PHOTOREACTIVATION STUDIES ON AZOTOBACTER

VINELANDII ATCC 12837

APPROVEDs

Major Pi»6fessor

Minor Professor irfTAin *• uf : , , \ n

Director of the Department of^\Biology

Dean of the Graduate School

PHGTOREA CT J. VAT ION STUDIES CN AZOTOEACTER

VINEIAJVDII ATCC 1283?

THESIS

Presented to the Graduate Council of the

North Texas State University in Fartjal

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Johnny Wayne Peterson, B. S,

Denton, Texas

August, 1968

TABLE OF CONTENTS

Page

LIST OP TABLES . . . . . . . . . . . . . . ill

LIST OF ILLUSTRATIONS iv

Chapter

I. INTRODUCTION 1

Phot or eac t at ion Defined. Previous Works Phot- or eac t %v si t ion He chaii i s sis

II. METHODS AND MATERIALS I?

Stock CtO.t'urca Preparation of the Inocula Ultraviolet reactivation PhotoreactivatIon Techniques and Preparation

of a Parle Control Determination of Survival

III. RESULTS . . . . . . . . . . . . . . . . . . . . 27

IV. DISCUSSION k?.

V. SUMMARY . . . . . , kr>

BIBLIOGRAPHY . . . . . .

ii

LIST OF TABLES

Table . Page

I. Constituents of Burk's Medina 18

II. Photoreac tivat ion Timing Schedule . . . . . . . . 25

LIST 05' ILLUSTRATIONS '

Figure Page

1. Diagram of tho Photoreactivation Apparatus . . . 23

• 2. Photomicrograph of the Vegetative Forms of Azotobacter vInelandli ATCC 1283? (1000X Phase Contrast) . . . . . 28

3. Photomicrograph of the Encysted Forms of Azotobacter ATCC 12837 (iOOOX Phase Contrast) 29

4, A Comparison of the Ins.ctivat.ton and the Photoreaetivation Curves of Afcoto-baoter llriele.ndii ATCC 1283?""""""""""" Vegetative and Encysted Cells . . . . . . . 32

5. In&ctivation and Photoreactivation Curves « of Azotobf c t er vlnglandll ATCC 12837 Demonstrated as a Function of the | Physiological Condition of the Cells ' | When Grown on Different Culture Media . , , 35

6. Inactivation and Photoreactivation of Azotobgxcter yinelandii ATCC 12837 When tha Cells Were Grown in Burk's Nitrogen-free Medium arid Plated on Burk's Agar With Varying Concen-trations of Axamoniusa Nitrate . . . . . . . 37

7. * Comparison of the Inactivation and Ph o t orea c t iva t ion of A >z c t oba c te r vine land i i ATCC 12 83 7~^hi¥Ts5Sidl&tely Plated Out After Irradiation and When'' Allowed to Dark Heal for Twenty-four Hours Prior to Plating , , , , , , , , , , 39

i v

CHAPTER I

INTRODUCTION

Phot or ea c t iva t ion IX; f m eel.

Photoreactivation refers to a phenomenon in which al-

terations, produced in biological systems by ultraviolet

light, nay be repaired or reversed by a*n e:rposv.r© of the bio-

logical system to light of a wavelength which is greater than

that of the damaging radiation. Ultraviolet light produces

numerous effects in biological systems. The most important

effects are morphological changes» delays in the onset of coll

division, decreases in vigor, increase in mutation rates, and

Increases in cell death rates (22). It is doubtf ul that any

single; ore of the above effects occurs separately. It is raore

probable that a combination of the above effects occurs when

cells are exposed to ultraviolet light. Ultraviolet light is

the only type of radiation which produces lesions in biological

systems which can be photoreactivated by light of greater x&ve-

length. Because of their ionising properties, electromagnetic

radiation with wavelengths smaller than the wavelength of ul-

traviolet light produces extensive dosage to the cell and hence

no phofcoreactIvacton• On the other Viand, electromagnetic,

radiations of very large wavelengths have little or no effect

of any kind on biological systensn The range of photoreacti- '

vating vavelengthss therefore, lies between 3150 and 5100 A (22),

2

There is a certain degree of specificity in various organisms

for photoreact ivatIng wavelengths which provide maximum

response, but apparently no distinction is made with regard

to interrelationships between photoreactlva tlon light wave-

length and type of 'ultraviolet light damage.

A search of the literature revealed several other terms

which have been applied to the same phenomenon. Pnotoresto-

ratlon, phot©reversal, and photoreeovery are among the terms

referred to in other reports (22). There h^s been a tendency

in recent years to use only the term photor&actIvation,

The general process for determining whether err not photo-

reactivation is possible in a given organism consists of four

steps. The first step involves the exposure of the c.rga;vL-iis

to a ciaaaging dose of ultraviolet light. The second step in-

volved In deaonstrafclng photoreactivafcien requires the incu-

bation of an aliquot; of the ultraviolet light^dacaged organises

in the dark. This aliquot serves as the control for the experi-

mental system involved. The third step involves the exposure of

another aliquot of the irradiated organism to the photoreaotl-

vating light. The fourth step involves the analysis of the two

arlquots for survlrors; Increased survivor rate or decrease in

mutation frequency in the light-treated aliquot indicates & re-

versal of the damage caused by the ultraviolet light, and

therefore, the quality of photore» c t ivat ion.

This review of the pertinent literature has been concerned

with wavelength of d a t i n g as well as photoreaetlvating lisrht

as the variables in the phenomenon„ These are not the only

factors which affect photoreactivation (22). Another impor-

tant factor is the quantity of energy, as damaging radiation,

which interacts with the biological system. Very large doses

of ultraviolet light will cause irreversible damage to the

biological -system and photoreactitration will not be demon-

strated in these cases. There are two explanations offered

for this dramatic loss of photoreactiv&tion capability; (1)

such extensive damage that the capacity of the photoreacti-

vat irig mechanism is exceeded, and (2) damage to the photoreac«

tivation mechanism itself. Another factor is the time interval

after exposure to ultraviolet light and before exposure to the

photoreactivating light, The organism must be exposed to the

photoreactivating light within five to thirty minutes after

ultraviolet light Jrradiation before a decrease in the photore~

activability occurs (9). Ke.Trier (2?) has reported that this

time m y be extended to twanty-four hours if the cells are kept

chilli ed in ice during the intervening period. The explanation

given for this observation is based on the theory that cells

with damaged deoxyribonucleic acid (IMA) will not be capable

of synthesizing protein at these lo>; temperatures, and there-

fore that the alterations induced in the DNA by ultraviolet

light will not be manifested by causing the synthesis of pro-

teins incompetent with the viability of the cell. The time of

exposure to the photoreactivating light also affects the amount

of photoreactiva tion. The amount of pbotoreactivation in-

creases proportionate];/ with an increase in total energy

delivered as photoreactivating light. Since photoreacti~

vation involves chemical reactions, the rate cf the reaction

is directly proportional to the amount of energy input, The

pH of the culture medium affects the photoreactinability of

the cells,, Ultraviolet light causes ionization of essential

cell constituents. The hydrogen ion concentration affects

the stability of those ionised structures. Ionization of the

structures will cause a change in the electronic configuration

of the molecules. As a result, the molecules will change with

respect to their attraction for other molecules. This change

in attraction w!31 alter cell constituents to the extent that

they will not function properly. It has been unaquivcoally

shown that photoreactivation In bacteria in greatly affected

by the; culture medium in which the organisms are groins prior

to irradiation, and also by the medium used for survival assays*

Roberts and Aldous,(33) reported that the ultraviolet light

sensitivity of cells of Escherichia coli B, irradiated and then

plated on nutrient broth was greater than those of cel]s plated

on a chemically defined medium,

A search of the readily available literature revealed the

fact that little or no work has been reported concerning the

photoreact ivation. of bacteria in different physiological states

other than those nertioned, Specifically, no report could be

found on the presence or absence of photoreactiration in the

5

Azotobacter in the encysted state. No information could be

found concerning: phot ©reactivation in the Azotobacter growing

in nitrogen-free madia as opposed to those growing in complete

media. The latter is of importance when the work of Whelden (kZ)

is considered. This investigator found that Ionizing radiation

has widely differing effects on the Azotobacter existing in

different physiological conditions,

Previous Works

Photoreactivation was first discovered by Kelner in 19^9 (2'v)

His report indicated the occurrence of photoreactivatlon in four

different genera of microorganisms, These genera were Escheri-

chia coli 3/r, Streptcjiyces grlseus ATCC 3326, Penecllllum nota~

turn, and Sacchayopycas cerevlsioe« The piorxser work done by

Kelner with the above organisms furnished other investigators

with information on the nature of ultraviolet-induced cell damage

and the means whereby this damage nay be diminished or completely

reversed.

After Kelner1a reports, a sextos of papers were written on

phot oreac t Ivat ion which demonstrated that the "Dhe11.oifle.non occurred

universally (22). An attempt to illustrate the universal nature

of photoreactiva13on is made in the following discussion, A

large volume of literature was written about photoreactivation

in many organisms ranging from the viruses to higher plants and

animals, Photoreacfcivation has been studied intensely in certain

viruses. Photoreactlvalion of the T bacteriophages of Escherl-

coll B vB.s reported by Dulbecco (11), Bsvr&en and

Kleczkowski (2) demonstrated the occurrence of photoraaoti-

vation of some plant viruses, specifically in tobacco necrosis

virus in the French bean and the spherical, tomato, bushy, stimb

virus of Nlcotlana glutinosa. The tobacco mosaic virus, on the

other hand, could, not be photoreactivated according to these

investigators, B&wden and Kleczkowski believed that there was

a direct correlation between the re3.at3.ve anovnt of nucleic acid

and the photoreactivabllity of the virus since the nucleic acid

content of the tobacco mosaic virus is about one-third of that

present in the other two viruses. Ort the other hand, Pfefferkorn

et al. (31) reported that a. pseudorabi.es virus and a Herpes

siiupj.cx virus are equally phctore&ctiv&hly, although the nucleic

acid content of these two viruses is not dissimilar.

Several genera of bacteria, have been Investigated and shorn

to possess the quality of photoreactivstlon. Extensive research

on this phenomenon has been reported using Escherichia coll and

ot?ier closely related organisms. Many investigators, including

Kelner (25), Witkin (^3), Hollaender (21), and Novick and

Szilard (30) participated in the research, Monod et al. (29)

observed that Escherichia coll K12, irradiated with ultraviolet

light in a citrate buffer and plated on a synthetic agar medium,

was not photcre&otivable. After the addition of catalase or

ferrous sulfate, photoreac titration could be demonstrated.

De Daurat e_t al, (8) in 1963 showed that Aerobaoter aerq^enes

could also be photoreactivated,, Several other bacterial species,

including Bacillus and Asotobact-or, have beers reported to possess a

capacity for phofcoreactlvatlori. The majority of quantitative

data found in the literature on photoreactivationwsre compiled

using the bacteria and viruses as experimental organisms.

The occurrence of the propensity for photoreactivation in

the protozoa and algae was reported by several investigators.

C h lamy d oirona s sioewussl (ho), ParaBeclum bursar la (14), ColpJL-

d iunt (15), Tetrahyaena (?)» and various other genera were shown

to be capable of being reactivated. Hill (20) discovered, that

when ultiaviolst light-irradiated cells of Eufilena ^racili,s

var. becillgris • were incubated in the darlc in a rmtr3ent medium

which permitted. cell divisionf they lost the ability to be

pho toreac t iva ted« The rate of this less increased with the

dose of ultraviolet light afimlnisbered.

Several fungi have been reported to be photoreactlv*s.bXe.

Among these are the molds# Perxecllllp.m notatum (26),

cllllt;s chr.vsoftenuia (3^)t and Ustilago maydus (6). Kelnr.r (?J••)

in 19^9 reported that the yeast Saccb&roaycea cerevisiae could

also 'be phofcoreactivated.

The insects can also be photoreactiv&ted under the proper

experimental conditions, Lethal and molt-retarding effects on

nymphs of Onoopeltos f^scjatus, the ailkweed bug (*tl), produced

by exposure to ultraviolet light, can bs pho feoreact iva feed.

Receesivo lethal mutations in the polar cap cells of Drosqphila

larvae have been phctcreac tivate.i (1), Yon Borstel and Wolff (39)

raported that "after an exposure to a photoreactivating 3 l.<nt

8 '

the hatching ratio of ultraviolet light-irradiated, wasp eggs

.produced by Kabrobracon ;jug;IandIs was increased.

Photoreactlvation ho.s been demonstrated in higher animals.

Rieck (32) reported significant photoreactlvation of the fore-

limb development in larvae of the salamanders, Amblystoma

maculaturn and Amblystoma opaciia. Pigmentation changes In tad-

poles of Rana plplens and Rana catosblam as a result of

exposure to ultraviolet light are also'subject to photoreac-

tlvation (kk). Griffen et &1. (39) observed that the induction

of ear tumors in Swiss nice is photoreactivable, but. only if

the ultraviolet light- and the photoro*:- ot-iv*.t--lng light are used

simultaneously, When the photor&uet5v«.t .tr.g light; Is applied

after the ultraviolet irradiation* the usual msthod in all

other systems, tumor induction increaces. Doubt has been cast

on these results„ It is believed that the photoreac t lv a t Irig

light used contained light in the ultraviolet wavelength region,

since Kelnsr and Taft (?&), using monochromatic ultraviolet

light, discovered photoreactiva tion of similar tumors in albino

mice, In 195S, Jaggar (22j reported that the photoreactlvation

of rumors was nor clearly demonstrated. Jaggar in 1967 stated

that the phenomenon appears not to exist In m&jsroalian tissues

(personal communication) in clear opposition to the above men-

tlened studie s.

Photc.reactivj.tion has been demonstrated in higher plants;

Balden and Kleezoweki (2) discovered that the first-formed

9

leaves of Fhaseolus vulgaris. the pinto bean plant, became

bronze colored and wilted upon exposure to ultraviolet light.

If the leaves are exposed to visible light after irradiation

with ultraviolet light, the bronze color will not appear and

the leaves will not wilt. Bubrov (10) demonstrated the occur-

rence of photoreae t i v& 11on in the epidermis of the onion,

Alllum cepa. Sokolov et aJL. (36) reported that leaves of the

beet, Beta vulgaris, irradiated with ultraviolet light would

turn greenish--brown. With an exposure to visible light, the

greenish-brown color would not appear ac lntexisely.

There are relatively few org&ni&M v/hich do not exhibit

some type of photoreactiva t ion (22), The greatest nuiober of

exceptions to photoreactivation occurs in the bacteria.

Johnson (23 5 failed to find photoreactivation in Bacillus

cereus when four other species of Bacillus and three other

strains of Baclilies cereus were found to be photoreactlvable

(3?» 38). Also, Bacillus subtilis. gaelllus polymyxa, and

Bacillus circutans could not be photoreactivated (37, 38).

Jaggar (22) believes that a possible reason for these diverse

responses is that the bacilli are very sensitive to destructive

action by the photoreac tivat iris light„ Bellamy and Germain (3)

were unable to photoreacbiv&te Streptococcus faecalIs and Streu-

Goodgsl et al. (1.6) could not photore.netivate

Influenzae or 2iplc>coccus pneumoniae. Goucher

iLl* (I? ) failed to photoreac11 va t e Azotobacter vineland!i

10

strain 0. They were able to photoreactlvate Azotobacter

chroocQocim, Azotobacter strain ,!Q", and Azotobacter agile

strain A^,*K The latter organ!sm is also designated Azoto-

bacter vine land il or Agotobacter agile variation vine land 11.

In 195*1-, Goucher and Kocholaty (18) photoreaotivated consti-

tutive and adaptive respiratory systems in vrhole CP.IIS of

Azotobacter agile A4,4.

A search of the readily available literature has re-

vealed that the photoreactivatior stv.dles on Azotobacter specie*

were done primarily or almost exclusively using nitrogert-freo

media, and that little or no work has 'been done regarding dif-

ferences in the physiological condition of the Azotobacter.

No reports could be found in the readily available literature

concerning the photoreactivstion of these nitrogen-fixing

bacteria in different physiological states. No reports coa.la

be found concerning pbotoreactiv&tlon of the Azotobacter in the

encysted state. No information could be foirnd concerning photo-

reactivation in the Azotobacter growing in nitrogen-free media

as opposed to those growing in complete media.

The purpose of this Investigation is to study photoreactl-

vation in different physiological conditions of the vegetative

cell as well as the phocoraactivation of the two morphologies 1

states of 'the Azotobacter cell: the vegetative cell and the

cyst,

11

Photoreac tIrat ion Mechani sms

Several mechanises for the photoreactivation of micro-

organisms have bean proposed. Novlck and Szllard (30) proposed

the poison theory,, They assumed that ultraviolet rays cause

the formation of a poisonous chemical cosiponnd which is produced

In proportion to the clone of ultraviolet light. The poison is

present In two forms: one is light sensitive and the other is

not, Wiren the cells coat&ining the Xiltravlolet-induced poison

are exposed to light, - part of the poison will deteriorate„ This

would allow the recovery of s-or-io of the organisms which vould

otharv/ise die. This explanation, while not "based on fact, is

compatible with laborsbory observations.

Another theory of photoreaetrvation '.i-aa reviewed by Jagg&r

(22). This theory involves a ohrcsophore end reactivable site.

A chroiru/pborc Is defined HS the .site of absorption of the photo-

reactivating lichfc. Several cxparIntents, support the existence

of the chromophore eicher in the nucleus or cytoplasm of the

cell., However, B3um et al. (5) asaert^d that the chroraopbora

probubly exists in the cytoplasm. In their report, it vas

pointed out that a delay In cell, cleavage can "be reversed by

photoreactivatirig light when the Irradiated sperm of ArbocXa

introduced into the enucleate half of an egg,

and the artificial zygote is illriaj.r.at-e-'l with visible light.

The sperm by Itself is not photcr cactM'&ble under the exoeri-

mental conditions stipulated* This is not presented as

12.

conclusive, proof that the chrciuophore exists in the cytoplasm

due to the nature of the experiment» Slrreb and Errera (35)

studied photoreactivatien of nucleate and. enucleate halves of

Amoeba proteus, Photoreactivation was approximately the same

in each half. This experiment was interpreted as demonstrating

the presence of the cbromophore In the cytoplasm, although it

does not exclude the possibility of the presence of the ehroiso-

phore in the nucleus, l'n co:iclus3on, It is believed that the

chroinophore exists in- the cytoplasm, but no proof exists for

the presence of a chroraophore exclusively in the nucleus,

A reactivable site is a crucial structure within the cell

that -suffers ultraviolet daiaage. Its composition is • thought to

be primarily DHA or RNA (22). Since ultraviolet radiation of

wavelengths of approximately 253? A is absorbed strongly by

nucleic acids, it Is believed that the D M molecule is pre-

vented from functioning due to the formation of heat-stable

bonds between the two DM. helices (22). These heat-stable

bonds are composed of two adjacent thymine or oytosine resi-

dues which are chemically bonded to each other to form thymine

or cytosine dimers (22). When these dimers are formed DNA

synthesis is blocked® The blockage of DJJA synthesis causes

cell division to halt, thereby preventing the formation of

bac i-erial colonies, The reac tivable sits and the chrouiopliore

comply with laboratory data, but their existence is theoretical.

CHAPTER BIBI:IOGBAPHI

1. Altenburg, L. S. and E. Altenburg, "The Lowering of the Mutagenic Effectiveness of Ultraviolet by Photore-activating light in Drosophlla," Genetics, XXXVII (February, 1952), 5^5^53/""

2. Bawden, F. C. and A. ICLeczkowslci, "Ultraviolet Injury to Higher Plants Counteracted by Visible Light," Nature, CLXJX (January, 1952) , 90-93.. _

3. Bellamy, W. D. and M. T. Germain, "An Attempt to Photore-activate Ultraviolet Inactivated Streptococci," Journal of Bacteriology, LXX (March, 1955)» 351-352,

Berger, H., F. L. Haas, 0. Wyss, and W. Si Stone, "Effect of Sodium Azide on Radiation Damage and Photoreacti-vation," Jouraal of Bacteriology-( LXV (October, 1953)» 538-5^3. — - ™ -

5. Blum, H. P., J, C. Robinson, end G. II, Logs, "The Loci of Action of Ultraviolet and X-radiation and of Photo-recovery in the Egg and Sp&oj of the Sea Urchin Arbaoia. punct-ulata," Journal of General Physiology, XXXV " *"""* TMay7"l951), ' —

6. Bro^'n, J. S., "A Study of Some Factors Concerned in Phot or ea c t iva t ion Including the Photoreversal of Mutation,55

Ph.D. Thesis, Department of Biology, Stanford University, 1952.

7. Christensen, E» and A. C. C-iese, "Increased Photoreversal of -Ultraviolet Injury by Flashing Light," Journal of General Physiology. XXXIX (July, 1956), 513-326/""""

8. De Laurat, Teresa, S., L. C. Verna, and Liliana Segre, "Influence of Ultraviolet Radiation and of Photoreacti-tration Upon Distinct Growth Phases of Aerobacter aerogenss," Microbiology Bsuana; XVI(2), 1963, 81-197 : —

9. Char, K. and A, C-uha, "Studies on Photoreactivation of g. ooli/G and its DMA Following Ultraviolet Irradiation," International Journal of Radiation Biologv, V T T ( 5 ) , 19o3, kZi-^Zb, **

-0. Bubrov, A. P., "Phetoreactivation of Plant Cells," 3-<o~ finite, M{k), 1963, 501.-509,

Ik

11. Dulbacco, R,, "Experiments on Photoreactivation of Bacteriophages Inactive t'ed with Ultraviolet Radis tier:t " Journal of Bacteriology, I,IX (October, 1950), 329-3^7*

12. , "Phofcoreactlvatlon," Radiation Biology, Vol. II, edited by A, Hollaender ("5 volwaesTritew^Yorl:, McGraw»KilI Book Co., I no., 1955.

13. ^ , "Reactivation of Ultraviolet-Inactivated • Bacteriophage by Visible Light," Nature, CLX1T (June, 19'-9), 5- 9-950. —

14. Ehret, C, F., "The Photcreactivability of Sexual Activity and Rhythmiclty In Paj 'aeciwm bursaria,11 Radiation Research. Ill (1955Tf"2221

15. Giese, A. C. R. M. Iverscn, D. C. Shepard, C. Jacobson, and C. Lc Brandt, "Quantum Relations in Photoreac-tlvation of Coltddiuia," Joitrml of General Physiol orv XXXVII (June7" 1953K ™ 2 5 8 7 ~ " *

16. Goodgal, S. H,, C, S. Rupert. and Re M» Kerriott, " Phot orea o t iva t i 021 of Anf -l.uenzao. " Tha Chemical Basin of Ileredl'ty.'" e^ted^^Tw7*D7 McElroy and B, Glass, Baltimore, The; John Hopkins Press, 1957„

17» uoucner,. C. R« and VI. Kocholaty , "A CoiaparIson of the Cytochrome Structure and RodSatIon Effects in Azcio-Mp^er,'» Archives of BloehcMstry and Biophysics,'™ IXVIII (1957T, "30-3B. ' ~

18. G'oucher, C. R.f D, A. Waldraan, and VI. Kocholaty, "Photo-inactivation and Photore&ctlvation of Constitutive and Adaptive Respiratory Sysvsras of Azotobacter," Journal 9l Bacteriology, IXIX (December,^9T5T7W3~?oTr~™~'

19 • Griff in, A. C» V. S, I) oilman s. EC. B. B or lee, P; Bourvsrt, and E. L. Tatars., "The Effect of Visible Light on the Carcinogenicity of Ultraviolet Light," Cancer Research XV (1955) » 523-523. — — .

20. Hill, Helens 2,, "Studies of Chlorcplast Development Selena," Bloghyjslcs VI (4) (July, 1966),

373-3^3.

21. Hollasnder, A., editor. Radiation 3ioloc;v, Vol „ IT (b, volumes), Mew Yorlv , JtcGr^HllT Fo5Tcol, Inc., "1955.

15

22. Jag-gar, John, "Photoreacfcivation,w Bacteri olng; 1 ca. 1 Reviews, edited by Wilson Perry, Vols.'21-23, (June, 1 9 & K '99-135.

23. Johnson, F. H., E. A. Flagler, and H. F. Blum, "Relation of Oxygen to Photoreactivation of Bacteria After Ultra-violet Radiation," Proceedings Society Experimental Biological Med ineTLl&Tril93<nT"'32-33V""*

2k. Kelner, A. and E. B. Taf t, "The Influence of Photoreacti~ vating Light on the Type and Frequency of Tumors Induced by Ultraviolet Radiation," Cancer Bertaarch. XVI (1956),, 860-666„

25. Kelner, A., ,}Ef feet of Visible Light on the Recovery of Streptonycos jfra*lseua Conidia from Ultraviolet Irra-diation Injury»" Proceedings National Academy Science United States. IXXY~'(l9¥9)'* Y3-79T" .....

26 . . , "Experiments on Photoreac tivation with Bacteria and Other Microorganisms," Journal of Cellular and Comparative Physiology, Suppl.* 1, XXXIX~T^rii,~"l9^2), 115-11?,."'

27 • » 15 Pho t or ea c t iv©. t ion of Ultr&v i ol e t~ ir rad la ted Escherichia coli, v,-ith Special Reference to the Boas Reduction Principle and to Ultraviolet-induced Mutation," Journal of Bacteriology« LV1I (July, 19^9), 51X~522.

28. Latarjot, R. and L. R, Caldas4 "Restoration Induced by Catalase in Irradiated Microorganisms»M Journal of General Physiology. XXXV (July, 1952), H"55~lyfQ~ "**"

29. Honod, J.. A. Torriani, and M. Jolit, ''Sur la Reactivation de Bact&ries St&rillsees par le Payonnsment UV,M Cowpie r&ndne. CCXXIX (1S&9), 557-559.

30. Novick, A, and L. S2Hard, ''Experiments on Light Reacti~ vation of Ultraviolet-inactivated Bacteria," Proceedings of the National Acadersy of Science United Statesrxx^"~"~ ( " W 9 ) , 591-600". : ~~ ' — • —

31. Pfeff erkorn, E» R.» Boyce Vf. Burse, and Helen M. Coady, "Characteristics of the Photoreactivation of Pseudorabies Virus," Journal of Bacteriology, XC11 (June, 1966), b56-Boi,

32. Sleek, A. F., f!The Effects of Ultraviolet and of Photo™ recovery on tne Develop Fore limb of Amblystoma, *' Journal of Korphnlogry, XCIV (Kareh, 195^;™'3^7«^0S.

16 .

33* Roberts, lu B. and E. Al&owa, ssEeeovery frost Ultraviolet Irradiation In Escherichia coll," J o u r m l of Bactsrl^-loo-y LVII (S e p t ets.berTs: W )7""363-»375* ~ **-»

34. Rosgner, P, R., "Reversal of the Lethal and Mutagenic Action of Ultraviolet Radiation on Penlclllima Chrise-s' enua,S! Bacteriology Proceedings » (1951 )T^2^?37'

35. Skreb, Y. and H, Errera, "Action des Rayons U. V. cur des Frapv-ants Nuclees et Anuclees d'amibes," Bxoer*-cental Cell Research, XII (1957), 649-656. —

36» Solcolov, In L., A. V. Gnrshii, and L, P, Ostapovich. "Photorcactivaticn in Either Plants,Biof3^lVa. VIII(l) (1963), 127-129. • —

37. Stuy, J«. H, , "Photoreaetivation of U1 travlolet~inactivated Baeil li, * Bic-c'msai etry et BtopbysJ es Acta, XXXJ (i v5*) 238-240. • ' . _

38. _ "Studies on the Mechanism of Radiation I m c « tivation of Microorganism, II. Photoreaetivation of Some Bacilli and of the Spores of Two Bacillus carers Strains," Biochemistry et Biophysics ActaT"XXIX""(WTd-) 238-240. " - — • — „ -- •

39. Von Borate."I s R. C* and S. Wolff, "Photorec-.c tivation Experiments on the Nucleus and Cj boplas© of the Babro-brae on Egg," Proceedings of the National AcadeBy"of Science United Stetess XL1 *( 19"55T,'~100 'lb"097,'"""'l"

40. Weis, Bo, "Photcreaetivation of Chlaraydcstonas," Microbial Genetics Bulletin. VI (1952)» 23T"

41. WellSj ?» Hv and T* J» Hamilton, "Photoreversal of Lethal and Molt Retarding Effects of Ultraviolet Radiation on" Hilfefsea Bug Nymphs," Anatomical Record, CXVII (3953). 644. " ~ - •

42* Wheldon, R. M, ,_E. V, Enssmarm, and C. P. Ha skins, "The Influence of Roentgen Hays Upon the Nitrogen Fixation hy « Journal of General Physiology, XXTVf6) (1941), 7 89-796 • ~ - — *.» • *

43. Vitkin, B. M,, "Inherited Differences in Sensitivity to Radiation in Esshsrlchlst coll," Proceedings of the

&9*A'£2£. 9l. SeAence'^ni t'e^^tateiT^XXXlTTl946), 59-68. ~

44, /JIVA&W j.nd, P. D. a no. lu Sc3iisg.aH, "Phofcorecovery frotc. 0 1 i,.tca.v xole'c--i?xu.'Joeci Pigmentation Changes in Anuran larva« t

n

°f Cellular and Ccraaarative Physiol opt~"'XT™V ' (April; 1955)7 "107:175: " — -

CEiiPTSR II

hSTHODS AND HATER IAL8

S took Cul t'; \ res

All of the experimentation was* carried out using the sell

organism, Agotcbacber YinclsrvJii, desigriatcd 12837 by the

American Type Culture Collection, This culture was obtained

from the Microti elegy StocV* Culture Collac cionv North Tejr .s

State University, Denton, Tcrss, This stock wa<! Kfdntained

on Tryptlease Soy slants && well as Trypticasa boj broth and

Burk's nitrogoii«freo broth with daily transfers. Both the

vegetative and encysted forms of Asotobaeter Xir^lan&j i were

utilized* The vc^etativo cells uers grown in twenty-five

milliliter quantities vhlah viere Incubated at thirty degrees

centigrade in a New Brimswiet incubator-shaker for a period

of twenty-four hours. The encysted for^s were grown on Burk*s

butane?! agar plates at thirty degrees centigrade for fourteen

days in order to obtain one hundred percent eneystment,

C ti .1 tur e K o d ia

Four types of media were utilized in the research reported

here. The first was Trypticasa Soy media (1). For broth prep-

aration, thirty grams of the dehydrated media were sdded to a

lifcsj.' ox distilled wa-ter &ivx e.u toeIsiT d at one hundred and

17

18

twenty-one degrees centigrade for a period of'fifteen minutes.

Twenty grams of a<jar per liter of tsefilatere added In order to

prepare agar plates arid culture tube slants. The second type

of culture medium that was employed tias Bu.rk' .<? nitrogen-free

medium (5). This medium was prepared by the addition of the

following cheiaicals to one' thousand milliliters of distilled

water:

TABLE X

CONSTITUENTS OP BUBK,:R MEDIUM

KH?PO^ 0.2 gia„

KgHPOj^ 0.8 gru

MgSQV?H20 0.2 gm.

CaSO^ 0.085 gra.

FeSO^*7H20 0.005 em.

Na2M00^•2H20 0.0003 gm

Sucrose 10 gm.

The third type of culture medium was Burk's ammonium

nitrate agar. The constituents of this medium are the same

as those listed in Table I, but wryIns quantities of ammonium

nitrate were added as a source of inorganic nitrogen. The

fourth type of medium utilized was Burk's butanol agar (^)„

This medium was prepared by the addition of all of the chemicals

listed ir? Table I (except the sucrose) to one thousand milliliters

19

of distilled water, Twenty gr&utas of agar were also added per

one thousand milliliters of medium. This medium was auto-

claved at one hundred and twenty-one degrees centigrade for

fifteen minutes. After sterilization, the medium was allowed

to cool to approximately forty-five degrees centigrade. At

this point, two milliliters of filter-sterilissed n~but&nol

were added to the medium and it was poured into petrt dishes.

The plates wore stored on the shelf for a period of four days

before use.

Preparation of the Inocula

Vegetative cells were prepared by allowing them to grow

in either twenty-five milliliters of Burls*s nitrogen-free broth

or tvien by-five milliliters, of Trypfcicase Soy broth for twenty-

four hours in the incubator-shalrer at thirty degrees centigrade,

The cells were washed, three tines by centrifug&tion and reaua-

pended in sterile distilled water. The optical density was

adjusted to approxiiriB.tely 0.8-0.9 at 5^0 millimicrons in ©

Bausch and LosCh "Spectronic 20" colorimeter using distilled

water as the blank. The cell suspension was then chilled in

Ice (2).

Cysts of .A zotpbacter were produced by cultur'ing the

organism for fourteen days at thirty degrees centigrade on

Burkss butanol plates. The degree of encystment in these

cultures was determined by microscopic observation of the

cysts. The cysts were removed from the agar and suspended

20

in sterile distilled water. The cysts were cashed three times

and resuspended in sterile distilled water. The optical den-

sity was adjusted to approximately 0,8-0.9 by dilution with

sterile water. The suspended cysts were then chilled in ice (3).

• ' Exposure to Ultraviolet Light

The vegetative and encysted cells were exposed to ultra-

violet light in the same jammer# All of the following

irradiations ware done in a rooa 3Illuminated with a twonty-five

watt, safety, yellow light bulb, The inactivating light was an

ultraviolet lamp (Mlner&llght) 'which was Mounted on a large

ring stand to facilitate the regulation of the distance of the.

ultraviolet lamp from the bacterial cells. The height of the

lamp was set at thirty--three centimeters fx cm the cells. The

energy output of the leuap was mea.su.red by a YS1 Kettering Model

3 9

"65" Radiometer and mis adjust'.'id to 1 X 10- ergs/cm --sec.' by

adjusting the height of the ultra•riolet lamp* The peak output

. of the lamp was 2537 A, The cells were exposed to the inacti-

vating light for the following periods of times jsero, thirty,

sixty, ninety, and one hundred and twenty seconds.

Four-milliliter ailiquots were removed from the chilled cell

suspension and placed in sterile glass petrl dishes where the

depth of the cell suspension was less than one millimeter. The

cells wore then exposed to the light for previous!y _ deterxsined

periods of time and agitated constantly in order to minimize

differences in exposure.

- 2 1

Photoreactlration Techniques and Preparation of a Dark Control

Immediately after exposure to ultraviolet light, three

milliliters of the suspension were removed from the petri dish

and divided equally between two sterile tent tubes. One of the

two tubes was completely wrapped in aluminum foil to block out

all light. This tube served as the control. The two tubes

were then inserted into the phot ore-activation apparatus which

•is illustrated in Figure 1, The photore&ctivatioii apparatus

consisted of a water bath assembly with a 500-watt Sylvan la

photoflood light placed approximately four inches from the

culture tubas. The energy output of the lamp at this distance

was measured and found to be approximately 1,13 X 10® ergs/cm^-

sec, A plexiglass shield was placed between the water bath and

the photoflood to prevent breakage of bulbs, All cells were

left in the photoreaetivation apparatus for the same period of

time, approximately thirty minutes. The temperature of the

water bath was maintained at approximately twelve to fifteen

degrees centigrade during the whole time of the experiment.

Determination of Survival

In order to determine the number of Azotobaeter cells

killed and the number of cells photoreactivated, a means of

counting the bacteria was needed. The spread-plate method was

chosen because of its accuracy and convenience. The cells were

plated in triplicate by making serial dilutions using sterile

22

Fig. l»~Diagr&m of the Photoreactivation Apparatus (3)

Thermometer

Water faucet

23

Ring stand

iiin|!i ? 4 ' a t ! 1 v j ,

t -4 'A /v kj:. \ n; j : ] L * -- ::U l r „

'tV J > • \ Cr . /

Running v^afer

\ -Gbss beaker

Support ring

Maximum distance about 2 iix«V$

Aluminum- wr0 pped tuba • with 2.6 m! ceils

Hose attached to faucel -

"11'l'q f | | ,41 i

U ? - - . A \ 2 X « m l | :2.tsm! C-'3? H,0 • — , f-

i >. * . uV? /

500--watt phot o f 1 ood

-ft k f e - - * - ' i I { > — \ r

(t Support ring

| f"" J | *— R;ng stand

Water bath essarofoiy for photoraac-l.lvatlon,

- • ' • 2 4

distilled water blanks. The plating medium was either TryptIn-

case Soy agar, Burk5s nitrogen-free agar, or Burk's medium,

plus ammonium nitrate. The pistes were incubated for forty-

eight hours in a dark incubator at thirty degrees centigrade.

Experimental Timing Schedule

To facilitate the operation of the experimentation, some

type of timing schedule rc&s required. There were three require-

ments of the timing schedule. First of all, it was necessary

that it allow the cells to stay in the photoreactiv&ting ap-

paratus for-precisely thirty minutes. Secondly, it was ncccssary

that the cells be plated out as soon as possible after the irra-

diation with ultraviolet light. Thirdly, it was necessary that

the schedule conserve time. The schedule utilized in this study

can be seen in Table II.

The left-hand column represents decreasing time in minutes.

Column two represents the control tubes and the phot©reactivation

tubes at various exposure periods. Column three represents the

amount of- exposure of each sample to ultraviolet light. Column

four represents the staggered times at which the samples were

put into the photores ct ivation apparatus. Column five represents

the staggered times at which the samples were removed from the

photoreactivation apparatus.

2 5

" TABLE II

PHO TGRTSAC T1VA.TION TIMING SCHEDULE

Clock Tube U . V . •In Out t Ime number* (In sec.) Po R. P. B . (min.)

90 30R 30" 90 60 90 30C 30" 90 60

7 5 OR QV 7 5 4 5 7 5 OC 0« 7 5 4 5

6o 6OR 60" 60 30 60 6 oc 6on . 60 30

9 0 S 90" 4 5 1 5 k5 90c 90" /j<5 1 5

30 12 OR 1 2 0 " 30 0 30 120C 1 2 0 " 30 0

*R = Photoreactivate C - Dark Control

CHAPTER BIBLIOGRAPHY

1. 1)Ifco Manual, Difco laboratories, Detroit, Michigan, I.966,

2. Kelrser, A., "Photoreactivation of Ultraviolet-irradiated Escherichia coll, with Special Reference to the Dose Reduction Principle and to Ultraviolet-induced Mutation," Journal, of Bacteriology, LVIII (July, 19^9), 511-522,,

3. I.ichsteirt, H. C. and E. L. Oginsky, "Effect- of Ultraviolet Irradiation and Photoreactiva t .Ion on Bacterial Viability and Mutation," Experimental Microbial Phvsiolo«;y, W. H. Freeman and Ccwparry, 19*65'. ~ —

Socolof sky, M. D. and 0. Wyss, "Re sis ts.nc e of the A so to-&£$?£ Cyst," Journal of Baoteriolo?,y, LXXXIV (Jslmiry, 1962 J, 119~12^". .

5. W ilr.cn, P. W. and S. G. Knight, Expej^iments in Bacterial IfkX I-. ogx , Minneapolis, Burgess^Publishing Company'T~ 1952 • . '

26

CHAPTER III

Results

This thesis may be divided into two Main divisions since

there are two morphological states of Azotobacter vlnelandii

ATCC I.283?. The first division involves an examination of

photoreactivation in the encysted form. The second division

involves a study of phctoreactivatlon in the vegetative form.

The division concerning photoreactivatIon in the vegetative

cells may further be divided into ssvei-al subdivisions since

phot,©reactivation was examined in various .physiological con-

ditions of the vegetative cells.

Figures 2 and 3 are phot©micrcgr&phs of the two morpho-

logical forms of Agotqbaoter vInelandii ATCC 12837, the vege-

tative form and the encysted form respectively, that were used

in this study. It is interesting to note that the vegetative

forms are pleomorphic rods, which are the smaller of the two

forms. The encysted forms possess the typical, spherical cen-

tral body with the large entlne and exine of the cyst surrounding

it. A further description cf these forms has been presented in

the litera ture (1).

28

4P A V ###. w

; / * : >

t • V .

Fig. 2—-Photomicrograph of the vegetative forms of Azptobacter vineiandii ATCC 12837 (1000X phase contrast).

29

• * . • • • « v

Fig. 3—-Photomicrograph of the encysted form of Azotobacter vineland11 ATCC 12837 (1000X phase contrast). ~~~~ "

q

• ty 1 '

. 3 0

The first study involves fclts photoreac titration of Azoto-

• bacter vlnelandil ATCC 1^33? cysts, • Since the Agotolpacter

cyst is a very resistant form (1) , one would anticipate a

survival curve from exposure to ultraviolet light to decrease

slowly as a function of time. Figure k illustrates the inact

vation curve and photoreactiva tion curve for Azotobacter ylns~

land11 ATCC 1283? when the cells were originally grown on Burk's

butanol agar and immediately plated out on Burk's nitrogen-free;

agar. It is evident from Figure 4 that the cysts of Agofrohaetor

vinelandii ATCC 12837 can be photoreactivated but that the amount

Of photoreactivation is not as great as that observed in the

vegetative cells.

The second study is concerned with photoreactivation of the

vegetative cells under various physiological growth condition**,

A comparison of photoreactivation of the encysted form with

that of the vegetative form raay be made If the vegetative cells

are treated as the cysts were in the previous section. Figure k

illustrates a comparison of the pho tor eac t lv a t i on curves and the

inactivaticr; curves of both the cysts and the vegetative cells

when Burk's nitrogen-free medium 'is used for the original cul-

ture medium and the piating medium.

31

Fig. —A comparison of the inactlvation and the photo-reactivation curves of Azotobacter vineland.il ATCC 12837 vegetative and encysted cells. The curve on the left repre-sents inactlvation of cells and the curve on the right represents photoreaotivatIon.

m >»

O

o o x: £L

i*: o

o o c

32

o

e> © >

O UJ CO «w»

us £

K-5 5 < 0: cc

1 VAiAilOS 1N 3 Oc! 3 d

33 •

Figure 5 illustrate:-; the Iractivo.tio.ri and pho'toraacti-

tration of Azotobact.ey vineland11 ATCO 1203? as a function of

'the physiological growth condition of the cells. When the

cells are grown in Burk*s nltrogen-fiee media and. after irra-

diation are plated out on Trypticase Soy agar, no significant

amount of photoreactivation occurs. In contrast, Figure ^

revealed that photoreactivation occurred when the cells were

grown in Burk's nitrogen-free medium and subsequently plated

out on Burk's nitrogen-free agar.

Furthermore, when the cells were grown in Trypticase Soy

medium and after irradiation were plated out on Trypticase Soy

agarc little or no significant amount of photoreactivation

occurred. When the cells were grown in Trypticase Soy medium

and after irradiation plated out on Burk1 s nitrogens-free agar,

again no significant amount of photoreactivation occurred.

An attempt was made to determine if ain.mon5.iim nitrate would

have an effect on the photoreactiv&bility of the vegetative

cells,, Ammonium nitrate was selected as a source of inorganic

fixed nitrogen, since the ammonium ion is one of the inter-

mediates in the nitrogen fixation pathway (2), Varying

concentrations of ammonium nitrate ware incorporated into the

plating medium. All cells were originally grown in Burk1s

nitrogen-free medium. The results of the analysis are found

in F igixre 6.

3if

Pig® 5—'Ift^ctivatlon and photoreactivat ion curves of Azoto'&acter yinelandii ATCC 12837 demonstrated as a function of"the physiologies 1 condition of the cells when grown on different culture media. The CUSTO on the left represents inactivation of cells and the curve on the right represents photorea o t ivatlozn

35

< CO

h~

c o X) Q> o

C l

JZ

2 03

to

3 CD

x I \

o Q 1

<

CO

Zs£ 4.,

3

CD

c o

• u ©

u

D-

CD CO H

< CO

H

c o

o

p

CO CO H

o Cv!

X

o

1 V A ! A d n S J N 3 D d 3 d

36

Fig, 6--Inactlvation and photoreactivation of Agotolacter vine land 11 ATCC 1283? when the cells were grown in Buric' s nitrogen-free medium and plated on Burk1 s agar with varying concentrations of amoniwn nitrate. The curve on the left repre-sents lnactivation of cells and the curve on the right represent;: ph o t or e a. c 11 va t i on,

37

x U! O

o cc H

m

tc < o < IU ill ct: UL I

z w o o

cc ill

H \

Ft o 33S

•Y*

J*s°

n n o /kv - '} U %JT*. r'"3 Cfi

X O:

r&fS-*'

±_

o Is* cr)

IM

O

H <C

o <

cr OS

o o o o o o o O & u 53* ro CO < >

r i a y o s i n 2 383d

38

Fig® 7--A comparison of the inac t iva t ion and photoreac t i -vat ion of Azojfcoba&ter viiielandia ATCC 1283? -when immediately pla ted out"aFkir™"ix^ when allowed to dark heal f o r twenty-four hours p r io r to p l a t i n g . The curve on the l e f t represen ts inevctivatlon of c e l l s and the curve on the r i g h t represents photoreactivat-ion.

39

O c o o D >

o o ©

O

£L cS c: O

o >

o a c:

& o

Si o ©

CJ x>

o

o >

o a m

o JC GL

O >

o c c

«p o

XI

n o c

o

sue

o e-io ID

1 V A I A u n s

o o < to

1 N 3 0 y 3 d

O

- • '*0 .

Figure 7 illustrates the effect of delayed PNA expression

on the photoreactivabillty of Agptobacter vineland11 ATGC

1283?. The delayed DMA expression was accomplished by keeping

the cells under refrigeration in the dark for a period of

twenty«-four.hours after photoreactivation attempts. Ths

original culture medium was Tryptlease Soy broth and the

plating medium was Tryptlease Soy agar. The results of the

analysis are found in Figure ?.

CHAi'TiiR BIBLIOGbA?H 1

1. Socolofsky, M. D. and 0. Vyss, "Resistance of the Azoto-bacter Cyst," Journal of- Bacteriology, LXXXIV (Jamiary t I962T, 119-12 *". ** ~ " —

2. Vela, Gerard R., unpublished notes, Department of Biology North Texas State University, Denton, Texas, 1968,

41

CHAPTER IV"

E> is cuss i on

The encysted cells ere ranch more resistant to ultraviolet

light than are the vegetative cells. This is indicated in the

research of Socolofsky and Wyss (k) arid also in Figure 5 of

this report. The same figure also illustrates that the en-

cysted state, which is almost net&bolically inactive9 can be

photoreactivated when exposed to the same treatment as the

vegetative cells„ A search of the readily available literature

reveals that this is the first rej>ort of the phoboreactivatlon

of cysts. Neverthelesss a greater amount of photoreactivntlon

occurs in the vegetative cell than in the cyst. It was pre-

viously reported that the vegetative cells could be photo-

react Iv l"ed on Burk1 s nitrogen-free media (2), No reports were

found in the readily available literature concerning any change

in the photoreactivability of Azotcbacter with a change in the

culture medium. It can be seen in Figure 6 that the culture

median. has a startling effect on the shape of the in&ctivation

and photoreactlvation curves. It in inferred from these data

that AKotohaoter vinelandli ATCC 1283? grown on Trypticase Scy

fcroth is core susceptible to ultraviolet light and that it has

no capacity for pho tore&.c t iva t Ion. Azotobacter vine land ii ATCC

'12837 grovm In Bark's nltrogen-^free broth and plated on

k-2

43

Trypfc lease Soy agar .Is more susceptible to ultraviolet light,

•and It also exhibits no capacity for photore&ctlvation.

The loss of ultraviolet resistance and photoreactiva-

bility as determined in the data presented in Chapter III can

be Interpreted in one of two ways;

(1) The phenomenon may be the result of the healing of

ultravlolet-induced lesions when the cells are plated en Bark's

nitrogen-free medium as opposed to plating on Trypt; lease Soy

agar where the PNA can express itself immediately. Healing

due to delayed protein synthesis is'a well knemn phenomenon,

(2) The phenomenon may also bo the result of a funda«

mental difference in the- physiological constitution of the

organ.! sm brought ©.bout by its growth as a mesotroph on Burk's

chemically defined medium as opposed to its growth on a hetero"

trophic basis on TryptJcaae Soy media,

A report -by Roberts and Aldous in l$~'-9 (3) presents

evidence which supports the second interpretation. They found

that in Escherichia coll the cells were more sensitive to

ultraviolet light when grown on Nutrient agar than when they

were grown on a chemically defined medium.

It was believed for a short time during this research

that the nitrogen-fixing mechanism participated in the increased

resistance to ultraviolet light and the increased rates of

photoreactlvation• If this were true, then the time required

for nitrogen fixation would allow the DMA time enough to repair

Itself. This viewpoint supports the first interpretation of

the loss of ultraviolet resistance and photoreactivability•

This viewpoint was demonstrated to be wrong, according to the

data presented in Figure 7. Anmonium nitrate was incorporated

into Burk's nitrogen-free nediraa in varying concentrations.

Regardless of the concentrations of ammonium nitrate used, the

photoreactivability of cells plated on nitrogen-free media and

those plated on arasonium nitrate media is approximately the

same* This indicates that ammonium nitrate has no effect, on

the photoreaetiv&billty of vegetative cells. Therefore, since

the ammonium ion is an intermediate in nitrogen fixation (5)»

the nitrogen fixation mechanism has little or no effect on the

sensitivity of the vegetative cells to ultraviolet light or the

photoreactlvab.il1ty of the vegetative cells.

It is well toiotm that if bacterial cells are refrigerated

in the dark to delay protein synthesis, a certain amount of

recovery from ultraviolet irradiation Bay be obtained. If this

is the case for Azbto'oaqterf then the first interpretation is

again supported. However, the data presented in Figure 8 do

not readily support this. The data appear to indicate that

only a small amount or no.dark repair has occurred. This data

would then lead one to support the second interpretation that

the phenomenon is the result of a fundamental difference in

the physiological constitution of the organism brought about

by its growth as a xaesctroph on Bark's chemically defined

^5

medlvnj, as opposed to Its growth on a heterotrophic basis

on Trypfcicase Soy media. It in concluded then that only

Agotchapter ylnelandli />TCC 1283? cells gro;m mesctro-

phlcally possess the capability for photor e&c t Iva t ion•

In 196^ Ben Gurin (1) described experiments with

Escherichia coll K12 strains in which the growth was in-

hibited., after irradiation with ultraviolet light, in a

miniwal media by the addition.of twenty-five micrograms of

cysteine per milliliter. This amino acid and other reducing

agents nay reveal a clue which will allow c.io to understand

the different sensitivities to ultraviolet light obtained en

different culture media.

CHAPTER BI3LI00RAPRY

1. Ben Gurion, Renana, ,{Photoreactivation and Cysteine Reversion of Lysogonic Induction,M Journal of Molecular Biology, IX(3) (Septembei-"*, 19S5)",* Sl.9-623.

2. Goueher,- C. R,, I. Kamei, and ¥. Kocholaty, "Ultraviolet Inac bivation and Photoreactivation of Azotobaofcer." Journal of Bacteriology, LXXII (January, 19$6), 18;4—138,

3. Roberts, R» B, and E«, Aldous, "Recovery from CJltravlolet Irradiation in Escherichia, coll," Journal of Bacterido$ry LVII (January , — ™~ —

4„ Socolofsky, H. D. and 0. Wyss, "Resistance cf the Azoto-baoter Cyst," Journal of Bacteriology, LXXXXV (Jsnijary, i962T,* 119-12*K ™ ~

5. Vela, Gerard R«, unpublished notes, Department of Biology, North Texas State University, Benton, Texas, 1968.

kf,

CHAPTER V

SUMMARY

This thesis was written to study photoreactivaticri in .

differe/it physj ologica'i conditions of the vegetative cell as

well as the photoreactivation of the two morphological states

of the A&otot&cter cell: the vegetative cell and the eyefc•

Vegetative cells gro*m in Burl?.1 s nitrogen-frec; broth and

T:r,;/ptioase Soy broth *r«re irradiated with •ultraviolet light

at time intervals between zero seconds and one hundred and

twenty seconds. They were than exposed to a high«intonsity

white light for thirty minutes. Trie cells grown in Bur\k:s

nitrogen-free broth' were plated out on limit's nitrogen-free

agar plates and Buries nedia, plu.fi varying concentrations of

ammonium nitrate as a source of nitrogen. These cells *rere

also plated ovl on - Trypticasc Soy agai-. The cells grown 3n

Tryptlease Soy broth were plated out on Burk's nitrogen-free

agar and Tryptic&se Soy agar. Some of the cells grown in

Trypticaso Soy broth were refrigerated in the dark after

photoreactivation to delay IJHA expression and were s u b s e -

quently plated out on Trypticase Soy agar. The encysted cells

were obtained by platins vegetative cells out on Burk's but&nol

ager for fourteen days. The cells were washed off the plates

l!-?'

k'6

and suspended in sterile cilst5.13.ed t?ater. These cysts were

also exposed to ultraviolet light, for the same periods, as

were the vegetative cells. They were exposed to visible light

for thirty minutes and thon were plated out on Burk's nitrogen-

free agar.

The results of the experimentation indicate that the cysts

of Azotobacter vlnelandii ATCC 12,837 can be photoreactivated.

The amount of phot©reactivation, however, did not appear to

be as great as that of the vegetative cells. Further work on

the photoreactivatlon of Agotobacter vegetative cells was done,

It was discovered that the amount of photoreactivation depends

on the physiological state of the vegetative cell. It vca.s in-

ferred from the data that vegetative cells grown on Tryptlease

Soy broth were more susceptible to ultraviolet light and had no

capacity for photoreactivation. Azotobacter cells grown in

Burk's nitrogen-free broth and plated on Trypticase Soy agar

are more susceptible to ultraviolet light and exhibit no capacity

for photoreactivation. The data also indicates that the nitrogen-

fixing mechanism is not involved in the loss of photoreactivation

on different culture media.

BIBLIOGRAPHY

Books

I)ifco TiinuaJ., DIfco laboratories, Detroit, Michigan, 19$r».

Dulcocco, E., "Photoreactl vat Ion," KMAeMeS Blolo^, Vol. XI, edited by A. Hollaendcr, (4 volumes)s Ne» York, IIcGrr.w-Hill Book Co., Inc., 1955.

Coodp,al» S. H, , C. 8. Rupert, and R. H. IJerriott, '-Photoro-"activation of Hemp;ghilv.R jbif jAtengae, " The, Chemical Bz sis of HeredYt>7 e 'itecl* "by T. "D".""HcKlf oy" and B. Glass, B&lfclmorc, The John Hopkins Press, 3 957.

Hollanders A,, editor, Radiation Biology, Vol, II, "(4 volumes), New York, KcGravr-Hili Book Co. „ Ino. t 1955.

t>J'31son4 P. V?» and S. G. Knight, in Physifilf). , Kinrifcapolis, Burgess Publishing Cowp&ny, 1952.

AT tiol.es

Altenlmrg, L. S. and E. Altenbnrg, "The lowering of the Mutagenic- Effectiveness of Ultraviolet by the Photo-react 3 vat ion Light it; •oph ila ,{' Genet Icq , XXXVII {February , 195?-)» 5^5-553.

Bawden, P. C. and A. Kdeozkowski, ''"Ultraviolet Injury to Higher Plants Counteracted by Visible Light/'1 Natures CI-XIX (January, 1952), 90-91. *

Bellamy, ¥« D. end M. T. Goii?ain, "An attempt to Phoforeajtiv*ate Ultraviolet Inactivated Streptococci," Journal of

I X K 1955), 351-3527'

Ben Curion, Rsnana, "Photoreactlv&t.ion and Cysteine Reversion of Lysoprenic Induction," Jo'?rnnl of Molecular Biology, IX(3) (September, 19c4), ' "

Bergcr, H,, F, L. Haas, 0. Vys?r;, and W. i>. Stone, "Bffcot of fiCrliu::; Allele on Radiation j armgc and Photovsnctiva t-i on.,i;

Jonrnni of Bact-sriof.o.tj,, LXV (October, 1953), 53S-5^3.

*50

Blum, H. F., J • C. Robinson, and R, M. Loos, "The Locx^oi on O"? Ultraviolet and X-r3-d lar ion and cu rno

recovery 'in" the Egg and Spei-m of the Sea Urchin Arbacla ttu^otulata',-" Journal of Geiisral ^-y;-X95i)t 32F3T2V*

Bro^n J. S. "A Study of Some Factors Concerned In Photo-'reactivation Including the Phot ore versal of Mutation,_ Pho D, Thesis, Department of Biology, Stanford University, 1952. '

Chri sbensen, B» and A. C« Gierke, '•Increased PtiOto*t»vt-.T.»vO. of Ultraviolet Injury by Flashing Light," Journal of General XXXIX (July, 1956/, 31

Be B-aurat, Teresa, S., L, C. Verna, and Lillo-m oogre, "influence of Ultravio? et Radiation and of P/iotoroaoti-vatlon Upon Distinct Growth Phases of AcroU-ctjgr

ISEHi;?;® XV"i(2), 1963, 81-69«

fihar K• and A» Guha, "Studies on Photoreactivation of B. coli/G and its D2JA Poll on ins Ultraviolet Irradiation^ In tr-rnationa 1 Journal of "V ii \3)» 19'a-s tzT^TzlT

Dubrov, A. P., "Pho tor e«ao t iv&tion of Plant Cells," (Transla.ted), V(^), 1963? 501 -509®

Dulbeeco, H., "Reactivation of Ultraviolet~< inactivated Bactev:'«ouhar;o by Visible Light," Nature, CLSII (June., 19^9)» 9^9-950.

, "Experiments on Phctoreactivation of Bactcrio-phagc™3 n a o tivs.tod with UltravJolet Radiat3.on," Jorjnial Pi, I^.y^:Ci2lSfiX» I I X (October, 1950), 329"3^?.

Ehret, Co F„, "The Photoaeactivafcility of Sexual Activity and Rhrthrtioity in Farsnooiiva Kvxsavla," Itediation uescaj/oh, i n (19557." zxzT"

Gieae, A. C., R, H. Ivorson, D, C, Shepard, C. Jacobson, and C. L. Brandt, -Qasntna Relations in Phofcoreaetivat1on of Colpldlara,11 Je;nns.) of General Physiplox>7.# XXXVII (June, 1953)« 2^25'iC

Concha a, C. B. an^ W„ Koe-nolaty, "A Comparison of the Cyto-chrome Same ta no an-?. Pad jet j en Effects in KoJ^pba^tor ? Archives of Biochemistry and. Bicybysies, LXV.1II (1957), 30~W.

51

Gouc'ner, C, R., D« A. Waldraan, and V. Kocholaty, "Photo-inactivation Photoreao fcivati on cf Constitutive and Adaptive Respiratory By a terns of Azotobac te r, " Journal of Bacteriology» LXIX (December, 19555 , ?03~?05»

Gouchsr, C. R., I. Kamei, and W. Kocholaty, "Ultraviolet Inactivation and Photoresctivafcion of Agotobaotsr." Journal of terloloa.y, LMII (January, 195&)> 184--188,

Griffin, A. C», V. S. Doln&n, E» B. Borlee, P. Bourvart, and Et L, Yatun, "The Effect of Visible Light., on the Carcinogenicity of Ultraviolet Light," Cancer Research, XV (1955)t 523-528.

Hi] 1, Helens Z., "Strd3.es of Chloroplast De-ve3ox>ment in Buglsna,v S Xt.7-. vi(*0 (July, 19*66}, 373-383,

Jaggar, John, ,,Phofcore*icfci'viition," Bactcr 1 o'logleal R^rl<rSr edited by Wilson Perry.,. Vols* (JuniV" W 99-135.

Johnson, F. H., E. A. Gleglor, and B, F» B'.Ivk, Relation of" Oxygen to Phc t or c*ac t Ivafc ion cf BartsrJr After Ultrayiol et Rfe dis.t i on , ff Pr ocada!;?h Sc-c iet-;v E? »er front o. 3 Riol AK lea 1 Medic ire, J.>±IvTl950)7 ~

Kelner-, A., "Effect of Visible Light on the Recovery of Strepto&yces &rdseu£ Conid 1a froa Ultraviolet Irradiation Injury*" Proceeding's of the National Academy of Science United Stat^V^XXW'"(1^9T7" ?>f9«"^

, "Experiments or Fhotorenctivatlctt vith Bacteria and Other Hicroor&eniirs*,v Journal of Cellular Corp .rativr Physiology, suppl» 3.„ XX^iX~rAf^iiri9j2 57"W-il7T""

, "Photoreactiwt ion of Ultravlolet-lrvad lr.ted Ksoherlch's coli, with Special Reference to the Dope Reduction Principle and to Ultra-/-iolet~in-*\iccd Mutation,11

Journal of Bacteriology, LVII (July, 19 -9), 511-522.

Keener, A, and E, B, Taft, u-fhe Influence of Photcnraot i-vabi'ng Light oa the Type and Frequency of • Tumors Induced, by ultraviolet Radiation*Cancer Research, XvT (1956) 860-866. — .. . . .—

Latarjei, Fie and L, R„ Caldss, f:R,esto.vation Induced by Catalan© in Irradintad K i or oorrr.nJ jibst, *•' Jourml of 0en-§T3l QiZ&iBlttSZ* XXKV (July, 1952), 455-5707

52

Lichsfcefui, H. C. i'l-nd E„ L, Oglns/lty, " E f f e c t of U l t r a v i o l e t I r r a d i a t i o n and PhofcovesxetiU&fcJon on B a c t e r i a l V i a b i l i t y and Ku ta t io i ) , » Bx^crSx-.antal n i c r o b i a l Phys3.nlo.ojar. Vf, H. Freeman ar,«d Company, 19&5»

Monofi, J . , A. T o r r i a n i , and K. J c l i t , (:8u.r l a R e a c t i v a t i o n de B a c t e r i a s Sfc&rt3is los c?*r l e e P&yonnemsnt UV," Costpfce r andug , CCXX1X (19**9). 557-559.

Koviclr, A. aril L. S z i l a x d , "Exper iments on L igh t R e a c t i v a t i o n of U l t r u v d o l e t ^ i n ^ e f i v a t o d B a c t e r i d P r o c ^ c i d l t ^ s of the n a t i o n a l Aoad-a.'";/ of Soicnc-.e U r l t e d S t a t e s , XXXV (19/5-0) f ¥5S-€'OT: * """"

Pfefferkox-ii , 1 . R«, Boyc# V. Burge, and Helen M» Coady, "0har>" io te r i s t i cs of t h e Photore,%ctIva1I on of P s e t u i « ' b 1 os Varus»" Jom-nfQ. of BacterJolorgy, XC1I (Jvji&» 1966) , 056-861,

Rico \ \ A. P . , "The E f f e c t s of U l t r a v i o l e t end of F h o t o i e c o v e r j o/; t he Developing; Poreli;nt> of .Aifblystoma t

, f J our no. 1 of KoyiJhslo&.v-XOTV ( l^ . rok, 195*0 • 367-H'OB,

Robe?/1 -v. s p» B, and E. A3 dot'?.; , "B&oovsry f r o n U l t r a v i o l e t I r r a d i a t i o n ir> E s c h e r i c h i a c o l l . , " Journal of B-r-.otoriolc~v, I,VII (Beptei ( 'ber7 ' l9 f^T7" '3?3«3?5.

Roe£s;e\ ? P . R . , " f taver t ia l of t h e Lethal and Ifata^ :io A otic-it of l:i."t'r-fc.v"iolet Radiat ion on Pe i i ion i iu ju cbr,%iyo^nvM,*' B&.ctex-iolokIcs.! PTj^^3d l«^£ , (1951 r , 62«63»

Skro'o* «?ml K» Ev.-rerfe, M o t i o n dew Rayons U„ VI SUP dec Fr3£.",siits Nriolee? e t Amxcl&es d ^ i i i b e s , , f F r^er imes i ta l p" i - i £te*S££3>t* XJ'I (195? ) , 6&9-C56.

Socolcf .sV:y, Ms I)„ and 0t. V/yiJ.s, " B e s i n t e n s e of t h e Agofc ~jjzj-c:'ypx Cy-otf" Jp-.iyr-rtl of i ^ c ^ r i p ^ . r ^ t J.XXXIV (Jar.u^xyi 19S'?-)'5

Sololo'i/ , Yn L , . A. V. G o r ^ h i l , ond .1. F . Ost&povich. t !i'h.jto-vso.c t ivf - t ion in El? her P l a n t s . " B i o f U d h t , , VHX( l ) (1963) , 127-1'19-

O 1-

* ? V ny„ 1 „ H, , 'i?h<,.toro.:i?.fci'?!f: t i o n of Uj t r&viole t - i rLVj . t iv .a tod ' B&.0illi ?

11 I t i o c h ^ i s t r y of Bicph./Pioo Ac ta , XXII (19^5) ,

"1 on ths. hecha?rl;i£: of R&.diatiort l y^ . c t iTa t ioo of hioroov.;--:i:1,s,vis , I I , Flv^ove^cfciv^tioi ' i of Soks Bo.c i l l i and of the '"porof; of t h e Tv,:> l a c i l ' r a s a•»?<•&<..* S t r a i n s . " 5.1 iv.s x i r i " 23s.-2.do,

53

V c n B o r s t e l , R, C . and-'S. W o l f f , "Fhofcoreacfclvation E x p e r i ~

w e n t s o n the R u c l e u s a n d C y t o p i s ss; of the Habrobaraqoii

Egf>," P r o c e e d i n g s of t h e ffetional A c a d e m y of S c i e n c e

yjiitedT 1955")7Tod£-~ioG9T~"~""'~

W e l s , P . , "Photorestotlvatiovj of C h l & m ' d o m o m s s , t t Microbial!

G e n e t i c s ^jn.sfciw, V I (1952Tr2"3':^

Wells-. P» H . and T» J , Fs.yiiltoi'i, " P h o t o r e v e r s a ! of L e t h a l

a n d H o l t R e t r a i n s E f f e c t s of U l t r a v i o l e t R a d i a t i o n on

Hilk.'-sed B u g N y m p h s , " A i m t o x i c a l R e c o r d , C X V 1 I (1953)»

6kk.

VTrieldc>>, R* Me a n d F.„ V , "The I n f l u e n c e of R o e n t g e n

'Hays U p o n the Ritrogtfte F i x a t i o n b y A ^ ^ o b a c t o r , w

j?X Gei;i3ral Pb>;slolpgy, XXIV(6)"" fi"9^V) ,* ? 8 9 « 7 9 6 »

Wltfclu, E . M * , !'lnhorited Difforo/ioes in S e n s i t i v i t y t o

R a d i a t i o n In K s o j u ^ j c h ^ i c o l l t " ProceediVif.s of the?

Koti o'nal Acadc-i-vy of S c i e n c e United"*S^Tfcs7*iXXXJl"Tlois.5) t 5 9 - 6 8 7 ™

Ziir:ft/i'i.:odt F 0 R . e n d B . K . S o b ? 1 1 " P h o t o r e c o v c r y frcru

U 1 t r a v i o l e t « i n d u c e d Pig/serttation Cnmi£;e,:> ir A ^ e r v a o t "

Joimo.-al of Cellufis/i' Cc^vpsret-'vu Phy.8jLcO.05> , X L V ( A p r i l .

1955ft 1 U 7 " 7 ? 5 7 " ""' ~ * ~ ~ " " "

Unptiblinb.od ?'Ja t o r i a l s

V e l a j G e r a r d R . , Wiptfoli -notes, DeparfcKsrfc of B i o l o g y ,

N o r t h T e x a s S t a t e U n i r e r s I t y , # D e n t o n , T e x a s , 1 9 6 8 .