medium affect the energy charge, the ratio of adenosine triphosphate to adenosine diphosphate, respiration and the quality of proteins in Azotobacter vinelandii 12837. These variables appear to be interdependent since a change in one greatly affects another. For example, the energy charge affects growth and the ratio of adenosine triphosphate to adenosine diphosphate and the latter varies with oxygen uptake in these bacteria. Our observations with

A. vinelandii (Fig. 3) agree with those of Chapman, Fall and Atkinson (22) who used Esherichia coli in that the energy charge of the adenylate pool in the two bacteria is between

0.9 and 0.8 in actively growing cultures. Measurements on cultures in Burk's medium supplemented with ammonium chloride and in Tryptic Soy Broth (Tables IV and V) show that the ratio of adenosine triphosphate to adenosine diphosphate tends to rise and fall with the energy charge. The reasons for this may rest in part on the increase in adenosine triphosphate concentration which is known to affect the value of the adenylate energy charge (25, 26). Cells

124 125 grown in Burk's medium supplemented with ammonium chloride had a lower energy charge (0.76) and 50% less ATP than cells from Burk's nitrogen-free medium supplemented with glucose.

The decrease could be due to a rapid release of nucleotides into the medium from the cells grown in Burk's medium supplemented with ammonium chloride, presumably as a consequence of continuous synthesis of ribonucleic acid (RNA). The other possible reason may be that the rate of utilization of adenine nucleotides is greater than the rate of biosynthesis.

The fluctuations in respiration rates are similar to those of the energy charge during these conditions of growth.

Since Azotobacter reportedly rely on the tricarboxylic acid cycle (TCA) and oxidative-level phosphorylation for the generation of energy, the oxygen patterns presumably reflect the adenosine triphosphate to adenosine diphosphate ratio and the energy charge. These fluctuations may be due to a special regulatory mechanism which enables the Azotobacter to produce energy by degrading their cellular components.

One of the most interesting results was obtained upon measurement of the adenylate energy charge during cyst formation. Cysts have been referred to as dormant structures

(105), but our results confirm the observation that the

Azotobacter cyst is capable of biological activity. The data reported here suggest that cysts of Azotobacter represent a form intermediate between the vegetative cell and the spore in terms of energy charge. The energy charge values measured during encystment were the lowest measured for 126 viable cells of Azotobacter. Although the total adenylate nucleotide pool in cysts is approximately the same as that in vegetative cells, the amount of adenosine triphosphate is

50% lower in cysts than in cells. The reverse is true for adenosine monophosphate. This is not surprising in view of the low level of oxygen consumption. It appears, then, that cysts can maintain life at adenylate energy charge values of

0.30 and at ATP/ADP ratios as low as 0.40. Even at these very low levels of available adenosine triphosphate, it is difficult to imagine maintenance of life by continuing oxidative phosphorylation, especially in the case of cysts which remain alive in dry soils for prolonged periods of time (131). In order to survive, they must either become dormant like spores or, indeed, maintain a low level of oxidative phosphorylation (i.e., energy charge = 0.30 and ATP/ADP = 0.38). If the former is true, it means that there is in the soil a phase of the Azotobacter cysts which has not yet been discovered. The stoichiometry of oxidation of hydroxybutyrate via the cytochrome system is commonly written as follows:

C4 H8 02 2CoA ------> 2CH 3 CH 20-CoA + 606 ------

4C02 + 6H 20 + 2CoA

From this, it is possible to say that if 1 x 108 cysts consume 2.8 x 10-10 moles of oxygen per min (Table VII), they will oxidize 4.7 x 10-11 moles of C 4 H8 02 per min. Since 127

cysts survive for 15 years (131) in soil, it can be reason-

ably assumed that 1 x 108 of these would require 3.7 x 10-4 moles of C4 H8 02 . Laboratory measurements reveal that 1 x 108 cysts contain 5.23 x 10-6 moles of hydroxybutyrate (Table

VIII), or only enough to last for 77 days.

It is obvious that the stoichiometric calculations cited

are invalid. Because of this, it seems certain that cysts in

dry soil maintain viability by some means other than the

oxidation of poly-beta-hydroxybutyrate as envisioned by the

reactions commonly known. One must assume that life is main-

tained in cysts in dry soil by reactions much more efficient

than the ones presented here or, alternately, that the commonly known cysts are simply a laboratory artifact and that soil

Azotobacter have some, as yet undescribed, life phase.

It is our opinion that cysts in laboratory cultures

maintain their adenylate energy charge by metabolizing n-

butanol or n-butanol related compounds. The data in Table

VII show that cysts, washed and dried on filter membranes

and without exogenous substrate were able to maintain an

energy charge of 0.30; consequently, the metabolic activity

must have resulted from endogenous respiration. It is there-

fore reasonable to assume that cell death does not appear to

result directly from reduced energy charge, as proposed by

Chapman, Fall and Atkinson (22); certainly, maintenance of life in laboratory cysts of Azotobacter may depend on

oxidative metabolism.

Table IX shows that the energy charge of cells grown in 128

phosphate-deficient media was slightly lower than that of the control cells. This alone would be sufficient reason for doubting that cells in phosphate-limited cultures die because the ratio of adenosine triphosphate to adenosine diphosphate becomes too high and inhibits respiration. Although this may be a mechanism well recognized in eucaryotic cells (37, 64), our data show that it is not one operative in A. vinelandii.

We performed measurements of the adenylate nucleotides

and showed that the reverse of the explanation offered by

Postgate and his co-workers (25, 26, 72) is actually true. Table IX shows that the energy charge of cells grown in

phosphate-deficient media was lower than that of the control cells. This shows that respiration is not inhibited by the ratio of ATP/ADP.

It can be deduced from Table XII that phosphate-limitation encourages the production of poly-beta-hydroxybutyric

acid. It is well established that glucose metabolism normally

proceeds by amphibolic pathways in which the carbohydrate

substrate is either shunted to biosynthesis or is oxidized via the cytochrome system to regenerate adenosine triphosphate

(76, 77). Organisms capable of storing excess energy-yielding

substrates (27, 28) possess an additional choice in metabolic routing. When A. vinelandii is grown in phosphate-deficient media, excess unoxidized substrate is routed to poly-beta- hydroxybutyric acid (109) via a pathway which does not involve 129 involve phosphate directly. Since phosphate is essential for conversion of substrate energy to the adenosine triphosphate form, it seems logical that the pathway leading to adenosine triphosphate regeneration would be closed or res- tricted due to a lack of inorganic phosphate. In this organism, the bifurcation in pathways is at the level of phosphoenol-

pyruvate (76, 77); if both the substrate pathway leading to biosynthetic reactions and that leading to adenosine triphos-

phate regeneration are inhibited by lack of inorganic phosphate

(100, 110), then the entire substrate load would be channeled

to poly-beta-hydroxybutyric acid synthesis (Fig. 27). Since

cysts grown in phosphate-deficient media could have stopped growing for many reasons other than lack of phosphate, and

since conversion from vegetative cell to the cyst form probably requires energy in the form of adenosine triphosphate (i.e.,

a high adenylate energy charge), biosynthesis could be as-

sessed by inducing encystment. Table X shows that encystment

in a phosphate-deficient medium was greatly inhibited. In addi-

tion, regeneration of adenosine triphosphate in cysts grown

in phosphate-deficient media was considerably lower than

that in cysts grown in the phosphate-sufficient control

medium. Also, phosphate-deficient cysts contain much larger

amounts of stored poly-beta-hydroxybutyric acid than control

cysts (Table XII). The three observations are in agreement

with the scheme suggested in Fig. 27.

Previous investigators (25, 26, 72, 146) have claimed

that oxygen uptake in phosphate-limited cultures is inhibited 130 because all available nucleotide phosphate goes to adenosine triphosphate. This is probably not so. It is difficult to accept the idea proposed by Dalton and Postgate (25, 26) because this is a reaction which requires the presence of readily available inorganic phosphate in the medium. Under conditions of phosphate limitation and sufficient oxygen (Table XI), adenosine monophosphate is converted to adenosine diphosphate; this is a well-known reaction mechanism which requires no exogenous phosphate. On the other hand, the conversion of adenosine diphosphate to adenosine triphosphate requires a readily available source of phosphate. The data in Table XI can be easily interpreted in terms of these reactions since the ATP/ADP ratios in cultures derived from cysts were much higher in a phosphate-sufficient than in phosphate-deficient medium. The same relationship has been observed in Streptomyces griseus (86). We suppose that oxygen consumption by phosphate-deficient cysts (Fig. 7) is inhibited because the lack of inorganic phosphate prevents conversion of adenosine diphosphate to adenosine triphosphate, and, as a consequence, the coupled cytochrome reactions also become inhibited. It is our opinion that bacteria in the genera Bacillus and Pseudomonas also employ this pathway.

We therefore propose that this may be a universal pathway available to organisms capable of storing substrate in the form of poly-beta-hydroxybutyric acid when these are in phosphate-limited conditions.

Senior and Daves (110) have reported a scheme for 131 poly-beta-hydroxybutyric acid synthesis in A. beijerincki.

Each of the substrates namely, beta-hydroxybutyric acid

(BHB), ethyl acetoacetate (EAA) and n-pentanol (np), utilized in our study has been shown to enter the above reaction scheme directly. It is evident that the substrates which promote poly-beta-hydroxybutyric acid formation can also serve as efficient inducers of cyst formation. Another explanation for cyst induction by these substrates could be that the breakdown of reserve material may preserve viability by providing a so-called "energy of maintenance" (84,85).

The data in Table XIV suggest that cysts grown in n- butanol related substrates have an energy charge between 0.25 -

0.35, a marked contrast to the endospores of B. subtilis in which the energy charge is approximately 0.1 (56). These energy charges are independent of the amount of encystment but it is influenced by changes in the rate of respiration.

The ratio ATP/ADP increased slightly when the cells were transferred to these encystment substrates. This was seen when the rate of synthesis of adenosine triphosphate became greater than the rate of utilization. Although it has been reported that respiration in A. chroococcum is controlled by adenine nucleotides, no actual measurements of adenine nucleotides were given to support the conclusions presented in that study (146). In our work, the ratio of adenosine triphosphate to adenosine diphosphate was found to be much greater in the control cells than in cells grown in phosphate- deficient media. In the earlier reports (25, 26) it was 132 postulated that oxygen uptake was inhibited because high ratios of adenosine triphosphate to adenosine diphosphate were attained. Quite the contrary, control cysts have much higher ratios of adenosine triphosphate to adenosine diphosphate than do those in phosphate-deficient media (Table XI).

The data presented here suggest that the high ratios of adenosine triphosphate to adenosine diphosphate play no directly discernible role in the rate of oxygen uptake in cysts of Azotobacter vinelandii. Clearly then, it is not the ratio of adenosine triphosphate to adenosine diphosphate which inhibits respiration in the cysts of A. vinelandii, but rather the lack of inorganic phosphate which prevents conversion of adenosine diphosphate to adenosine triphosphate.

During germination, the energy charge rose rapidly because of the availability of an energy source (Table XV).

With energy charge in its normal range, respiration rose, reaching nearly the rate observed in actively growing cells.

No doubt the rate of ribonucleotide synthesis increases with the initiation of germination, until the cysts become vegetative cells, fully self-sufficient in energy metabolism and biosynthetic operations.

Variability in morphology of A. vinelandii as a function of growth medium has been shown in many reports over the years, and the data presented in this study further support this idea. When A. vinelandii was grown in Burk's nitrogen- free medium supplemented with glucose or ammonium chloride or in Tryptic Soy Broth, definite differences in the protein 133

patterns resulted (Figs. 10-13). This is a clear indication that the carbon source greatly influences the biochemical and physiological make up of A. vinelandii. Although the

protein bands recorded from cells grown in Burk's nitrogen-

free medium with glucose were more dense than those from

cells grown on Burk's medium with ammonioum chloride. The possible reason for the differences between protein bands of

cells grown in Tryptic Soy Broth and those grown in Burk's nitrogen-free medium supplemented with glucose may lie in

the fact that Tryptic Soy Broth produces a fungoid morphology

in this bacteria. As with other criteria that have been

used in the description of the Azotobacter cyst, the protein

pattern of their extracts cannot be described in absolutes.

Many of the protein patterns are distinguishable with varying

degrees of resolution for there is a gradation from those

protein bands whose identity is always unmistakable to others

that are too faint to make their identification certain.

Definite changes were observed in the protein patterns

as cyst development progressed. Figure 15 shows a steady

decrease in the protein bands as the cell goes into cyst formation. These shifts in the protein bands are indicative

of biochemical and physiological changes which the cell

undergoes during the stages of cyst development. The flow

microfluorometry (FMF) technique has recently been used in

the measurement of protein and nucleic acid in microbral

systems. We have compared the protein and deoxyribonucleic

acid (DNA) contents of A. vinelandii during different stages 134 of the life cycle. The data presented in Fig. 19 represent a sampling of flow microfluorometry data of Azotobacter growing on encystment media. The data in Figure 19 show the nucleic acid distributions in populations of vegetative cells and cysts. The relative nucleic acid distribution in the vegetative cells showed a single peak "b" (Fig. 19).

Estimated by the channel ratio, the vegetative cells contained approximately four times as much nucleic acid as did the cysts depicted "a" on the first day of encystment (represented as

1NB). After the fixed cells were stained in fluorescein isothiocynate (specific for protein) and propidium iodide

(specific for deoxyribonucleic acid), a sample of each was observed under the light microscope for cell counts and morphology change. Through microscopic examination of these samples, the vegetative cells accounted for over 90% of the total population at this period. The results presented in this study show the nucleic acid distribution of cyst populations to be between channels 30 - 60, and the relative protein content decreased as cyst formation increased (Table XVIII).

The results obtained in this study indicate that the relative amount of protein remains fairly constant at different stages

(vegetative, pre-cyst and cyst) in Azotobacter regardless of the growth substrate. Furthermore, our results support the findings of Lin and Sadoff (79) in that the protein contents of the cell decreased as cyst development progressed and that the vegetative cells contained three times the amount of protein reported for cysts. 135

In summary, the effect of growth substrates on the physiological activities of Azotobacter was determined.

Three criteria, energy charge, respiration and protein concentration, were used to determine the response of the organism to each of the substrates. Maximal ATP/ADP ratio and energy charge values occured concurrently with maximal rates of respiration indicating the rate of respiration to be controlled by intracellular ATP/ADP ratio. From the results presented, it is apparent that cells grown in nitrogen-free media and those grown in media containing organic nitrogen show different responses to the three parameters (energy charge, ATP/ADP and respiration). Furthermore, the rate of protein synthesis and the capacity for growth are much more sensitive to changes in the value of the energy charge than changes in concentration of adenosine triphosphate. While the viable endospores of many bacteria have been shown to possess an adenylate energy charge very close to zero, it is assumed that this is commensurate with physiological dormancy. Since cysts are capable of physiological activity, it is reasonable to assume that they have an energy charge between that of inactive but viable endospores and vegetative cells. The energy charge of the cyst was measured and found to range between 0.35 - 0.3; this value does not lead to cell death as previously reported by Chapman, Fall and Atkinson (22).

The data presented reveal that the major effect of phosphate-limitation on the growth of A. vinelandii is seen as an inhibition of oxidative phosphorylation. This is ac- 136 companied by channeling of substrate energy to poly-beta- hydroxybutyric acid storage, decreased adenylate energy charge, low ratio of ATP to ADP, decreased cell viability, abnormal cell wall structure and inability to form cysts.

It is our opinion that cyst has the capacity for rapid responsiveness to specific germinating agent and self-suf- ficiency in energy metabolism and biosynthetic operations during the early stages of germination. During the early hours of some physiological events in laboratory cysts which germination, the exogenous energy and nucleotide reserves were utilized to maintain high energy charge, and other physiological activities. The specific nature of cyst survival in the soil is not fully understood at this time, but some physiological events in laboratory cysts which may bear on this have been observed and described in this report. BIBLIOGRAPHY

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