EFFECTS OF SELECTED PHYTOHORMONES ON THE GROWTH

AND MORPHOLOGY OF ESCHERICHIA COLI

APPROVED:

/ A\ /UJ-IUAA • ^ CUA*J^ Major Professor

L L't ' /\ Mindir Professor

\ ' / ;. i-

rVr"~ V ) I Director of the Department of Biology

Dean of the Graduate School

EFFECTS OF SELECTED PHYTOHORMONES ON THE GROWTH

AND MORPHOLOGY OF ESCHERICHIA COLI

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Lynn Mallory Little, B. S.

Denton, Texas

January, 1968

TABLE OF CONTENTS

Page

LIST OF TABLES .iv

LIST OF ILLUSTRATIONS v

Chapter

I. INTRODUCTION 1

II. MATERIALS AND METHODS 8

III. RESULTS 14

IV. DISCUSSION . . . . . . .22

BIBLIOGRAPHY • . »32

ill

LIST OF TABLES

Table Page

I. Concentrations of Phytohormones added to trypti-case soy broth cultures of Escherichia coli . 11

IV

LIST OF ILLUSTRATIONS

Figure Page

1. Growth curves for cultures treated with IAA. . . . 15 *

2. Growth curves for cultures treated with GA3. . . . 16

3. Growth curves showing that a lower concentration of IAA has negligible effect on growth, but that higher concentrations of GA3 and es-pecially IAA are inhibitory 17

4. Growth curves for cultures containing a stimula-tory concentration of GA3 and ineffective concentrations of kinetin, or a stimulatory concentration of GA3 and an inhibitory con-centration of IAA .18

5. Growth curves for inhibitory concentrations of GA3 and IAA tested separately and together. . 19

6. Representative photographs of untreated cells and cells treated with 1.0 mg/ml IAA, 5.0 mg/ml IAA, or 1.0 mg/ml IAA + 1.0 mg/ml GA3 . . . . . . . . . . . . . . . . . . 2 0

CHAPTER I

INTRODUCTION

Growth in plants is regulated by groups of substances

known as phytohormones. On the basis of their structural

similarities and modes of action, three major groups of

phytohormones have been identified and characterized,

auxins, gibberellins, and kinins.

Kogl, _et a l. (13) first described indoleacetic acid

as an auxin, and this chemical was purified from plant

materials by Kogl and Kostermans (14) in 1934 and by

Thimann (24) a year later. It is now known that in the

individual plant cell, auxins affect the plasticity and

elasticity of the cell wall, cytoplasmic viscosity,

protoplasmic streaming, respiration rates, metabolic

pathways, changes in oxidative states, content of nucleic

acids, and the activities of many enzymes (17).

In 1926, Kurosawa (16) in Japan was studying a disease

of rice caused by the fungus Gibberella fujikoroi which

produced a characteristic excessive growth of the rice

plant.. The separation of the responsible growth stimulant

closely preceded the first separation of auxin from plants

in 1928, and in 1935 Yabuta (25) crystalized the compound

and named it gibberellin. In general, it appears that the

gibberelliris are involved in the regulatory systems which

bring about the developmental response of the plant to envi-

ronmental cues (17).

The generic name "kinin" was proposed for chemical

substances which stimulate cytokinesis. The purines and

other plant kinins began to receive attention in physio-

logical literature in the 1940s, and in 1955 Miller and

his team (21) separated an active stimulant of cell division

from yeast DNA. This material, which they named kinetin,

was identified as 6-furfuryl aminopurine or 6-furfuryl-

adenine (19, 20). In addition to its effects on cell

division and bud differentiation, kinetin has been found

to have a variety of other growth effects, although the

overall plant responses to kinins have been much less

dramatic than those to auxins or gibberellins (17).

The effect of the presence of an auxin on the growth

of bacterial populations was first studied by Ball (1), who

reported that indoleacetic acid in concentrations of 0.0001

mg/ml to 0.001 mg/ml increased by more than two-fold the

final stationary phase titer of Escherichia coli suspensions.

Later, Handler and Kamin (7).could not detect a stimulation

of Escherichia coli growth using the same concentrations

of the auxin. According to Beckwith and Geary (2), indole-

acetic acid at concentrations of 0.0003 mg/ntl to 0.2 mg/ml

will stimulate the growth of Escherichia coli cells taken

from the logarithmic death phase, while a concentration of

10.0 mg/ml is completely inhibitory. Fletcher (5) reported

that low concentrations of auxins can stimulate the growth

of certain bacteria, but, as he pointed out, other workers

have found that auxins have little or no effect on micro-

organisms or that at higher concentrations they are definitely

toxic. An interesting finding by Dubos (4) is that peptone

and especially tryptophan can partially or completely

reverse the bacteriostatic effect of auxins.

Saono (23) tested the effect of gibberellic acid on the

growth of thirty-eight species of bacteria and two species

each of the algae Chlorella and Scenedesmus. Except for

two strains of Azotobacter chroococcum and Actinomycetes

coelicolor, the bacteria did not react to the acid at con-

centrations of 0.0005 mg/ml to 0.05 mg/ml. However, lower

concentrations of the acid stimulated multiplication of all

four algal species.

Kennell (12) reported that Escherichia coli cultures

grown' in a salts-glucose medium showed accelerated growth

when kinetin was present at a concentration of 0.001 mg/ml.

Maruzzella and Garner (18) found that concentrations of

kinetin from 0.002152 mg/ml to 0.00000002152 mg/ml exerted

a marked stimulatory effect on the growth of Bacillus mega-

terium and Agrobacterium tumefaciens and to a lesser degree

on Escherichia coli, Staphylococcus aureus, and Erwinia

carotovara. The growth of Corynebacterium michiganense was

partially inhibited by kinetin at most concentrations used.

Interestingly, no change in cell morphology or Gram stain

reaction was observed in any of the tests.

While numerous investigators have studied the effects

of auxins, gibberellins, and kinins on the growth of various

microorganisms, there have been no reported attempts to

determine whether treatment with more than one phytohormone

could cause a synergistic response in either enhancing or

inhibiting the growth of a bacterial population. Such

synergism with these groups of phytohormones has been often

reported on the growth of higher plants (3, 6, 8, 9, 10, 11,

15, 22, 26). The present investigation was undertaken as a

preliminary study to learn the response of Escherichia coli

cells grown under identical experimental conditions to

various concentrations of indoleacetic acid, gibberellic acid,

and kinetin alone, and in combination with one another.

CHAPTER BIBLIOGRAPHY

1. Ball, Earnest, "Heteroauxin and the Growth of Escherichia coli," Journal of Bacteriology, XXXVI (1938), 559-565.

2. Beckwith, T. D. and E. M. Geary, "Effect of Indole 3 Acetic Acid on Multiplication of Esch. coli and J3. typhosa," Journal of Infectious Diseases, LXVI (1940), 78-79.

3. Brian, P. W., "Role of Gibberellin-like Hormones in Regulation of Plant Growth and Flowering," Nature CLXXXI (1958), 1122-1123.

4. Dubos, Rene J., "Inhibition of Bacterial Growth by Auxins," Proceedings of the Society for Experimental Biology, New York, LXIII (1946), 317-319.

5. Fletcher, Wm. W., "The Effect of Herbicides on Soil Micro-organisms," The Physiology and Biochemistry of Herbicides, edited by L. J. Audus, London, Academic Press, 1964, pp. 20-62.

6. Galston, A. W. and H. Warburg, "An Analysis of Auxin-Gibberellin Interaction in Pea Stem Tissue," Plant Physiology, XXXIV (1959), 16-22.

7. Handler, P. and H. Kamin, "Indole Acetic Acid and Growth of Bacteria with Varying Requirements for Nicotinic Acid and Tryptophan," Proceedings of the Society for Experimental Biology, New York, LXVI (1948), 251-254.

8. Hillman, W. S. and W. K. Purves, "Does Gibberellin Act Through an Auxin-mediated Mechanism?," Plant Growth Regulation, edited by R. M. Klein, Ames, Iowa, Iowa State University Press, 1961, pp. 589-600.

9. Kato, J., "Physiological Action of Gibberellin with Special Reference to Auxin," Plant Growth Regulation, edited by R. M. Klein, Ames, Iowa, Iowa State Univer-sity Press, 1961, pp. 601-609.

10. , "Studies on the Physiological Effect of Gibberellin II. On the Interaction of Gibberellin with Auxins and Growth Inhibitors," Physiologia Plantarum, XI (1958), 10-15.

11. Kefford, N. P., "Auxin-Gibberellin Interactions in Rice Coleoptile Elongation," Plant Physiology, XXXVII (1962), 380-386.

12. Kennell, D. E., "The Effects of Indoleacetic Acid and Kinetin on the Growth of Some Microorganisms," Experimenta1 Cell Research, XXI (1960), 19-33.

13. Kogl, F., A. J. Haagen-Smit and H. Erxleben, "Uber ein Neues Auxin ('Heteroauxin') aus Harn. XI.," Zeitschrift Physiologisch Chemie, CCXXVIII (1934), 90-103.

14. Kogl, F. and D. G. F. R. Kostermans, "Hetero-auxin als Stoffwechselprodukt Niederer Pflanzlicher Organismen. XIII.," Zeitschrift Physiologisch Chemie, CCXXVIII (1934), 113-121.

15. Kuraishi, S. and R. M. Muir, "Increase in Diffusible Auxin After Treatment with Gibberellin," Science, CXXXVII (1962), 760-761.

16. Kurosawa, E., "Experimental Studies on the Secretion of Fusarium heterosporum on Rice Plants," Trans-actions of the Natural History Society of Formosa, XVI (1926), 213-227.

17. Leopold, A. Carl, Plant Growth and Development, New York, McGraw-Hill Book Company, 1964.

18. Maruzella, Jasper C. and James G. Garner, "Effect of Kinetin on Bacteria," Nature, CC (1963), 385.

19. Miller, C. 0., F. Skoog, F. S. Okumura, M. H. von Saltza and F. M. Strong, "Isolation, Structure, and Synthesis of Kinetin, a Substance Promoting Cell Division," Journal of the American Chemical Society, LXVIII (1956), 1375-1380.

» 5 IJ L : '' "

20. "Structure and Synthesis of Kinetin,"

Journal of the American Chemical Society/ LXXVII (1955), 2662-2663.

21. Miller, C. 0., P. Skoog, M. H. von Saltza and F. M. Strong, "Kinetin: a Cell Division Factor from Deoxyribonucleic Acid, "Journal of the American Chemical Society, LXVIII (1955), 1392.

22. Phillips, I. D. J., A. J. Vlitos and H. Cutler, "The Influence of Gibberellic Acid Upon the Endogenous Growth Substances of the Alaska Pea," Contributions to the Boyce Thompson Institute, XX (1959), 111^-120.

23. Saono, Susono, "Effect of Gibberellic Acid on the Growth and Multiplication of Some Soil Microorganisms and Unicellular Green Algae," Nature, CCIV (1964), 1328-1329.

24. Thimann, K. V., "On the Plant Growth Hormone Produced by Rhizopus suinus," Journal of Biological Chemistry, CIX (1935), 279-291.

25. Yabuta, T., "Biochemistry of the 'Bakanae' Fungus of Rice," Agriculture and Horticulture (Tokyo), X (1935), 17-22.

26. Yoda, S. and J. Ashida, "Effects of Gibberellin and Auxin on the Extensibility of the Pea Stem," Plant Cell Physiology (Tokyo), I (1960), 99-105.

CHAPTER II

MATERIALS AND METHODS

Escherichia coli ATCC 11303 cells were grown in the

dark at 37° ^1° C in 10-rtil volumes of trypticase soy broth

(TSB, Baltimore Biological Laboratory) contained in 13 x 150

mm colorimetrically-matched screw-cap Pyrex test tubes. The

tubes were inoculated by transferring 1„0 ml from a 12-hr

culture through three 9-ml sterile distilled water dilution

tubes and pipeting 0.1 ml from the third dilution tube into

each culture tube. A modification of the method of Scott

and Chu (1) of obtaining synchronized cultures was employed.

This consisted of refrigerating the dilution tubes and hold-

ing the cells at 5° ^1° C for one hour before inoculating

into the 37° culture tubes. Synchrony of division, however,

was not achieved, as the generation time was found to be

approximately forty minutes. Some cultures were treated by

including in the growth medium various concentrations of

indole-3-acetic acid (IAA), gibberellic acid (GA^), and/or

kinetin, all of which were obtained from Nutritional Bio-

chemical Corporation, Cleveland, Ohio.

8

The number of cells present in a given culture was

determined in two ways. First, replicate plate counts

were made on cultures which did not contain hormone after

0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48, and 96 hours of

incubation. At the same time intervals, the tubes were

read colorimetrically on a Bausch and Lomb Spectronic 20

Colorimeter at a wavelength of 650 mp.. A regression line

was plotted from the data. Second, on other cultures not

treated with hormone, replicate direct counts were made at

these same time intervals using a Petroff-Hausser Counting

Chamber. Colorimetric readings were made as before, and a

regression line was plotted from the data. Other untreated

cultures were both plated out and read directly to jfcompare

the two methods of counting. It was found that the number

of cells as determined by direct counts equalled approxi-

2

mately 1.6 x 10 times the number of cells as determined by

plate counts. In all subsequent experiments the number of

bacteria present was determined colorimetrically on the

basis pf the turbidity-versus-direct counts regression line, r 'I A

which had a standard error of -1.3953 x 10" cells/ml at a

confidence level of P = 0.05.

In experiments employing IAA and/or GA^, the desired

quantity of phytohormone was added directly to the culture

10

broth. These tubes were then heated and shaken to facilitate

dissolution of the hormones before the culture tubes were

autoclaved at 121° C for fifteen minutes. Kinetin, insoluble

in water, was dissolved in 10% NaOH before being added to the

broth. Ten per cent HCl was then added to the tubes to

bring the medium back to its original pH of 7.3 -0.2 (2).

All .experiments involving phytohormones were performed

in triplicate, and all concentrations were tested at least

twice. Controls consisted of culture tubes containing 10 ml

TSB but no hormone. Also,, in experiments employing kinetin,

additional controls consisted of tubes containing TSB plus

the same quantities of 10% NaOH and 10% HCl as were added to

the kinetin-containing culture tubes. These tubes received

no hormone.

Culture tubes were inoculated as previously described

from inoculum cultures which were checked for Escherichia

coli growth by Gram staining and streaking on eosin wethylene

blue agar plates. The culture tubes were read coloripietric-

ally upon inoculation and after 3, 4, 5, 6, 7, 8, 10, 12,

48, and 72 hours of incubation. Before each reading, each

tube was shaken on a Super-Mixer (Lab-Line Instruments, Inc.)

for twenty seconds to bring about a homogeneous dispersion

of cells. Tubes were held in a 37° water bath while out of

11

TABLE I

CONCENTRATIONS OF PHYTOHORMONES ADDED TO TRYPTICASE SOY BROTH CULTURES OF ESCHERICHIA COLI

Phytohormones Employed Singly*

IAA GAi Kinetin

0.0001 0.0001 0.0001 0.001 0.001 0.001 0.01 0.01 0.01 0.1 0.1 0.1 1.0 1.0 5.0 5.0

Phytohormones in Combination*

1.0 GA3 +0.1 IAA 1.0 GA3 + 1.0 IAA 5.0 GA3 + 1.0 IAA 0.1 GA3 +0.01 IAA

0.1 GA3 + 0.1 Kinetin 1.0 GA3 + 0.01 Kinetin 1.0 GA3 + 0.001 Kinetin 0.01 IAA +0.1 Kinetin

0.1 IAA +0.1 Kinetin +0.1 GA3

*All concentrations are given in milligrams phyto-hormone per milliliter trypticase soy broth (mg/ml) .

the incubator for reading. Gram stains were made on all cul-

tures after 8, 24, 48, and 72 hours of incubation; these

stains were examined by phase contrast microscopy under oil

immersion at 1000 x magnification using a Nikon Microflex

12

Model EFM microscope so that the morphology of hormone-

treated cells could be compared with that of cells from

control cultures. Photographs of representative cells were

taken on Kodak 35 mm TRI-X Pan film with a Nikon M-35 dark

box and Type B camera adapter.

The actual concentrations of the phytohormones which

were tested singly and in combination are listed in Table 1.

The method employed in dissolving kinetin did not make it

possible to prepare concentrations of this compound as high

as were used of IAA and GA3.

CHAPTER BIBLIOGRAPHY

1. Scott, D. B. M. and E. Chu, "Synchronized Division of Growing Cultures of 12. coli," Experimental Cell Research, XIV (1958), 166-174.

2. Skinner, C. G., personal correspondence. North Texas State University, Denton, Texas, July 31, 1967.

13

CHAPTER III

RESULTS

Growth responses greater than plus one or less than

minus one standard deviation from the control mean were

considered to be attributable to the phytohormone(s) present,

The results of experiments in which stimulation (growth

response of more than plus one standard deviation from the

control mean or inhibition (growth response of less than

minus one standard deviation from the control mean) occurred

are presented in Figures 1-5. In each case, the vertical

lines represent -1 standard deviation from the mean for

each growth curve at 24, 48, and 72 hours of incubation.

It should be noted that the numbers of cells reported

are based on the size of cells occurring in control cultures,

In cultures having larger cells, the numbers reported are

erroneously high, although accurate in terms of total cell

density per culture.

It can be seen in Figure 1 that growth was inhibited

in cultures containing 1.0 mg/ml IAfl». Less growth occurred

in cultures treated with 0.1 mg/ml IAA than in the control

cultures, but this difference was not appreciable at 48 or

72 hours.

15

u CD -P •H trH *H r-4 i—1 *H a

a. to

a; o M-l O U a)

3 !3

2 x 10 10

1 x 10" 10

1—TSB

12 24

2—0.1 mg/ml IAA

3—1.0 mg/ml IAA

36 48 60

Hours

72

Fig. 1—Growth curves for cultures treated with IAA. Vertical lines represent +1 standard deviation from the mean.

Figure 2 shows that more growth occurred in cultures

containing either 0.1 mg/ml GA3 or 1.0 mg/ml GA3 than in

TSB cultures. Only the latter GA3 concentration elicited a

level of growth greater than one standard deviation above

the control mean.

In Figure 3 it can be seen that 0.01 mg/ml IAA had no

appreciable effect on Escherichia coli growth, but that both

5.0 mg/ml GA3 and 5.0 mg/ml IAA caused inhibition. The

16

u Q) -P "H rH

•H a U <D Ok

w i~H rH <l) o

o

<D

2 x 10 10

1 x 10 10

12

1—1.0 mg/ml GA^

2—0.1 mg/ml GA3

3—TSB

24 36 48

Hours

60 72

Fig. 2—Growth curves for cultures treated with GA3. Vertical lines represent ^1 standard deviation from the mean,

latter IAA concentration proved to be so inhibitory as to

prevent the rapid growth characteristic of bacterial cultures

during the early hours of incubation.

The concentrations of kinetin used had no discernable

effect on growth when tested alone. Figure 4 shows that

combinations of 1.0 mg/ml GA-j + 0.01 mg/ml kinetin and

1.0 mg/ml GA3 -f 0.001 mg/ml kinetin gave growth responses

greater than one standard deviation above the control mean

17

u Q)

•H rH -HI H iH -H a M CP O. CTJ

<D U

o n <D

3 S3

2 x 10

1 x 10 -

1—TSB

2—0,01 mg/ml IAA

3—5*0 mg/ml GA3

4—5.0 mg/ml IAA

Hours

Fig. 3—-Growth curves showing that a lower concen-tration of IAA has negligible effect on growth, but that higher concentrations of GA3 and especially IAA are in— hibitory. Vertical lines represent ±1 standard deviation from the mean.

response. Cultures treated with 1.0 mg/ml GA3 + 1.0 mg/ml

IAA gave an inhibitory response. 4

Figure 5 shows the effect of subjecting Escherichia

coli cultures to two inhibitory concentrations of phyto-

hormones, alone and together. The level of inhibition

demonstrated in cultures which received both hormones seems

to indicate that the effects of the two were synergistic.

18

2.4 x 10 10

u <y -p -H

•H a n <u a 03

r - i iH a; o m o u ai

2.2 x 10 10

2.0 x 10 10

1.8 x 10 10

12 24

1—1.0 GA3 + 0.001 Kinetin 2—1.0 GA3 + 0.01 Kinetin 3—1.0 GA3 +0.1 IAA 4—TSB 5—0.1 NaOH + 0.21 HCl 6—1.0 GAo + 1.0 IAA

36 48 60 72

Hours

Fig. 4—Growth curves for cultures containing a stimu-latory concentration of GA3 and ineffective concentrations of kinetin (1 and 2) or IAA (3), and for cultures containing a stimulatory concentration of GA3 and an inhibitory concen-tration of IAA (6). Values given in the figure are in mg/ml except for 5, the latter representing ml 10% acid or base. Vertical lines represent *1 standard deviation from the mean

The only cells that were morphologically distinguishable

from control cells were those treated with 1,0 mg/ml IAA,

5.0 mg/ml IAA, or 1.0 mg/ml IAA + 1.0 mg/ml GAg. All cultures

19

u Q) -P • H

-H g

U Q) a. CQ r—i .H <P o in o u QJ

3 53

2 x 10 10

1 x 10 10

1—TSB

2 — 5 .0 mg/ml GA3

3—1.0 mg/ml IAA

4—5.0 mg/ml GA3

+ 1.0 mg/ml IAA

12 24 36 48

Hours

60 a 72

Pig. 5—Growth curves for inhibitory concentrations of GA3 and IAA tested separately and together. Inhibition is seen to be synergistic in the latter case. The dashed line represents the growth curve that would be expected if the inhibition were only additive. Vertical lines represent ±1 standard deviation from the mean.

which received these concentrations of phytohormones contained

many cells that were larger than those found in any other

cultures. The greatest over-all increase in cell size was

seen in cultures treated with 1.0 mg/ml IAA. Many of these

cells were approximately one-and-one-half to two-and-one-half

times longer and larger in diameter than control cells, and

tended to be more nearly coccoid in shape. Cultures containing

20

mm r

i U

• o

Fig. 6—Representative photographs of untreated Escherichia coli cells (A) and of cells treated with 1.0 mg/ml IAA (B), 5.0 mg/ml IAA (C), and 1.0 mg/ml IAA + 1 . 0 mg/ml GA3. All photographs were taken using phase contrast microscopy after seventy—two hours incubation.

5.0 mg/ml IAA produced cells that were generally larger in

diameter than control cells (though not as large as cells

treated with 1.0 mg/ml IAA), and longer by one and one-half

to several times the length of control cells. Those cells

treated with 1.0 mg/ml IAA + 1 . 0 mg/ml GA3 were generally

about the same size as control cells, to slightly larger,

and tended to be more nearly coccoid.

21

No important differences in Gram stain reaction were

noted. Representative photographs of cells occurring in

cultures treated with the three concentrations of hormones

mentioned and in control cultures are included in Figure 6,

CHAPTER IV

DISCUSSION

While some of the phytohormone concentrations used may

appear unusually high in comparison with those which have

been reported to affect plant growth, they were chosen on

the basis of results of experiments performed preparatory to

this investigation in which Escherichia coli cultures were

grown in the presence of graded concentrations of the

hormones for twenty-four hours and were then plated out on

trypticase soy agar plates so that colony counts could be

made. The particular concentrations of the three phyto-

hormones chosen to be tested in combination with one another

were selected on the basis of their effects on the growth

of Escherichia coli when used singly.

The results indicate that when indole-3-acetic acid was

included in the culture medium of Escherichia coli cells,

growth was slightly inhibited by a concentration of 0.1 mg/ml,

increasingly inhibited by a concentration of 1.0 mg/ml, and

almost completely inhibited by 5.0 mg/ml. When gibberellic

acid was included in the culture medium, 0.1 mg/ml showed no

22

23

effect on growth, 1.0 mg/ml gave a substantial increase in

growth, and 5.0 mg/ml was definitely inhibitory. Kinetin

had no apparent effect on either the growth or the morphology

°f Escherichia coli in any concentration used? the same is

true for the quantities of 10% NaOH and 10% HCl which were

used.

Since 1.0 mg/ml GA^ proved stimulatory when used alone,

an experiment was designed to determine whether this concen-

tration of GA^ could still increase growth when tested in

combination with concentrations of IAA and kinetin which had

not evidently affected growth. It is interesting that both

1.0 mg/ml GA3 + 0.001 mg/ml kinetin and 1.0 mg/ml GA^ + 0.01

mg/ml kinetin stimulated growth (Figure 4), but that the

combination of 1.0 mg/ml GA^ + 0.1 mg/ml IAA gave a rate and

extent of growth which was not different from the control.

It appears that the potentially stimulatory effect of GA3

was in some way masked by the presence of IAA.

Another experiment was set up to determine whether two

hormone concentrations which were inhibitory alone might

produce a synergistic inhibitory response when tested

together. Such synergism would be apparent if the level of

inhibition (i,.e., the difference in the number of cells

between control cultures and experimental cultures) could

24

be shown to be greater in cultures containing both hormones

than the sums of the levels of inhibition caused by each

hormone alone. Synergistic inhibition did occur in cultures

containing 1.0 mg/ml IAA + 5.0 mg/ml GA3 as is shown in

Figure 5. The dashed line in this figure is the growth

curve that would be expected for a culture containing these

concentrations of hormones if the inhibitory effects were

additive, not synergistic.

The purpose of a final experiment was to determine

whether various concentrations of the three phytohormones

which had had no apparent effect on growth when tested alone

could produce an appreciable effect on growth when tested

together. The highest concentration of each hormone which

had shown no effect on growth (0.01 mg/ml IAA, 0.1 mg/ml GA3,

and 0.1 mg/ml kinetin) was selected, and these were tested

separately, in pairs, and all three together. Through

seventy-two hours of incubation, none of these cultures gave

a growth response different from the control mean by as

much ate 1 standard deviation.

Again it may be pointed out that in cultures containing

larger than normal cells, the total number of cells present

must actually have been less than the number reported since

cell-number was determined by optical density measurements

25

based on the size of cells occurring in control cultures.

Thus, the larger the individual cells in a culture, the fewer

cells needed to account for any particular optical density,

assuming that these larger cells were as dense throughout as

were control cells. This is, however, not necessarily an

entirely valid assumption since the larger cell-size may

have been a result of increased cell wall elasticity

rather than increased protoplasmic content (increased

density) per cell.

Until recently, there had been no reported explanation

of the mechanism of IAA-induced inhibition in bacterial

cells. In 1964, Fukuyama and Moyed (4) announced that ribo-

flavin-catalyzed photooxidation products are the inhibitory

agents, rather than the acid itself. One of these products

was tentatively characterized as 3-hydroxymethyloxindole,

and the other was identified as 3-methyleneoxindole. The

former compound was thought to be non-enzymatically converted

to the latter, which was in turn converted to 3-methyl-

oxindole, a non-toxic substance.

Later in the same year, Still, Fukuyama, and Moyed (15)

presented a possible mechanism of inhibition. The effect

of 3-methyleneoxindole was attributed to its high reactivity

with sulfhydryl-containing enzymes. Since inhibition of any

26

one of the large number of essential sulfhydryl enzymes

could result in inhibition of growth, these workers con-

sidered it unlikely that 3-methyleneoxindole has a single

major site of action. Instead, it is probable that the

functions of at least several critical systems are suspended.

Thus, these authors concluded, the enzymatic reduction of

3-methyleneoxindole to 3-methyloxindole may account only in

part for the transient nature of the bacteriostasis. Dubos

(3) pointed out in 1946 that peptone could partially or com-

pletely reverse the bacteriostatic effect of auxins. The

peptone content of the trypticase soy broth employed as the

growth medium in these experiments may have played a part

in reversing lAA-induced inhibition, but to what extent and

by what mechanism is not known.,

Conn and Stumpf (2) have commented that hormones have

not been recognized to function as either enzymes or co-

enzymes, but that they probably function in controlling

either;the synthesis or activation of enzymes. While it

has not been reported, it might be feasible that GA^, or

one of its catabolic products, could function by inducing

the production of an enzyme which could catalyze this

product's conversion to another substance. If this latter

substance were a substrate common to some metabolic pathway

27

of the cell, then the cell might conceivably be able to

carry on a higher rate of metabolism using this increased

supply of substrate. The fact that GA3 is stimulatory in

one concentration and inhibitory in another might suggest

that the inducing substance could cause the formation of a

product which is an inhibitor of the over-all reaction.

Thus, if too much substrate were present, the inhibitor

produced might slow the reaction by competing with the sub-

strate for some essential enzyme's active site.

In the case of kinetin, since none of the concentrations

used had any detectable effect, it cannot be.concluded that

this phytohormone altered the biosynthesis of Escherichia

coli cells.

It is of note that the only cultures which produced

cells morphologically distinguishable from untreated cells

were those containing either 1.0 mg/ml or 5.0 mg/ml IAA.

In these cultures, cells appeared which were both longer

and larger in diameter than control cells. It has been re-

ported (1, 8, 16) that the action of auxins, including IAA,

softens the walls of plant cells by increasing their plas-

ticity. With a softening of the wall, there ensues a

swelling of the cell by simple osmotic water uptake until

the restraining forces of the wall balance the osmotic

28

values of the cytoplasm. It seems reasonable that the

same phenomenon could occur in bacterial cells, thus

accounting for the enlarged appearance of cells grown in

the presence of IAA.

Gibberellin was discovered because of its ability to

increase growth in plants, and in this quality its effects

are more impressive than those of auxins (9). Whether the

gibberellin effect is due to a stimulation of cell division

or of cell enlargement has been extensively debated; "various

workers have presented data which seem to support one con-

clusion or the other (6, 7, 13, 14). In the case of bacterial

cells, the present investigation would tend to support the

argument for increased cell division, at least at some GA3

concentrations, but not for increased cell enlargement since

the only cultures treated with GA3 which produced cells larger

than control cells were also treated with IAA.

Since Miller's team separated kinetin from yeast DNA

and found it to be an active stimulant of cell division,

numerous reports have appeared on its ability to increase

cell division in plants. Kinetin also causes marked altera-

tions ;in the protein and nucleic acid components of plant

tissue (10,11), a circumstance which could be a basic part

of its effect on cell division (5). Partheir and Wollgiehn

(12) have reported that protein content, RNA, and DNA in

29

Nicotiana plants are all increased markedly by kinetin

treatment. An argument for similar activity in Escherichia

coli is not given credence by the present work, as kinetin

was not seen to have any effect on either growth rate or I

cell size.

The conclusions reached in this investigation may be

summarized by pointing out that IAA showed no effect on

growth rate at lower concentrations and was inhibitory at

higher ones, that GA3 showed no effect at lower concentrations,

stimulated growth at a higher concentration, and was inhibi-

tory at the highest concentration tested, and that kinetin

did not appear to affect Escherichia coli growth or morpho-

logy at all. GA^ alone did not seem to affect cell morphology,

while those concentrations of IAA which inhibited total

growth also caused the formation of larger than normal cells,

this latter phenomenon being a possible consequence of

increased cell wall elasticity. Disparate results have

been reported by earlier workers who have investigated the

general question of phytohormone effects on microbial cells.

While the present preliminary study provides more information

to add to that already accumulated, the underlying principles

involved in hormone-cell relationships are yet to be learned.

CHAPTER BIBLIOGRAPHY

1. Cleland, R., "A Separation of Auxin-induced Cell Wall Loosening Into Its Plastic and Elastic Components," Physiologia Plantarum, XI (1958), 599-609.

2. Conn, Eric E. and P. K. Stumpf, Outlines of Biochemistry, 2nd ed., New York, John Wiley and Sons, Inc., 1966.

3. Bubos, Rene J., "Inhibition of Bacterial Growth by-Auxins, " Proceedings of the Society for Experimental Biology, New York, LXIII (1946), 317-319.

4. Fukuyama, T. T. and H. S. Moyed, '"Inhibition of Cell Growth by Photooxidation Products of Indole-3-acetic Acid," Journal of Biological Chemistry, CCXXXIX (1964), 2392-2397.

5. Guttman, R., "Effects of Kinetin on Cell Division with Special Reference to Initiation and Duration of Mitosis," Chromosoma, VIII (1956), 341-350.

6. Haber, A. H. and H. J. Luippold, "Effects of Gibberellin on Gamma-irradiated wheat," American Journal of Botany, XLVII (1960), 140-144.

7. Hayashi, T., Y. Takijima and Y. Murakami, "The Bio-chemistry of Bakanae Fungus. 28. The Physiological Action of Gibberellin. IV, " Journal of the Agricul-tural Chemistry Society of Japan, XXVII (1927), 672-675.

8. Heyn, A. N. J., "Der Mechanismus der Zellstreckung," Rec. Tray. Bot. Neerl., XXVIII (1931), 113-244.

9. Leopold, A. Carl, Plant Growth and Development, New York, McGraw-Hill Book Cortipany, 1964.

10. Mothes, K., "Uber das Altern der Blatter und die Moglichkeit ihrer WiederverjungungV" Naturwissen-schaften, XLVII (1960), 337-350.

30

31

11. _ , L. Engelbrecht and 0. Kulajewa, ,rUber die Wirkung des Kinetins auf Stickstoffverteilung und Eiweisssynthese in isolierte Blattern. Flora (Jena), CXLVII (1959), 445-464.

12. Partheir, B. and R. Wollgiehn, "Uber den Einfluss des Kinetins auf den Eiweiss-und Nukleinsaure Stoffwecksel isolierten Tabakblattern," Ber. Deut. Botan. Ges., LXXIV (1961), 47-51.

13. Sachs, P. M., C. F. Bretz and A. Lang, "Short Histo-genesis: The Early Effects of Gibberellin Upon Stem Elongation in Two Rosette Plants," American Journal of Botany, XLVI (1959), 376-384.

14. Sawada, K. and E. Kurosawa, "On the Prevention of the Bakanae Disease of Rice," Experimental Station Bulletin of Formosa, XXI (1924), 1-19.

15. Still, Cecil C., T. T. Fukuyama and H. S. Moyed, "Inhibitory Oxidation .products of Indole-3-acetic Acid," Journal of Biological Chemistry, CCXL (1964), 2612-2618.

16. Tagawa, T. and J. Bonner, "Mechanical Properties of the Avena Coleoptile as Related to Auxin and to Ionic Interactions," Plant Physiology, XXXII (1957), 207-212.

BIBLIOGRAPHY

Books

Conn, Eric E. and P. K. Sturapf, Outlines of Biochemistry, 2nd ed., New York, John Wiley and Sons, Inc., 1966.

Fletcher, Win. W., "The Effect of Herbicides on Soil Micro-organisms, " The Physiology and Biochemistry of Herbicides, edited by L. J. Audus, London, Academic Press, 1964, pp . 2 0 - 6 2 .

Hillman, W. S. and W. K. Purves, "Does Gibberellin Act Through an Auxin-mediated Mechanism?," Plant Growth Regulation, edited by R. M. Klein, Ames, Iowa, Iowa State University Press, 1961, pp. 589-600.

Kato, J., "Physiological Action of Gibberellin with Special Reference to Auxin," Plant Growth Regulation, edited by R. M. Klein, Ames, Iowa, Iowa State University Press, 1961 , p p . 6 0 1 - 6 0 9 .

Leopold, A. Carl, Plant Growth and Development, New York, McGraw-Hill Book Company, 1964.

Articles

Ball, Earnest, "Heteroauxin and the Growth of Escherichia coli," Journal of Bacteriology, XXXVI (1938), 559-565.

Beckwith, T. D. and E. M. Geary, "Effect of Indole 3 Acetic Acid on Multiplication of Esch. coli and E. typhosa,,r

Brian, P. W., "Role of Gibberellin-like Hormones in Regulation of Plant Growth and Flowering," Nature CLXXXI (1958), 1122-1123.

Cleland, R., "A Separation of Auxin-induced Cell Wall Loosening Into Is Plastic and Elastic•Components," Physiologia Plantarum, XI (1958), 599-609.

32

33

Dubos, Rene J., "Inhibition of Bacterial Growth by Auxins," Proceedings of the Society for Experimental Biology, New York, LXIII (1946), 317-319.

Fukuyama, T. T. and H. S. Moyed, "Inhibition of Cell Growth by Photooxidation Products of Indole-3-acetic Acid," Journal of Biological Chemistry, CCXXXIX (1964), 2392-2397,

Galston, A. W. and H. Warburg, "An Analysis of Auxin-Gibberellin Interaction in Pea Stem Tissue," plant Physiology, XXXIV (1959), 16-22.

Guttman, R., "Effects of Kinetin on Cell Division with Special Reference to Initiation and Duration of Mitosis," Chromosoma, VIII (1956), 341-350.

Haber, A. H. and H. J. Luippold, "Effects of Gibberellin on Gamma-irradiated Wheat," American Journal of Botany, XLVII (1960), 140-144.

Handler, P. and H. Kamin, "Indole Acetic Acid and Growth of Bacteria with Varying Requirements for Nicotinic Acid and Tryptophan," Proceedings of the Society for Experimental Biology, New York, LXVI (1948), 251-254.

Hayashi, T., Y. Takijima and Y. Murakami, "The Bio-chemistry of Bakanae Fungus. 28. The Physiological Action of Gibberellin. IV," Journal of the Agricultural Chemistry Society of Japan, XXVII (1927), 672-675.

Heyn, A. N. J., "Der Mechanismus der Zellstreckung," Rec. Trav. Bot. Neerl., XXVIII (1931), 113-244.

Kato, J., "Studies on the Physiological Effect of Gibberellin II. On the Interaction of Gibberellin with Auxins and Growth Inhibitors," Physiologia Plantarum, XI (1958), 10-15.

Kefford, N. P., "Auxin-Gibberellin Interactions in Rice Coleoptile Elongation," Plant Physiology, XXXVII (1962), 380-386.

Kennell, D. E., "The Effects .of Indoleacetic Acid and Kinetin on the Growth of Some Microorganisms," Experi-mental Cell Research, XXI (1960), 19-33.

34

Kogl, F., A. J. Haagen-Smit and H. Erxleben, "Uber ein Neues Auxin ('Heteroauxin') aus Harn. XI.," Zeit-schrift Physiologisch Chemie, CCXXVIII (1934) 90-103.

Kogl, F. and D. G. F. R. Kostermans, "Hetero-auxin als Stoffwechselprodukt Niederer Prlanzlicher Organisraen. XIIi:.," Zeitschrift Physiologisch Chemie, CCXXVIII (1934), 113-121.

Kuraishi, S. and R. M. Muir, "Increase in Diffusible Auxin After Treatment with Gibberellin," Science, CXXXVII (1962), 760-761.

Kurosawa, E., "Experimental Studies on the Secretion of Fusarium heterosporum on Rice Plants," Transactions of the Natural History Society of Formosa, XVI (1926), 213-227.

Ma'ruzella, Jasper C. and James G. Garner, "Effect of Kinetin on Bacteria," Nature, CC (1963), 385.

Miller, C. O., F. Skoog, F. S. Okumura, M. H. von Saltza and F. M. Strong, "Isolation, Structure, and Synthesis of Kinetin, a Substance Promoting Cell Division," Journal of the American:Chemical Society, LXVIII (1956), 1375-1380.

"Structure and Synthesis of Kinetin," Journal of the American Chemical Society, LXXVII (1955), 2662-2663.

Miller, C. O., F. Skoog, M. H. von Saltza and F. M. Strong, "Kinetin: a Cell Division Factor from Deoxyribonucleic Acid," Journal of the American Chemical Society, LXVIII (1955), 1392.

Mothes, K., "Uber das Altern der Blatter und die Moglichkeit ihrer Wiederverjungung," Naturwissenschaften, XLVII (1960), 337-350.

_ , L. Engelbrecht and O. Kulajewa, "Uber die Wirkung des Kinetins auf Stickstoffverteilung und Eiweisssynthese in isolierte Blattern. Flora (Jena), CXLVII (1959), 445-464.

35

partheir, B. and R. Wollgiehn, "IJber den Einfluss des Kinetins auf den Eiweiss-und Nukleinsaure Stoffwecksel in isolierten Tabakblattern," Ber. Deut. Botan. Ges., LXXIV (1961), 47-51.

Phillips, I. D. J., A. J. Vlitos and H. Cutler, "The Influ-ence of Gibberellic Acid Upon the Endogenous Growth Substances of the Alaska Pea," Contributions to the Boyce Thompson Institute, XX (1959), 111-120.

Saono, Susono, "Effect of Gibberellic Acid on the Growth and Multiplication of Some Soil Microorganisms and Unicellular Green Algae," Nature, CCIV (1964), 1328-1329.

Sachs, F. M., C. F. Bretz and A. Lang, "Short Histogenesis: The Early Effects of Gibberellin Upon Stem Elongation in Two Rosette Plants," American Journal of Botany, XLVI (1959), 376-384.

Sawada, K. and E. Kurosawa, "On the Prevention of the Bakanae Disease of Rice," Experimental Station Bulletin of Formosa, XXI (1924), 1-19.

Scott, D. B. M. and E. Chu, "Synchronized Division of Growing Cultures of 12. coli," Experimental Cell Reaearch, XIV (1958), 166-174.

Still, Cecil C., T. T. Fukuyama and H. S. Moyed, "Inhibitory Oxidation Products of Indole-3-acetic Acid," Journal of Biological Chemistry, CCXL (1964), 2612-2618.

Tagawa, T. and J. Bonner, "Mechanical Properties of the Avena Coleoptile as Related to Auxin and to Ionic Interactions," Plant Physiology, XXXII (1957), 207-212.

Thimann, K. V., "On the Plant Growth Hormone Produced by Rhizopus suinus," Journal of Biological Chemistry, CIX (1935), 279-291.

Yabuta, T., "Biochemistry of the 'Bakanae' Fungus of Rice," Agriculture and Horticulture (Tokyo), X (1935), 17-22.

Yoda, S. and J. Ashida, "Effects of Gibberellin and Auxin on the Extensibility of the Pea Stem," Plant Cell Physiology (Tokyo), I (1960), 99-105.

36

Unpublished Materials

Skinner, C. G., personal correspondence, North Texas State University, Denton, Texas, July 31, 1967.