71-7518

MILLER, Marcia Ann, 1942- GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATIS.

The Ohio State University, Ph.D., 1970 Microbiology

Please Note: School lists name as Marcia Ann Wolfe Miller. University Microfilms, A \ERO\Company , Ann Arbor, Michigan

Copyright by

Marcia Ann Miller

1971 Copyright by

Marcia A n n Miller

19 71

71-7518

MILLER, Marcia Ann, 1942- GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATIS.

The Ohio State University, Ph.D., 1970 Microbiology

Please Note: School lists name as Marcia Ann Wolfe Miller. University Microfilms,A \ER0X Company , Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE

IN STAPHYLOCOCCUS AUREUS AND MYCOBACTERIUM SMEGMATI3

DISSERTATION

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

By

Marcia Ann Miller, B.S., M.A.

The Ohio State University 1970

Approved by

Adviser Academic Faculty of Microbial and Cellular Biology ACKNOWLEDGMENTS

I wish to express my appreciation to my advisor,

Dr. Melvin S. Rheins, for his guidance throughout the course of my graduate study.

I also wish to express my gratitude to fellow graduate students Helen M. Pollock and Robert Z,

Maigetter and to Mr. Ivan Kapotenovic for his technical assistance.

Most of all, I would like to thank my husband

Larry for his constant encouragement, patience, and halpfulnoss, without which this degree would not have been possible.

ii VITA

November 23, 1942. . . . Born - Fremont, Ohio

1956-1960...... Attended Salem-Oak Harbor High School, Oak Harbor, Ohio

1960-1962...... Attended Michigan State University, East Lansing, Michigan

December 27, 1962. . . , Married - Larry Reed Miller

1963-1965...... B.S. Ed., Bowling Green State University, Bowling Green, Ohio

1965-196 6 ...... M.A., Bowling Green State University, Bowling Green, Ohio

1966-1968, ...... Research Biologist, Genetics Division, Department of Biology, Bowling Green State University, Bowling Green, Ohio (Drosophila Genetics and NASA Biosatellite Project)

1968-1970...... Ph.D., The Ohio State University, Columbus, Ohio

PUBLICATIONS

Marcia A. Miller and Shirley A. Harmon, "Genetic Associ­ ation of Determinants Controlling Resistance to Mercuric Chloride, Production of Penicillinase and Synthesis of Methionine in Staphylococcus aureus." Nature. 215:531-2, 1967.

Marcia A. Miller and Irwin I. Oster. "Further Observations on Temperature Sensitive Mutations in Multicellular Forms." Proceedings XII International Congress of Genetics.' 1; l4l~. 1968.

iii VITA (Continued)

FIELDS OF STUDY

Major Field: Microbiology

Area of Specialization.Microbial Genetics

iv TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iii

LIST OF TABLES ...... vii

LIST OF ILLUSTRATIONS ...... viii

INTRODUCTION ...... 1

REVIEW OF THE LITERATURE ...... 5

Drug Resistance and Microbial Genetics ..... 5

Mutagenesis and Extrachromosomal Inheritance . . 11

Genetic Studies: Transduction and Lysogeny . . 18

MATERIALS AND METHODS ...... 22

Source and Description of Cultures ...... 22

Basic Propagation Media ...... 25

Iodometric Method for Activity of Penicillinase 26

Detection of Penicillinase Activity by a Bio-Assay Method ...... 27

Isolation of D-Cycloserine Resistant Mutants of S. aureus ...... 27

Determination of Spontaneous Mutations ..... 28

Acridine Orange and Temperature Treatment . . . 29

Induction of Mutants by Ultraviolet Irradiation ...... 31

Propagation and Titration of Bacteriophages . . 32

Transduction Technique ...... 34

v TABLE OF CONTENTS (Continued)

Page

Harvesting of Cells for DNA Extraction .... 34

DNA Extraction and Determination of DNA .... 36

Determination of DNA Base Composition ..... 39

RESULTS ...... 41

Culture Characteristics and Techniques for Cultivation of M. smegmatis 607 B and .§• aur'eus U 9 W ...... 41

Bacteriophages: Host Range, Plaque Formation, and Propagation ...... 50

Methods for Detection of Penicillinase Activity ...... 60

Characterization of D-cycloserine Resistant Cells ...... 66

Lack of Reversal of D-cycloserine Inhibition by Mycobactin, a Growth Factor for Specific Mycobacteria ..... 74

Spontaneous Mutation Rates ...... 78

The Effect of Acridine Orange and Elevated Temperature on Penicillin and D-cycloserine Resistant Cells ...... 78

The Mutagenic Effect of Ultraviolet Radiation on Penicillin and D-cycloserine Resistant Cells 82

Transduction of Penicillin and D-cycloserine Resistance ...... 85

Extraction of DNA . 87

Determination of DNA Base Composition ...... 87

DISCUSSION ...... 103

SUMMARY ...... 119

BIBLIOGRAPHY ...... 121

vi LIST OF TABLES

Table Page

• 1. Composition of Synthetic Medium ...... 23

2. Reagents used in Deoxyribonucleic Acid Extraction Procedures ...... 35

3. Amino Acid Requirements for M. smegmatis 607 B and S. aureus U9W in a Synthetic Medium ...... 51

4. Titer of Various Mycobacteriophage After Six Hours of Incubation for Propagation at 37 C with M. smegmatis 607 B in Varying Concentrations of Tween 80 .... . 55

5. Phage Typing Patterns Exhibited by Various Mycobacteriophages on Several Strains of Mycobacteria Employing the Spot-Plate M e t h o d ...... 56

6. Degree of Resistance to D-cycloserine Demonstrated by Various Organisms on Several Types of Semi-Solid Laboratory Media 70

7. Spontaneous Mutation Rate to Drug Sensitivity of D-cycloserine Resistant Cloned Cells of S. aureus U9W and M. smegmatis 607 B ...... 79

8. The Effect of Mutagenic Agents Upon Loss of Penicillin and D-cycloserine Resistance in Strains S. aureus U9W and smegmatis 607 B ...... 81

9, Transduction of the Penicillinase and D-cyloserine Determinants to Penicillin and D-cycloserine Sensitive Cells of Strain U9V/ of S. aureus ...... 86

vii LIST OF ILLUSTRATIONS

Figures

Figure Page

1. Effect of Tween 80 in varying concentrations on the propagation of mycobacteriophage D29 with host M. smegmatis 607 B as indicated by a drop in phage titer with increase in per cent of Tween 80 incorporated into the culture medium...... 54

2, The effect of various broth media on the degree of resistance to D-cycloserine demonstrated by S. aureus (penicillin resistant), S. aureus (penicillin sensitive) and M. smegmatis 60T~B ...... 73

3, Antagonism of D-cycloserine inhibition of aureus growth by heated preparations containing L-alanine ...... 76

4. Survival curve of M, smegmatis 607 B after irradiation with ultraviolet rays ...... 84

Plates

Plate Page

I M. smegmatis 607 B. Typical rough colony formation on TSA after 72 hours incubation at 37 C in the dark ...... 43

II M. smegmatis 607 B on Lowenstein-Jensen m e d i u m ...... 46

III S. aureus U9W on TSA, demonstrating differences in pigmentation ...... 49

IV Plaques of various mycobacteriophage plated on M. smegmatis 607 B...... 59

V Phage susceptibility of S. aureus U9V/ (penicillin resistant variant) to staphylococcal and mycobacteriophages .... 62

viii LIST OF ILLUSTRATIONS (Continued)

Plate Page

•VI Agar-plate modification of the iodometric assay for production of penicillinase in the penicillin resistant variant of S. aureus U9W 65

VII Detection of penicillinase activity determined by a bio-assay method, using a penicillin sensitive variant of S. aureus for noting inhibition by residual penicillin , 68

VIII Densitometer trace and corresponding ultraviolet photograph of DNA from M. smegmatis 607 B ...... 91

IX Ultraviolet photographs of mycobacterial DNA coming to equilibrium during a 24 hour run . . 93

X Ultraviolet photographs of mycobacterial DNA coming to equilibrium during a 24 hour run . , 95

XI Densitometer trace and corresponding ultraviolet photograph of DNA from S. aureu3 U9W, Pase+ D-CSr ...... 98

XII Ultraviolet photographs of staphylococcal DNA coming to equilibrium during a 24 hour run ...... 100

XIII Ultraviolet photographs of staphylococcal DNA coming to equilibrium during a 24 hour run ...... 101

ix INTRODUCTION

The discovery of numerous antimicrobial agents during the past 30-40 years has greatly altered both the treatment and relative importance of many diseases. Following the spectacular discoveries of many antimicrobial agents, however, was the disturbing recognition of the emergence of drug resistant pathogenic microorganisms. These strains have become important not only in clinical medicine but to the molecular biologist as well where these organisms have served as tools for genetic research. Our knowledge of the function and regulation of nucleic acids has paralled g advancements in antimicrobial therapy. Studies on the mechanism of drug resistant strains have contributed to the clarification of nucleic acid function in normal metabolism and under the influences of these inhibitors.

The first evidence of the genetic origin of drug resistance was obtained with Staphylococcus aureus and 20 penicillin. Following this initial observation, the genetic mechanism of drug resistance has been studied employing the microbial systems of recombination; transduction, transformation, and conjugation. By employing these systems, the genetic determinants (genes) can be investigated with regard to their location in the cell, that is, chromosomal or extrachromosomal. Mutagenic

studies, employing both physical and chemical mutagens, have also been employed to characterize the chromosomal

or extrachromosomal nature, of drug resistant genetic

determinants, due to their selective action on extra-

chromosomal material. Some mutagens selective for

extrachromosomal genetic material are the acridine dyes 82 and elevated temperatures.

An example of genetic studies to determine the

chromosomal or extrachromosomal nature of a drug resistant

determinant concerns penicillin resistance, demonstrated 9 75 by S, aureus, due to penicillinase production, * It has

been indicated that this determinant is extrachromosomal 32 69 in nature. * Various non-chromosomal genetic deter­ minants are well known in this organism and these

established markers serve as references for heretofore

unstudied loci.

The stepwise development of resistance to D-cycloserine 41 17 in S. aureus and Escherichia coli have been reported,

but to date no further genetic studies have been carried

out. Both D-cycloserine and penicillin are cell wall 97 inhibitors. It is considered that related series of

functions (drug resistance) may be determined by a closely 35 linked cluster of genes. It was hypothesized therefore

that the genetic determinant(s) governing resistance to

D-cycloserine in S, aureus was extrachromosomal and closely associated with those determinants controlling resistance to penicillin.

Because extensive work concerning drug resistance has been carried out on S, aureus. the organism was used in this study as a reference genetic model. Although drug resistance has by no means been studied completely in this organism, it was felt that new Information could be contributed in the area of the genetic aspects of

D-cycloserine resistance in this microorganism,

Mycobacterium smegmatis 607 B also was studied for mutagenesis because of the fact that genetics of mycobacteria has been little studied by modern methods despite practical importance of the mycobacteria. There­ fore many problems solved with other bacteria remain unclear with the mycobacteria, Mycobacteria exhibit a number of simple characteristics such as colonial morphology, and complex characters as induced enzyme systems, which could be useful in a study of their genetic nature.

Improper treatment to prescribed regimens for tuberculosis with the primary therapeutic agents has contributed to the emergence of drug resistant organisms.

Increasing resistance to these primary drugs has augmented the use of secondary chemotherapeutic agents such as 98 D-cycloserine either singly or in combination with other drugs. Better knowledge of strain resistance warrants investigations for better control of administration of drugs in anti-tuberculosis chemotherapy.

M , smegmatis 607 B is considered a non-pathogenic microorganism (Kim, personal communication) and is there­ fore only an approximate model of the more pathogenic mycobacteria for genetic studies. However the history implies its derivation from the original Koch strain of

Bacillus tuberculosis (Human), and it therefore may serve a closer relationship to a causitive agenc of clinical tuberculosis. In addition it is more suitable to invest­ igation because of its growth characteristics and ease of manipulation. It was for these reasons that M. smegmatis

607 B was chosen as a new model for the ensuing genetic studies.

This particular investigation was undertaken to obtain a clearer understanding of the genetic nature and linkage of the determinants controlling penicillinase production and resistance to D-cycloserine in H. smegmatis 607 B and

^ §.• aureus U9W, The effect of the mutagenic agents acridine orange, elevated temperatures, and ultraviolet irradiation, along with results of transductional studies served to characterize these determinants as being chromosomal or extrachromosomal where possible. Buoyant density studies also were employed to gain additional information concerning these genetic factors. REVIEW OF THE LITERATURE

Drug Resistance and Microbial Genetics, Analysis of the development of drug resistance can be determined by the techniques of microbial genetics. Studies of chemo­ therapeutic agents and microbial genetics have contributed extensively to the eluciation of genic interaction and control of biochemical pathways in microorganisms. In the past twenty years, research in these areas has been fruitful in clarifying the relationships which exist between control mechanisms and structural (gene) aspects of enzyme formation.

Mutants constitute unique tools in both genetic and biochemical analysis. The fundamental ideas of biochemical genetics originated in the light of the function of genes enhanced by knowledge in the chemistry of intermediary 35 metabolism. The biosynthesis of the peptidoglycan of bacterial cell walls is a complex sequence of enzymatic reactions. This synthesis occurs in three general stages and antibiotics are known which interfere at each specific 97 point. The elucidation of the structures and mechanisms of the biosynthesis of the major macrcmolecular components of peptidoglycan sysnthesis has been greatly facilitated by the analysis of various drug resistant and sensitive organisms. Studies on two antibiotics, penicillin and

D-cycloserine, have contributed much to these findings.

Both antimicrobial drugs interfere with bacterial 34 cell wall synthesis or maintenance. While D-cycloserine and penicillin both block cell synthesis, this occurs at different points in the metabolic sequence with the two agents,^ The uridine nucleotides that accumulate in the presence of these inhibitors have been shown to be 34 different. Penicillin interferes with crosslinking reactions (transpeptidation) in which D-alanine is linked to the free amino group of the peptide from a neighboring chain, resulting in the accumulation of precursors quite distinct from those brought about by D-cycloserine, Thus, both weaken bacterial cell walls but obviously through different modes of action.

D-cycloserine, identified as D-4-amino-3-iso::asolidone has its greatest activity against Gram positive cocci and the tubercle bacillus, although it also demonstrates activity against many other bacterial genera and is there­ fore considered a broad spectrum antibiotic.Resistance to the antibiotic D-cycloserine exhibited by various microorganisms has been reported and this has included 41 observations with such organisms as the staphylococci 73 and the mycobacteria.

Evidence has been presented which indicated that D-cycloserine is more effective in vivo than in vltro1^ * ^ and in a report by Bondi et al. 11 it was concluded that

D-cycloserine interferred with the metabolism of alanine.

The conversion of L-alanine to D-alanine by the enzyme

D-alanine racemase and the biosynthesis of D-alanyl-

D-alanine, which depends on D-alanyl-D-alanine synthetase, are known to be two essential steps in the biosynthesis of 18 bacterial cell walls. The two enzymatic reactions were shown to be competitively inhibited in vitro by

D-cycloserine and most efficiently reversed by D-alanine, the structural analogue of D-cycloserine. D-alanine:

D-cycloserine antagonism has been shown to occur in many 39 organisms such as S, aureus and numerous mycobacteria, 118 including M, smegmatis. L-alanine has been found to be inactive in reversing the effects of D-cycloserine and additionally, it was observed that the amino acid, DL-serine, a degradation product of cycloserine, had no effect upon the antibiotic.11 38 Hoeprich indicated that the apparent resistance to

D-cycloserine, encountered when many bacteria were tested employing conventional culture media, was in actuality a reflection of the presence of D-alanine in the medium.

He demonstrated that even before final preparation of some commercially available media, there existed sufficient amounts of D-alanine to account for the reversal of growth inhibition by D-cycloserine, Synthetic medium supplemented with L-alanine had a barely detectable amount of D-alanine prior to autoclave sterilization, but contained a signif­

icantly greater amount after the heating due to racemization.

This emphasized the difficulty in evaluating susceptibility tests prior to this time, and stressed the need for 38 employing synthetic medium devoid of DL-alanine.

The emergence of resistance to D-cycloserine has been observed in numerous situations in which the results demonstrated that the acquisition of resistance is a genetic 7 4 property. Resistant mutants of Streptococcus strain

Challis have demonstrated elevated amounts of both alanine 80 79 racemase and D-ala-D-ala synthetase. Perry and Slade

indicated, however, that the unusually high susceptibility of the tubercle bacillus to the drug could not be explained

solely on the basis of its action on the D-alanyl-D-alanine

synthetase. This was based on the evidence that the

D-cycloserine sensitivity of the partially purified enzyme

obtained from the H37r _ strain of M. tuberculosis was c i * * ■ 1 1 _ “ indeed similar to that of the enzyme obtained from other

and less susceptible bacterial species,

Fleming, as early as 1929, noted that bacteria differ markedly in their sensitivity to penicillin. As penicillin

became more available for therapeutic use (1946), its mode

of action in destroying bacteria was ascribed to inhibition

of protein synthesis, nucleic acid synthesis, formation 27 of cell walls, etc. Early studies revealed that penicillin interferred with the formation of cell walls. 59 Lederberg and St, Clair demonstrated that osmotically labile bodies were formed when actively multiplying cells of several Gram negative rods were exposed to penicillin, but reverted to normal form when penicillin was removed from the environment, 78 Park isolated several uridine-linked compounds from growing staphylococci treated with penicillin. The compounds contained an amino sugar which was found to be 95 muraraic acid unique to bacterial cell walls. The peptide was a precursor of the cell wall which accumulated due to inhibition of wall formation by penicillin.

Various types of microorganisms demonstrated resistance to penicillin due totheir production of a penicillin destroying enzyme termed penicillinase,*" The penicillin-destroying action was shown to be due to the opening (hydrolysis) of the g-lactam ring of the penicillin molecule with subsequent formation of penicilloic acid.

The enzyme was subsequently found to be present in both

Gram negative as well as Gram positive bacteria.

Kirby^ first reported the extraction of a highly potent penicillin inactivator from penicillin resistant staphylococci which proved to be the enzyme penicillinase.

This enzyme was found in S, aureus to be partially extra- 29 cellular and, later, to be inducible in that the enzyme was formed only in traces until the organisms were treated 10 with penicillin, Weinberger^^ reported that the amount of enzyme produced in vivo was quantitatively sufficient to account for the high degree of penicillin resistance observed in infections caused by penicillinase producing staphylococci.

Reports have also shown inactivation of penicillin by the enzyme penicillinase in various mycobacteria. The first evidence for the penicillinase enzyme in mycobacteria 112 was presented by Woodruff and Foster in a strain of 63 M. tuberculosis and later in M. smegmatis by Lutkenhuse,

This enzyme, when associated with the mycobacteria has been recognized as being intracellular and partially extra- 42 92 cellular and inducible. Penicillin inactivation by the mycobacteria apparently occurs by an enzyme reaction, 48 analagous to that found in penicillin resistant S. aureus, and which occurs by the hydrolysis of the {3-lactam ring.

Naturally occuring penicillin resistant strains produce penicillinase, whereas sensitive staphylococcal strains rendered resistant to penicillin by subculturing repeatedly in increasing sub-inhibitory concentrations of 82 penicillin do not contain penicillinase. It is of importance to note that in the latter instance, the organisms tend to revert to a large extent, to the original state of penicillin sensitivity when maintained in the absence of penicillin. However such is not the case with naturally occurring penicillin resistant strains. 11

Naturally occurring penicillin resistant strains retain their original degree of resistance to penicillin when subcultured in its absence, however penicillinase-less variants are thrown off at a specific rate indicating a 82 certain degree of instability. This instability has been attributed to the extrachromosomal nature of the genetic determinant which governs this characteristic.^

Mutagenesis and Extrachromosomal Inheritance.

Bacteria tend to mutate^ wherein a variant type of bacterium arises through some change in the nucleotide sequence. Most known bacterial genes multiply as part of a single linkage structure, the bacterial "chromosome".

However, some groups of genes can multiply independently 58 of it and have been termed plasmids. Nonchromosoma1 or extra-nuclear elements are well recognised with many organisms, including the staphylococci^”* and the mycobacteria, where in a Mycobacterium species it was suggested for one of the first times in a report by Jones 44 and White. Their suggestion was based on the high frequency loss which occurred spontaneously of a determinant for a characteristic pertaining to colonial morphology.

Plasmid genes do not necessarily demonstrate linkage with any special part of the bacterial chromosome however they may in rare instances show association with chromosomal loci. In addition, nonchromosomal inheritance manifests itself as an apparently high mutability of some character 12 4 44 71 resulting in its irreversible loss. * 9 This mutability mu3t then be distinguished from that of a chromosomal gene.

The elimination of many plasmids or plasmid genes is greatly enhanced by certain chemical and physical mutagens.

These include acridine dyes,^^ elevated temperature,^ 30 and ultraviolet radiation. The "curing” effect demon­ strated by these mutagens is presumptive evidence for the extrachromosomal nature of a genetic determinant. However

the failure to demonstrate such an effect would certainly

leave question as to its location. It has been found that 82 not all plasmids are cured with equal ease.

The first report of the selective loss of cytoplasmic particles following treatment with an acridine dye 23 (Euflavin) occurred in a population of yeast cells.

The rate at which petite colonies arose, attributable to the absence of respiratory enzymes residing in the mitochondria, was greatly increased following treatment with 37 acridine dyes. The well documented experiments of Hirota on the "curing" of the F (fertility) factor from E. coli by acridine dyes stimulated many workers to try to cure other organisms of genes which were though:to be extra- chromosomal in nature.

The mycelial factor in Aspergillus and the multiple drug resistance factor in Shigella^"* have been reported to be lost following acridine dye treatment. Hashimoto 33 et al, noted erythromycin resistance was lost jointly with penicillin resistance upon treatment of staphylococci with acriflavine. Additional work with yeast populations in Saccharomycss cerevisiae revealed petite mitochondrial populations induced by acriflavine,^ The spontaneous loss of streptomycin resistance in E. coli was greatly enhanced 31 by treatment with acridine dyes. Various virulence markers in S. aureus were reportedly to have been "cured'1 93 22 by treatment with acridines. Dyke and Richmond reported similar loss of the penicillinase plasmids in various hospital isolates of staphylococci, Hiller and

Harmon^® further noted that in addition to the determinants governing resistance to penicillin in S. aureu3, resis­ tance to the mercury ion was also found to be lost following acridine orange treatment. Early reports on studies with various mycobacteria have indicated that acridines are are highly inhibitory to these organisms,^ however no data have been reported to date linking the selective action of acridine to the elimination of specific genetic determinants in the mycobacteria. Certain inferences however have 89 been made along this line in a report by Salevev et. al.

It was reported that a marked decrease in the limiting concentration of various antibiotics, including

D-cycloserine, occurred when cells were grown in culture media containing various acridine dyes.

To the contrary, Novick^"* was unable to eliminate penicillinase plasmids by acridine dye treatment in 14 4 82 aureus. Asheshov and Richmond also have reported

the failure to demonstrate penicillinase-less variants

from a resistant parent strain of S. aureus. Stocker 94 et al. showed that the multiplication and transfer

Of the col factor in E. coli was unaffected by acriflavine

treatment. It may be that sensitivity to acridine dyes

is not an obligate consequence of the extrachromosomal

state, or that chromosomal counter parts of various factors

such as resistance to penicillin etc. do indeed exist.

As early as 1954, Fairbrother et al,^ noted that penicillin resistance in S. aureus was eliminated by growth at elevated temperatures (44 C). This was subsequently 69 5 confirmed by May et al, and by Asheshov. Miller and

Harmon^® more recently have reported loss of the determ­

inants governing resistance to penicillin and the mercury

ion following growth at 44 C. It was hypthesizcd by

May et al.^ that penicillinase production was determined by an autonomously replicating particle(s) (plasmids) whose doubling rate was equal to the cell doubling rate at

37 C, but the replication of such was slowed down or suppressed at higher temperatures (44 C). The plasmids would then be diluted out in subsequent generations resulting in penicillinase-less variants.

The elimination of other genetic determinants following growth at elevated temperature has been reported, 15 although this purging effect was not universal. Mutants, in which plasmid replication fails at high temperature, have been isolated for known extrachromosomal elements, 43 such as the F factor in E. coll. Elevated temperature was used to study the hereditary nature of plastids in the 30 bleaching phenomenon in Euglena gracilis strain Z.

It was found that elevated temperature brought about the permanent loss of the capacity to develop chloroplasts although organisms were not cured of plastids (leucoplasts).

The elevated temperature was found to effect mutations of 87 30 plastids and since DNA and RNA have been found in plastids, it was suggested that plastids themselves carry genetic material, extranuclear in nature.

A third method of determining the extrachromosomal nature of an element concerns the degree of its sen­ sitivity to ultraviolet irradiation is perhaps the least understood and studied, although when used in combination with acridine dyes and/or elevated temperatures a clearer picture of the chromosomal or extrachromosomal nature of a genetic determinant is obtained. Sensitivity of extranuclear material to ultraviolet irradiation is not limited to bacteria, 63 Studies of chloroplast development in Euglena have demonstrated that albino cells produced through ultraviolet irradiation form colorless clones which have not been reported to revert to green, indicating this to 16 be a heritable effect. The action spectrum for ultra­ violet inactivation of chloroplast formation was found to peak at 260 and 280 mu implicating nucleoprotein as the probable site of ultraviolet action. The irradiation of nuclei alone never resulted in bleached 30 cells thus indicating specificity for extranuclear genetic material.

Additional evidence in determining whether a gene is chromosomal or extrachromosomal was reported by 3 Arber and is based on the sensitivity of bacterial genetic material to ultraviolet irradiation while under­ going transduction, Arber1s observations, which were made on E. coli. have been reported to occur in other 56 organsisms such as S. aureus. It was observed that a high rate of spontaneous loss of the marker and a decrease of transducing efficiency upon irradiation are char­ acteristic of an extrachromosomal gene.

A confirming report on these findings was issued by

Ashe3hov.^ She employed a strain of S. aureus which was resistant to mercury salts and synthesized the enzyme penicillinase. Mercury sensitive variants were obtained at a relatively high spontaneous freqeuncy, whereas penicillinase-less variants were not recovered from this strain. This would tend to indicate the extrachrom­ osomal nature for the mercury genes while the penicillinase genes would be chromosomal. Ultraviolet irradiation of a 17 transducing phage preparation for these markers demon­

strated that the transduction efficiency of the mercury marker fell exponentially whereas transduction of the penicillinase genes increased approximately ten-fold.

The reason(s) is not well understood for the differences exhibited by the transducing phage prep­ arations where the transduction rates of various transduced genes was influenced by their physical state

in the cell, i.e. chromosomal or extracrhromosomal. It 82 has been suggested that ultraviolet irradiation may

activate repair enzymes either in phage or cause them to

be formed in the recipient cell. This process may then

facilitate integration of transduced chromosomal genes

but would have no effect on extrachromosomal genes.

The greater sensitivity of extrachromoscmal genetic material to acridine dyes, elevated temperature, and ultraviolet irradiation have been confirmed by many, and

in several well documented cases denied by others. Not

only is there disagreement as to the interpretation of the results following exposure to reported selective mutagens

for extrachromosomal genetic determinants, but the mechanism(s) of the interactions remain unsolved.

Additional investigation is needed to further the under­

standing of the select sensitivity of extrachromosomal

genes to these agents. G en etic Studies: Transduction and Lysogony. Perhaps one of the most fruitful areas of research since the early

195C's for both the geneticist and bacteriologist has been the study of the realtionshlp of bacteria and bacterio- 85 phages. A study of physiologic effects on the host bacterium by phage infection has produced valuable infor- mationwhich has implication in the study of the transfer 14 of genetic information, The investigation of various systerns has yielded information with regards to synthesis of DNA, RNA, phage specific enzymes, bacterial enzymes, and a number of other physiologic functions. It was found that genetic determinants governing drug resistance could be transferred from resistant donor strains to certain sensitive recipient strains by bacteriophages.11^ This process was termed transduction and was reported for the first time in these experiments.

Numerous transduct.ionai studies of various genetic 35 determinants have been reported. So far, the only method available to transfer staphylococcal markers from donor to

recipient cells has been transduction, inasmuch as all attempts to discover a mating system in S. aureus have 82 failed. Furthermore transformation in the staphylococci has not been reported to date.

Transduction of the penicillinase marker among the pathogenic staphylococci was first observed by Ritz and 84 Baldwin. The frequency with which this determinant is 19 C Q transferred Is 1:10 -10 , and is independent of whether the recipient is naturally sensitive or a derived sensitive 75 via mutagenic agents. The size of the genetic material

transferred from donor to recipient cells is limited by

the amount of DNA accommodated in a phage head. No

available data exist for bacteriophages of S, aureus. however studies on two of the T-even phage3 of E. coli 5 82 approximates this size at 10 base pairs.

The following markers may also be present in the

plasmid state in S .aureus; chloramphenicol resistance,

erythromycin resistance, resistance to mercury salts,

ca,dmium salts, and arsenate i o n s . ^ Co-transduction of

factors, which indicates they are closely linked, has

further been found to occur in S. aureus^ * ^ but not

necessarily to every transductant. This process has been

referred to as "plasmid dissociation",^ and has enabled

the determination of the order for various genetic markers

in several strains.

No confirmed reports of transduction in the myco­

bacteria have been reported to date although it was sug­

gested as the mechanism of genetic transfer in studies 45 carried out by Juhasz in 1960 and by White et al. in 108 1961. Transduction attempts were made by Russell 86 et al. in 1963 but these were not successful due to

unsuitable selective markers. No further reports of

attempted transduction have been sited to date. Transformation has been conducted in various species of the mycobacteria,^®* *®^ A report by Tsukamura and

Hashimoto has indicated transformation of isoniazid and streptomycin resistance in Mycobacterium avium by DNA extracted from resistant cultures. Inasmuch as numerous reports of lysogenization exist in the mycobacteria, there can be little doubt that transduction could occur.

Transduction of other bacterial genetic determinants may accompany lysogenization although this is not a pre- 13 requisite for lysogenization.

Many of the genetic studies which have been carried out in the mycobacteria have employed M. smegmatis 607 B as the test organism. The isolation of the first mycobacteriophage active on a strain of M. smegmatis was 28 by Gardner and Weiser, Many strains of phage, lytic for mycobacteria have been isolated but few have been found to

lyse pathogenic mycobacteria. This is perhaps not too unusual since most phages have been isolated from the soil where saprophytic but not pathogenic human strains would 85 more likely be found, M, smegmatls 607 B however, is

lysed by many of these phages which makes it a valuable test

system because of its reported derivation from the original

Koch strain of Bacillus tuberculosis (Human), Many additional phages specific however for M. smegmatis 607 B 12 have also been found to lyse M. tuberculosis. 21

Colony changes from rough to smooth were observed in strains of M. smegmatis 607 B following exposure to several phages.^ The smooth colonies were found later to be lysogenic for the infecting phage, and hence resistant to phage lysis. They were not resistant to other phages to 109 which the organism was usually susceptible. Reversion to rough colony morphology was further obtained from 44 smooth colonies which had lost lysogeny. In addition to changes in colonial morphology, variation in plaque types, new protein synthesis, and altered enzyme activity have been reported in lysogenized cells. * This would seem to infer that the preceding changes were brought about through lysogenization.

The usefulness of bacteria as genetic tools cannot be doubted. Certain test organisms have predominated in these areas of research for numerous reasons, usually because of their ease in manipulation, and have established guidelines for initiating studies with other organisms.

Expansion of investigations to systems in unstudied organisms may further lead toward a better understanding of fundamental genetic principles. MATERIALS AND METHODS

Source and Description of Cultures, The strains of

Staphylococcus aureus employed in this study were obtained from a collection of cultures in the Department of Biology,

Bowling Green State University, Bowling Green, Ohio, The strain designated as U9W was initially isolated at Temple

University, Philadelphia, Pennsylvania, The strain U9W was susceptible to lysis by phages 80 and 81 of the

International Typing Series, and initially was found to be resistant to at least one unit of penicillin G Potassium

(E.R, Squibb and Sons, New York) per ml in Trypticase Soy

Agar (TSA, Baltimore Biological Laboratory), A penicillin sensitive variant of strain U9W was recovered as a spon- 32 taneous mutant in an earlier study. In this work both the penicillin resistant and sensitive forms of strain U9W subsequently were found to be sensitive to at least 1 ug per ml of D-cycloserine (D-CS, Lilly Laboratories,

Indianapolis, Indiana),

A synthetic medium devoid of the amino acid alanine was used to determine drug resistance or sensitivity to

D-CS, The D-CS synthetic medium was prepared using the 1 flfi components listed in Table 1, Penicillin resistant

22 TABLE 1

COMPOSITION OF SYNTHETIC MEDIUM

Component Amount

K 2HPO4 7.0 g KH2PO4 2.0 g Sodium Citrate*5 H 2O 0.5 g MgS04 *7 H2O 0.1 g (NH4)2304 1.0 g L-Arginine»HCL 50.0 mg L-Aspartic acid 90.0 mg L-Cystine 20.0 mg Glycine 50.0 mg L-Glutamic acid 100.0 mg L-Histidine»HCl 50.0 mg L-Isoleucine 30.0 mg L-Leucine 90.0 mg L-Lysine*HCl 50.0 mg L-Methionine 50.0mg L-Phenylalanine 40.0 mg L-Proline 80.0 mg L-Serine 30.0 mg L-Threonine 30.0 mg L-Tryptophan 10.0 mg L-Tyrosine 50.0mg Nicotinic acid 1.0 mg Thiamine»HC1 1.0 mg Agar-purif ied 1.2 g Distilled water 1,000.0 ml 24

strains wore designated as Pase+ and sensitive strains were designated as Pase*’, D-CS sensitive strains were designated as D-CS".

The strain of Mycobacterium smegmatls termed 607 B was obtained from Dr. W.B, Redman, Veterans Admin­

istration Hospital, Atlanta, Georgia, Strain 607 B was a buff colored variant of the original parent strain

607 as described by the American Type Culture Collection,

Rockville, Maryland, The original parent strain, 607,

is reputed to have been received as Mycobacterium

tuberculosis horninis (Koch) in November 1925 from

Dr, R,D, Herrold. Dr, Herrold reported that the culture

was the original Koch strain of Bacillus tuberculosis

(Human) brought over by F.J. Novy in 1888 and maintained

on artificial medium during which time it lost its

virulence and has assumed the ability to grow quickly on

artificial media,(B,A, Brandon, personal communication)

The strain now is considered to be saprophytic and is 12 avirulent for guinea pig. Strain 607 B initially was

found to be resistant to at least 1 unit of penicillin

per ml and 200 ug per ml of D-CS, This strain also serves

as the multiple host for mycobacteriophages DSP^, DS6A,

Rlf LEO, D29, GS4, and GS7.

The Tice strain of M. tuberculosis (BCG) was obtained

from Dr, H. Kim, the Trudeau Institute, Saranac Lake,

New York, 25

The strains of Mycobacterium phlel obtained from the

American Type Culture Collection (ATCC) were designated as

11728, which was the host for mycobacteriophage 11728-B, and 11758 which was the host for phage 11758-B,

Additional M. smegmatis strain were ATCC 14468, 11727 serving as host for phage 11727-B, and ATCC 11759 with accompanying mycobacteriophage 11759-B.

The stock cultures of S. aureus were maintained on

TSA slants at 4 C and subcultured at monthly intervals.

The mycobacteria stock cultures were cultivated on

Lowenstein-Jensen Medium (Difco, Detroit) at 4 C and subculturcd every two months. All stocks were phage- typed following subculture to insure derivation from the parent strain,

Basic Propagation Media. The following media were employed in propagating cultures and phages.

(i) D-CS Synthetic Medium. The list of components used are found in Table 1, An amino acid mixture was prepared and sterilized using an S-l (#14) Seitz sterilizing pad. The pre-sterilized amino acid mixture was added aseptically to an autoclaved solution of salts and agar (purified agar, Difco, Detroit). The pH was adjusted with 1 N NaOH prior to the addition of agar and before sterilization. One per cent glucose was added aseptically prior to pouring of plates. 26

(U) Middlebrooks 7H9 Medium. The standard medium was supplemented, following sterilization, by adding aseptically one per cent glucose solution and Bovine

Serum Albuimin, Fraction V ( 0,5 per cent, Sigma Chemical,

St. Louis, Missouri).

(iii) Trypticase Soy Agar and Glycerin, Trypticase

Soy Agar was supplemented with five per cent glycerin

(Fisher Scientific, Fair Lawn, New Jersey).

(iv) Nutrient Broth-3. Nutrient broth was sup­ plemented with IM CaC^.

(v) Mycobacteria Bottom Agar. Bottom agar was composed of nutrient broth and agar, supplemented with one per cent glycerol and 0.5 per cent NaCl.

Iodometric Method for Activity of Penicillinase.

Pure cultures of S. aureus and M. smegmatis were streaked for isolation on to nutrient agar plates containing 0,2 per cent soluble starch and 1 unit per ml of penicillin

G, After overnight incubation, each plate was flooded with

3 ml of freshly prepared phosphate-buffered saline (pH 6.4) containing iodine (3 mg per ml), potassium iodide (15 mg per ml) and aqueous penicillin G (50 units per ml) and observed after 30 minutes for the characteristic decolor- ization that occurred around penicillinase-producing colonies. 27

Detection of Penicillinase Activity by a Bio-Assay i i _ . r ~ . ti----r i irri.i ~ ~ i I ~ ~ 1 i - ■ i - m m -rn m w * «i m i —— ' i i i

Method, Penicillinase activity was determined by a bio­

assay for residual penicillin in a cell free suspension.

An agar-supported column was made by slanting tubes

horizontally which contained TSA, One militer of the

solution to be assayed for residual penicillin was pipetted

into the bottom of the diffusion tube and allowed to diffuse

for 24 hours at room temperature. The liquid was then

removed with a sterile pipette and the TSA column inoc­

ulated with a 1:20 dilution of a 24 hour culture of a

penicillin sensitive S. aureus, After overnight incubation

at 37 C, the column was examined and the zone of inhibition

of growth, due to the upward diffusion of penicillin, was

measured between the foot of the column and the point of

confluent growth of the penicillin sensitive S. aureus.

When the penicillin was inactivated or destroyed by

penicillinase activity in the cell filtrate, the zone of

inhibition observed was shorter than that in a control

tube, which contained an equivalent concentration of

penicillin but no cell filtrate.

Isolation of D-Cycloserine Resistant Mutants of

S, aureus, D-CS resistant mutants of S. aureus strain U9W

were isolated as follows: The parent strain of the

bacterium, sensitive to at least 1 ug per ml of D-CS, was

inoculated and incubated on TSA slants for 18-20 hours at

37 C, The growth on the slant then was harvested into one 28 ml of Trypticase Soy broth (TS-broth, Baltimore Biological

Laboratory), One tenth ml samples of the suspension then

were seeded onto the surface of the D-CS synthetic medium

containing 1 ug per ml of the antibiotic, incubated at 37 C

and observed daily for growth of single colonies. Nine­

teen D-CS resistant mutant colonies were recovered 72 hours

post-inoculation. The mutants were retested on fresh D-CS

synthetic media containing 1, 5, 10, and 20 ug per ml of the

antibiotic and phage typed to insure their derivation from

the parent strain. Fresh medium was prepared and stored at

4 C for a maximum of one week.

Determination of Spontaneous Mutations, To determine

the spontaneous mutation rate of the penicillin and D-CS

resistant strains of ««■S, ■aureus i ■ - m and M, ■■ smegmatis w»i i t it in n i in i" to drug

sensitivity, the strains obtained from cloned cells were

inoculated into appropriate broth media which v?ere

incubated at 37 C, S, aureus was inoculated into TS-broth

and incubated for 18-20 hours, whereas M. smegmatis was

grown in Middlebrooks 7H9 medium (Difco, Detroit) on a

magnetic stirrer for 24 hours. Constant mixing with a

magnetic stirrer insured proper aeration of the myco­

bacterial culture. Appropriate dilutions of the cultures

were prepared, and 0,1 ml amounts were streaked onto TSA

plates supplemented with 5 per cent glycerin (TSA-G),

Following incubation for 24-48 hours at 37 C, master plates

containing less than 200 colonies were replicated onto 29

TSA-G plates containing 1 unit of penicillin per ml or

D-CS synthetic medium containing 10 ug per ml of the antibiotic. Colonies which did not grow on the media containing either penicillin or D-CS were retested, employing the original colonies on the master plates, for sensitivity to those agents and were phage-typed to insure their derivation from the parent strains.

Acridine Orange and Temperature Treatment. Acridine orange (AO, Matheson Coleman and Bell, Cincinnati, Ohio) was dissolved in distilled water to a concentration of 500 ug per ml and maintained as a stock solution in the dark at

4 C. Tubes containing 10,000 units per ml of penicillin or

5,000 ug per ml of D-CS were prepared separately and stored

in a -20 C freezer for a maximum of two weeks.

Procedures for determining the effect of AO and ele­ vated temperatures upon penicillin and D-CS resistance in

§> a^reus strain U9W and M. smegmatis strain 607 B were as

follows: Penicillin and D-CS resistant cultures of strain

U9W were inoculated into TS-broth containing 10 ug of acridine orange per ml, whereas strain 607 B was grown in

Middlebrooks 7H9 medium containing 10 ug of AO per ml.

Prior to inoculation the medium was adjusted to pH 7,3 or

7.6, using 1 N NaOH,, The cultures were subsequently

incubated in light for 18-20 hours. The procedures for exposing cultures to elevated temperatures consisted of

the following. S. aureus strain U9W was inoculated into 30

TS-broth and incubated at 44 C in a water bath for 18-20 hours, M , smegmatls strain 607 B was inoculated into

Middlebrooks 7H9 medium and incubated at 44 C on a magnetic stirrer with an attached heating element.

Six cultural conditions were employed for obtaining mutants of strains U9W and 607 B: 1) Control cultures

inoculated into the approriate medium for each strain and

incubated at 37 C. 2) Cultures inoculated into the

appropriate medium containing 10 ug per ml AO, previously

adjusted to pH 7.3 and incubated at 37 C. 3) Cultures

inoculated into appropriate medium containing 10 ug per ml AO previously adjusted to pH 7.6 and incubated at

37 C. 4) Cultures inoculated into appropriate medium and

incubated at 44 C. 5) Cultures inoculated into

appropriate medium containing 10 ug per ml AO previously

adjusted to pH 7.3 and incubated at 44 C. 6 ) Cultures

inoculated into appropriate media containing 10 ug AO

per ml adjusted to pH 7,6 and incubated at 44 C.

Following incubation, dilutions were prepared and 0.1 ml aliquots were plated on TSA plates and incubated for 18-

48 hours at 37 C. Master plates were subsequently repli­

cated onto the appropriate medium containing either 1 unit

of penicillin per ml or 10 ug per ml of D-CS. Colonies on

the master plate which did not grow on the penicillin or

D-CS medium were retested on fresh antibiotic medium and

phage typed to insure derivation from the parent culture. 31

Mutants of strain U9W were maintained on TSA slants at 4 C,

Subcultures of the mutants were prepared at monthly inter­ vals and each transfer was phage typed.

Induction of Mutants by Ultraviolet Irradiation. The germicidal lamp G15T8 (General Electric Company), emitting light primarily (more than 74 per cent) of 2537 Angstroms served as the source of ultraviolet irradiation. The distance of the lamp from the bottom of the dish was

15 oil. From this working distance the G15T8 lamp yielded 2 the intensity of 80 erg/mm /sec. (Germicidal Lamps, General

Electric Manual TP 122, 1969),

A culture in the exponential phase of growth (24 hours incubation) was centrifuged and twice washed with sterile

0.85 per cent NaCl solution. Three ml portions of the washed suspension, giving a 1 mm layer, were transferred to sterile plastic Petri dishes and each sample was sub­ jected to the required dose of ultraviolet irradiation

(120 seconds). Following irradiation, 0.1 ml samples were streaked on TSA-G plates and incubated at 37 C for 48 hours or until isolated colonies appeared. Master plates containing less than 200 colonies were replicated onto TSA plates containing 1 unit of penicillin and/or D-C3 syn­ thetic medium plate3 to which 10 ug per ml of the antibiotic had been added.

Control samples were treated in the same manner before irradiation was begun. All procedures including 32 irradiation and post-irradiation treatment were carried out under yellow light (100 VI insect repellent lamp) in order to prevent photoreactivation.

Propagation and Titration of Bacteriophages. The bacteriophage of S . aureus strain U9W used in this study was phage 80 of the International Typing Series. The phage was obtained from a collection of staphylococcal phages in the Department of Biology, Bowling Green State

University, Phage 80 was also used as the transducing phage after propagation on the appropriate penicillin and

D-CS resistant donor strain. Phage 80 propagated on strain U9V/ was designated as phage 80/U9W,

The mycobacteriophages employed were kindly supplied by Dr, VI.B, Redman, Veterans Administration Hospital,

Atlanta, Georgia and had been previously shown to be lytic for the host bacterium 607 B. These phages included D29,

R^, DS6A, GS4, and GS7. Phage D29 propagated on strain

607 B was designated as D29/607 B; R^/607 B; DS6A/607B;

GS4/607 B; GS7/607 B.

The bacteriophage were propagated by a modification of the soft-agar overlay method. The propagating strain of

S, aureus U9W was inoculated and incubated on TSA slants for 18 to 20 hours at 37 C. The growth on the slant then was suspended in one ml of TS-broth. The propagation strain of M. smegmatis 607 B was inoculated in Middle- brooks 7h 9 medium and incubated at 37 C for 18-20 hours on a magnetic stirrer. Five ml of the broth culture was centrifuged for five minutes at 3,000 rpm in a Sorvall centrifuge. Following centrifugation, the clear super­ natant was removed and the cells resuspended in 1 ml of fresh Middlebrooks 7H9 medium.

A 0,2 ml aliquot of the appropriate bacterial sus­ pension was added to two ml of melted and cooled soft agar containing 0.4 per cent Bacto agar. In addition,

0.15 ml of a phage lysate was added to the cooled soft agar. The cell suspension and the phage lysate were gently mixed and pured over the surface of a TSA plate for strain U9W or mycobacterial bottom agar for strain

607 B. Following incubation at 37 C for 6 hours or at room temperature for 12 hours, the soft agar was sus­ pended in 5 to 7 ml of TS-broth and centrifuged at 3,000 rpm for 10 minutes. The supernatant fluid was removed and filtered through a 0.45 u Millipore filter. The resulting filtrate of phage was stored at 4 C in sterile screw- capped tubes.

To determine the titer of phage in the filtrate, serial ten fold dilutions of the phage suspension frcm

— 1 -8 1 x 10 through 1 x 10 were prepared in nutrient broth containing 0.01 per cent 1 M CaC^ (NB3). From each of the dilutions, 0 .0 . ml was deposited onto different sections of the survace of a TSA-G plate previously inoculated with the appropriate propagating strain of 34 bacteria. After the inoculum had absorbed, the plate was incubated at 37 C for 18 to 20 hours. The titer of the phage suspension was calculated from the number of plaques formed on sectors where 1 x 10" through 1 x 10“ dilutions had previously been deposited.

Transduction Technique, Bacteriophage 80 of strain

U9W was propagated on appropriate penicillin and D-C3 resistant donor strains and various penicillin and D-C3 sensitive mutant strains were used as recipients, A phage suspension containing approximately 2 x 10^ plaque forming g units (PFU) per ml was combined with approximately 1 x 10 sensitive cells. The mixture was incubated at 37 C for one hour and 0,05 ml amounts were inoculated onto the surfaces of media containing either penicillin or D-CS and spread with sterile bent glass rods. Penicillin transductants were selected by spreading the transduction mixture onto the surface of Brain Heart Infusion agar plates (BHI, Difco) containing 0,15 units of penicillin per ml, A concen­ tration of 1 ug per ml of D-CS was added to the synthetic medium for transduction purposes to recover D-CS resistant recombinants. Incubation of the penicillin and D-CS plates was at 37 C for 24-48 hours.

Harvesting of Cells for DNA Extraction, A list of the reagents employed are found in Table 2, Strain U9W of

S. aureus was grown in a 1 liter volume of TS-broth at 37 C for 18 to 20 hours. Strain 607 B of H. smegmatis was grown TABLE 2

REAGENTS USED IN DEOXYRIBONUCLEIC ACID EXTRACTION PROCEDURES

Reagents Concentration

Saline-EDTA 0.15 M NaCl plus 0.1 M ethylenediaminetetraacetate pH 8 Sodium Lauryl (NaCi2^26s®4)» ^5 Per cenC Sulfate Lysozyme 3X crystalized, 10 mg Sodium Chloride 5 M solution, pH 7.2 Chloroform- 24:1 (v/v) isoamyl alcohol Ethyl Alcohol 95 per cent Dilute-Saline 0.015 M NaCl plus 0.0015 M Citrate Trisodium Citrate. pH 7.2 Standard-Saline 0.15 M NaCl plus 0.015 Citrate Trisodium Citrate, pH 7.2 Concentrated- 0.285 NaCl plus 0,0285 Saline Citrate Trisodium Citrate, pH 7.2 Acetate-EDTA 3,0 M Sodium Acetate plus 0.001 M EDTA, pH 7 Ribonuclease crystalline, in 0.15 M NaCl pH 5.0 Isopropanol 36 in a 1.5 liter volume of Middlebroolcs 7H9 medium for 48 hours at 37 C on a magnetic stirrer. The harvesting procedure was designed to yield from 2 to 3 g, of wet packed cells. Bacteria were harvested by centrifugation and washed once with 50 ml of Saline-EDTA, Flask cultures were examined for purity by staining smears with acid fast and Gram stains before continuing with DNA extraction procedures.

DNA Extraction and Determination of DNA. A modified method of Marmur^ was used for cell disruption and DNA extraction. Following the harvesting of cells, the cells were suspended in a total volume of 20 to 30 ml of saline-

EDTA. Lysis was effected by the addition of 10 mg of lysozyme (3x crystallized, Calbiochem) to the mixture with incubation in a 42 C water bath for 18 hours. A 10 mg sample of pronase (B grade, Calbiochem) then was added to the mixture which was further incubated at 42 C for three hours. Subsequently 1.5-2.0 ml of sodium lauryl sulfate were added and the mixture incubated for one additional hour in a 60 C water bath. Following cooling of the mixture to room temperature, lysis was observed by an increase in viscosity of the mixture, accompanying the release of the nucleic acid components.

Sodium chloride was added to a final concentration of

1 M to the viscous lysed suspension with the entire mixture being shaken (wrist action shaker) with an equal 37 volume of chloroform-isoamyl alcohol in a ground-glass stoppered flask for 20 minutes. The emulsion was separated into three layers by a 5 minute centrifugation at 10,000 rpm in a Sorvall RC2-B refrigerated (2 C) centrifuge. The upper aqeous phase was removed by a 1 ml pipette fitted with a rubber bulb and carefully placed in a 25 ml graduate.

The initial precipitation technique of the nucleic acid with ethyl alcohol was varied for both S. aureus strain

U9W and M. smegmatis strain 607 B as follows. For strain

U9W approximately two volumes of cold ethyl alcohol were gently layered on the collected aqueous phase and allowed to precipitate overnight followed by collection of the DNA with a 5 minute centrifugation at 10,000 rpm. In the precipi­ tation procedure for strain 607 B an equal volume of cold ethyl alcohol was vigorously added to small volumes (5 ml) of the aqueous phase. The method was changed with both strains as the layering technique employed by Marmur^ did not work with these two strains. The precipitated nucleic acid was immediately removed by winding the thread-like precipitate onto polished glass rods.

The precipitates from both strains then were trans­ ferred to approximately 5 ml of dilute saline citrate for dispersion following which the solutions were adjusted to standard saline citrate by adding equal volume of concen­ trated saline citrate, shaken as before with an equal volume of chloroform-isoamyl alcohol for 20 minutes, 38 centrifuged, and the supernate removed. The supernatant fluid was repeatedly deproteinized with chloriform-isoamyl alcohol until very little protein was seen at the interface.

Following the last series of deproteinizations, the supernate was precipitated with ethyl alcohol and dispersed in saline citrate (one half the supernatant volume).

Ribonuclease was added to a final concentration of 50 ug per ml and the mixture incubated for 30 minutes at 37 C.

Following another deporteinization the supernate again was precipitated with ethyl alcohol and dissolved in 4,5 ml of dilute saline citrate. After solution had occurred, 0.5 ml of acetate-EDTA was added and while the solution was stirred rapidly, 0.27 volume of isopropyl alcohol was added drop- wise until the DNA threads precipitated out of solution.

The precipitated DMA was washed once in 70 per cent ethyl alcohol and dissolved immediately in 5 ml of dilute saline citrate which was then brought up to standard saline citrate by adding an equal volume of concentrated saline citrate. All samples were used within 18-20 hours following extraction.

The final dispersed solution of DNA was examined spectrophotometrically to estimate the quantity of DNA and amount of protein present. A 50 ug per ml sample of salmon sperm DNA was used as the standard sample comparing the absorbancy ratios reading at 260 mu and 280 mu. Samples were placed in 1 cm quartz cuvettes and read on the 39

Shimadzu Recording Spectrophotometer.

Determination of DNA Base Composition. Equilibrium ultracentrifugation in a density gradient was employed in 90 this study. The method of Schildkraut et al. was employed to determine the DNA base composition. A concen­

trated cesium chloride (optical grade, Harshaw Chemical

Company) stock solution was prepared by dissolving 10 g of cesium chloride in 5.5 ml of 0.02 M tris-buffer

adjusted to pH 8,5. To 0.84 ml of the prepared concen­

trated cesium chloride stock solution, 0.23 ml of a standard

saline citrate solution containing 5 to 10 ug of test DNA and 0.5 ug of a standard reference DNA (E. coli K 12) were

added. The resulting solution had a buoyant density (^)

of 1.710 to 1.713 as calculated from its refractive

index 0l) by the following equation:

^>25.0 C = l O ^ O l ^ D 25,0 G - 13.4974

Approximately 0.75 ml of the DNA-cesium chloride

solution was placed in a 12 mm 4° sector centrifuge cell having a Kel F centerpiece and plain quartz windows. The

sample was centrifuged in a Spinco Model E analytical

ultracentrifuge (AnD rotor) at 40,000 rpm at 20.0 C. An

initial ultraviolet absorption photograph was taken when

full speed was reached (zero time picture) with subsequent

photographs taken at 2 hour 8 minute intervals for 20 to

24 hours on Kodak commercial film with 25 sec. exposure 40 time. Exposed films were developed in Kodak D 19 developer for 12 minutes and fixed in Kodak Fixer for 10 minutes.

After washing for 30 minutes the film was bathed for 30 seconds in diluted Photo-Flo Solution (Kodak), drained briefly and allowed to dry. Tracings of the films were subsequently made on a Beckman analytrol densitometer.

Test DNA densities were calculated from the position of the reference DNA by the following equation:

? = ? Q + 4 ‘2U)2 ( r2 - rQ2 ) x 10'10 g cm*"3

= density of the standard DNA

& = speed of rotation in radians sec*"*

r = distance of the standard DNA from center of rotation

r = distance of the unknown DNA from the center of roation

19 DeLay reports an association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. The relation has been shown to be valid for animal, plant, and viral DNA. The following equation permits calculation of:

GC-content: % GC = 1020.6 ( - 1.6606 ) RESULTS

Culture Characteristics and Techniques for Culti­ vation of M. smegmatis 607 B and S. aureus U9W. Imperative to the genetic studies was the characterization of the test organisms. Preliminary work was concerned with growth characteristics of the mycobacterial strain in a liquid medium in an attempt to eliminate pellicle for­ mation which would limit its usefulness in phage 12 propagation, resulting in anomalous adsorption patterns* and mutagenic work where clumping of cells have been shown to affect the susceptibility of this organism by limiting 52 53 the accessability of single cells to various agents. *

Cultural characteristics, such as colonial morphology, were investigated due to reports indicating changes of these characteristics spontaneously at low frequencies.

It was necessary to establish the spontaneous frequency with which these occurred in comparison to the rate which may have been induced by the mutagens.

Colonies of M. smegmatis 607 B exhibited serrated margins and irregular surfaces when cultivated on semi­ solid agar plates of TSA plus glycerin and D-cycloserine synthetic medium and also Lowenstein-Jensens medium

(Plate 1), A total of 2,500 colonies were visualized by

41 42

Plate 1. 51 • sraegmatls 607 B. Typical rough

colony formation on TSA after 72 hours

incubation at 37 C in the dark. subculture onto 150 agar plates of each type of medium and without exception all colonies demonstrated this same morphology; i.e. no smooth colonies were observed. At an incubation temperature of 37 C the strain grew well, with isolated colonies arising two to three days post­ inoculation; at room temperature, growth was slower with individual colonies appearing in six to seven days.

Colonies grown in the dark were buff colored, but turned orange on prolonged cultivation in the light as illustrated in Plate II. No white variants were noted, as have been reported, when colonies were examined for change in pigmentation. Further, no cord formation was observed on examination of smears stained with acid fast stains. The organism reacted Gram positively and stained irregularly, providing a banded appearance.

Various broth media were employed for growth charac­ teristics of the organism in a suspension devoid of Tween

80. This was necessary due to the effect of Tween 80 upon adsorption of phage to the bacterial cells. These media included TS-broth, TS-broth plus 5 per cent glycerin, nutrient broth and nutrient broth plus 5 per cent glycerin,

Brain Heart Infusion (BHI) and BHI plus 5 per cent glycerin, and Middlebrooks 7H9 broth supplemented with glucose and Bovine Serum Albumin (Fraction V). Extensive pellicle formation was observed in all broth cultures, rendering them unsuitable for mutagenic work and phage 45

Plate II. M. smep^matls 607 B on Lowenstein-

Jensen medium. Both tubes were

initially incubated for three days at

37 C in the dark. Left: absence of

orange pigment when grown in the dark.

Right: orange pigment produced by

subsequent incubation in sunlight.

47 propagation in which uniform broth cultures are desirable.

Aeration of these broth cultures with an air stone was not satisfactory because of excessive foaming of the media.

Qefoaming agents were not investigated because of their questionable effect upon adsorption of phage to host cells.

Also, air was bubbled through a hollow glass tube, at the rate of 120 bubbles per minute, to which was attached a sterile glass wool filtering unit. Although relatively uniform broth cultures were achieved this required an extension of incubation (96 hours). The additional use of a magnetic stirrer, however, resulted in homogeneous

cultures obtained in 18-20 hours post-inoculation. The medium of choice was Middlebrooks 7H9 broth since it

afforded the most luxuriant growth for this period.

Colonies of S, aureus U9W were smooth and glistening with entire edges. Standard laboratory media, such as

TSA and BHI, afforded luxuriant growth with isolated

colonies at 24 hours at 37 C or at room temperature.

Homogeneous broth cultures were obtained without aeration

or the addition of surfactants. Colonies of the penicillin

resistant parent strain exhibited a golden color on both

TSA and D-cycloserine synthetic medium, while the penicillin

sensitive variant was white (Plate III). The difference in

pigmentation was not investigated.

The amino acid requirements of both organisms were

tested by using a medium consisting of the components 48

Plate III. S, aureus U9W on TSA, demonstrating

differences in pigmentation. Above:

Gold pigment exhibited by penicillin

resistant variant. Belov?: No pigment

exhibited by penicillin sensitive

variant,

50 previously listed in Table 1 under Materials and Methods,

The media were varied by eliminating amino acids singly and observing for growth of isolated colonies at 24, 48, and

72 hours. The results are represented in Table 3,

S. aureus U9W required the following amino acids:

L-histidine, L-leucine, L-phenylalanine, and L-threonine, whereas M, smegmatls 607 B required only L-asparagine.

Bacteriophages: Host Range. Plaque Formation and

Propagation, It has been shown that Tween 80 prevents the propagation of mycobacteriophage D29,*"^ Further it has been reported that various diluents, employed in the titration of raycobacteriophagefresult in irreversible inactivation of certain phages,^® Since difficulty was encountered in the early studies on the propagational technique it was necessary to investigate the effect of various external agents on the cells and bacteriophages.

Mycobacteriophage3, While attempting to propagate phage for routine typing in the laboratory, it was observed that the bacteria grew profusely with minimal lysis on media with Tween 80, When M, smegmatls 607 B and myco­ bacteriophage D29 were incubated for propagation of phage in media containing varying concentrations of Tween 80,

0,002 per cent or greater, little or no increase in phage titer occurred upon harvesting of the phage over the 2 original phage titer which was 4 X 10 . At concentrations of the Tween below this per cent value (0,002), phage 51

TABLE 3

AMINO ACID REQUIREMENTS FOR M. SMEGMATIS 607 B AND S. AUREUS U9W IN A SYNTHETIC MEDIUM

§ .• aureus U9W M, smegmatis 607 B Hours Post-Inoculation 24 48 72 24 48 72 Amino Acid Eliminated

L-alanine +a + + _ b + + L-arginine + + + - + + L-asparagine + + + - -- L-aspartic acid + + + - + + L-cystine + + + - + + L-glutamic acid + + + - + + Glycine + + + m m + + L-histidine mm __ + + L-isoleucine + + + - + + L-leucine -_ m • - + + L-lysine + + + mm + + L-methionine + + - + + L-phenylalanine mm -- mm + + L-proline + + + - + + L-serine + + + — + + L-threonine —_ • - + + L-tryptophan + + + - + + L-tyrosine + + + - + + L-valine + + + — + +

£ k + = growth with isolated colonies - = no evidence of any growth 52 titers approximated those systems without Tween 80 present

(Fig. 1). Of the other mycobacteriphages tested, slight inhibition of phage propagation by Tween 80 may be indi­ cated on phages GS4 and GS7, however marked inhibition of propagation by Tween 80 was demonstrated on phage DSP^

(Table 4).

During several phage titrations, the inactivation effect of several diluents on the mycobacteriphages was observed. Preliminary studies indicated that diluting of phage in twice-distilled demineralized water, or , to even a greater degree with physiologic saline, resulted in inactivation of the phages as indicated by a reduction in plaque forming units (infectivity). Parallel studies utilizing nutrient broth plus 0.002 M CaC^ (N33 broth) indicated that phage infectivity was fully retained.

These adverse effects were observed with mycobacteriphages

GS4, G37, and DSP^ although phages D29 and LEO did not seem greatly, if any, affected. Subsequently, titration of all phages was carried out employing NB3 medium.

A list of hosts and the host ranges of the various mycobacteriophages empbyed in this study is found in Table

5. It was necessary to determine the phage typing pattern of each Mycobacterium strain since this pattern would subsequently have been used throughout the study for identification procedures if mutants had been obtained.

A 10*^ titer of each stock phage solution was employed in 53

Figure 1, Effect of Tween 80 in varying concen­

trations on the propagation of

mycobacteriphage D29 with host

Jl* sniegmatls 607 B as indicated by a

drop in phage titer with increase in

per cent of Tween 80 incorporated into

the culture medium. Log Phage Titer Per Ml 10 11 0.00 9- - - .02 000 001 0.002 0.001 0.0005 0.00025 e Cn Ten 80 Tween Cent Per .0 0.008 0.004 54 TABLE 4

TITER OF VARIOUS MYCOBACTERIOPHAGE AFTER SIX HOURS OF INCUBATION FOR PROPAGATION AT 37 C WITH M. SMEGMATIS 607 B IN VARYING CONCENTRATIONS OF TWEEN 80

Per Cent Tween 80 0.00 0.00025 0.0005 0.001 0.002 0.004 0.008 Phage

LEO 2Xl09a 3X10 2X10 8X109 10XI08 1X109 12X108 GS4 6X1010 4X1010 3X1010 10X109 9X1010 4X109 1X108 GS7 4X1010 1XI010 3X1010 6X1010 8X109 1X108 3XL07 d s p l 7X109 8X109 1XI010 3X107 6X106 4X103 7X102

atiter of phage in plaque forming units TABLE 5

PHAGE TYPING PATTERNS EXHIBITED BY VARIOUS MYCOBACTERIOPHAGES ON SEVERAL STRAINS OF MYCOBACTERIA EMPLOYING THE SPOT-PLATE METHOD

Phages

D29 LEO GS4 GS7 DSP^l 11727-B 11728-B 11758-B 11759-B Strains

_b M. tuberculosis * a * — - am mm - — (BCG) Tice strain

M. smegmatis +c + + + + - - - m m 607 B

M. smegmatis * - mm - - + - am - 11727 ATCC

M. smegmatis * mm am - - + m m am 11728 ATCC

M. smegmatis - - mi ------14468 ATCC

M. phlei * - - - - - + - 11758 ATCC

M. phlei — — — —- mm am — + 11759 ATCC

?* = semi-confluent lysis - = no lysis c+ = confluent lysis 57 the testing. It is apparent that the phages have specific affinities for each of the strains of bacteria tested.

Phage D29 appears to be a broad spectrum phage in that it has the ability to lyse, to varying degrees, diverse groups of mycobacteria such as M. tuberculosis (BCG) Tice strain, M. smegmatis. and M, phlei. A duplicate series of experiments confirmed the phage-typing pattern demon­ strated by each strain of bacteria.

The plaques produced by the different phages specific for M. smegmatis 607 B varied considerably in size from pin-point plaques for LEO phage to large three mm. plaques for DSP^ phage. Some plaques were relatively clear as exemplified by phages D29, DSP^, and LEO, whereas others were turbid as in the cases of phages GS4 and GS7. Each phage was a single true breeding type, with no variants being observed, A total of 1,000 plaques were observed for each mycobacteriophage tested in a single experiment.

Duplicate platings from the same and different stock sample confirmed these findings. The plaque types for the five phages are illustrated in Plate 4.

Staphylococcal phage 80, Propagation of phage 80 was carried out using the soft agar overlay method which employed routine laboratory media (TSA and TS-broth) and gave consistent reproducible values in phage titration. No phage inactivation effects were noted when various diluents

(TS-broth, tvice-distilled demineralized water, or 58

Plate IV, Plaques of various mycobacteriophages

plated on H, smegmatis 607 B (Redman

Overlay Method), (a) D29 (b) LEO

(c) G34 (d) G37 (e) DSP1

60 or physiologic saline) were used in the titration pro­

cedures. Lysis with phages 80 and 81 occurred with both the penicillin resistant and sensitive variants. Neither o.f these two strains was susceptible to lysis by the various mycobacteriophage3, and conversely, none of the various mycobacteria strains were susceptible to either phage 80 or 81. Plate V demonstrates the lysing of the penicillin resistant variant of strain U9W of S. aureus with phages

80 and 81, but no lysis or even partial lysis is evident with any of the mycobacteriophages specific for

sinegmatis 607 B,

Methods for Detection of Penicillinase Activity.

Evidence for the elaboration of the enzyme penicillinase was important in establishing the basis for resistance to

the antibiotic penicillin in both the mycobacterial and

staphylococcal strains employed in this study. Organisms which do not elaborate this enzyme lose their resistance and revert, to a large extent, to original penicillin

sensitivity if subcultured in the absence of penicillin.^

Such organisms would not be suitable for genetic studies of

the determinant governing resistance to penicillin,

lodometrlc Method for Detection of Penicillinase

Activity. When plates for bacterial cultures of S. aureus

U9W were flooded with phosphate buffered saline (pH 6,4),

containing iodine, potassium iodide and aqueous penicillin

G, characteristic decolorization occurred around the 61

Plate V. Phage susceptibility of S. aureus U9W

(penicillin resistant variant) to

staphylococcal and mycobacteriophages.

(a) staphylococcal phage 80 (b) staphy­

lococcal phage 81 (c) mycobacteriophage

DSP^ (d) mycobacteriophage GS7 (e) myco­

bacteriophage GS4 (f) mycobacteriophage

LEO (g) mycobacteriophage D29. Arrows

point to areas where phage suspension was

added but wherein no lysis occurred. 62 penicillinase producing colonies. The surface of the agar developed a bluish-purple color from the formation of starch iodine complexes, but the colonies retained their usual pigment. Within five to ten minutes the area immediately surrouding penicillinase producing colonies became decolor­ ized and a transparent colorless zone was apparent. This probably was attributable to the action of penicilloic acid with iodine, forming complexes which lead to dissociation of the blue starch iodine complexes. Testing indicated that the penicillin resistant variant of S. aureus U9W was a penicillinase producer. The results of the test are illustrated in Plate VI, No such zones appeared around penicillin eensitive colonies and thus this technique constitutes another method for distinguishing the two variants. Extracellular penicillinase production by the mycobacterial strain was also indicated by this test however the zones of clearing were minimal in comparison to those produced by the staphylococcal strain.

Detection of penicillinase activity by a bio-assay for residual penicillin. The results of this experiment are summarized below. The following formula was used to calculate the per cent reduction of the zone of inhibition of a penicillin sensitive organism by residual penicillin remaining in broth in which a penicillinase producing 21 organism had previously been grown. 64

plate VI. Agar-plate modification of the iodo-

metric assay for production of

penicillinase in the penicillin

resistant variant of S. aureus U9W,

66

ram Inhibition control - mm inhibition test ------x 100 mm inhibition control

The length of the zone of inhibition recorded for the control tube was 35 mm. No zone of inhibition of the penicillin sensitive variant of S. aureus was observed when filtrate from a penicillin resistant variant was employed of S. aureus. This indicated the penicillin in this culture was destroyed by the action of penicillinase.

A zone of inhibition approximately two mm in length was recorded for the filtrate removed from the penicillin resistant M. smegmatis. Using the above formula, the per cent reduction of the zone of inhibition was 94 per cent. This indicated that not all of the initial penicillin in the broth culture was broken down by the mycobacterial strain, thereby inhibiting to a limited degree the penicillin sensitive strain of S. aureus.

(Plate VII)

Characterization of D-cycloserino resistant call3.

The influence exerted by D-alanine, present in certain commercial media, in antagonizing the action of D- cycloserine has been demonstrated by various investigators.^ 21 39 40 * * Autoclave sterilization of synthetic medium containing L-alanine has also been shown to subvert the inhibitory action of D-cycloserine by racemization of

L-alanine to D-alanine.These reports necessitated 67

Plate VII. Detection of penicillinase activity

determined by a bio-assay method, using

a penicillin sensitive variant of

S. aureus for noting inhibition by

residual penicillin, (a) filtrate

containing 50 units of penicillin per

ml (note zone of inhibition) (b)

filtrate of TS-broth only (c) filtrate

in which penicillin resistant S. aureus

was grown (d) filtrate in which

penicillin resistant M. smegmatis was

grown (e) filtrate in which penicillin

resistant variant of S. aureus was

grown with 50 units of penicillin per ml

in TS-broth (f) filtrate in which

penicillin resistant M. smegmatis wa3

grown with 50 units of penicillin per ml

in TS-broth (note minimal inhibition). 68 69

studies to establish what effect L-alanine exerted on resistance to D-cycloserine demonstrated by the test organisms.

The organisms used throughout this work were initially tested for drug resistance to D-cycloserine, using

standard laboratory media (TSA and BHl-agar) to which was added, subsequent to sterilization, the appropriate amount

of the antibiotic. The penicillin resistant and sensitive variants of S. aureus U9W and M, smegmatls grew on the two media containing a maximum of 50 ug per ml of D-cycloserine.

Plates of S. aureus were observed for growth at 24 hours post-inoculation, while the mycobacterial strain was observed for growth 48 hours post-inoculation. When these

same organisms were streaked for isolation on D-cycloserine

synthetic medium devoid of L-alanine, growth was not

evident for either of the two variants of staphylococci even at the lowest concentration of antibiotic used.

Incubation for an additional 48 hours failed to yield any

growth. Growth did occur however on the same synthetic medium in the absence of D-cycloserine at the end of the

initial 24 hours of incubation. These data are summarized

in Table 6.

Resistance to the antibiotic D-cycloserine also was tested employing the tube dilution method. Similar results were obtained when resistance was measured by employing various broth media with growth being quantitated TABLE 6

DEGREE OF RESISTANCE TO D-CYCLOSERINE DEMONSTRATED BY VARIOUS ORGANISMS ON SEVERAL TYPES OF SEMI-SOLID LABORATORY MEDIA

D-cycloserine: ug per ml 0.0 0.5 1 5 10 20 30 40 50 60 100 Organism Medium

b S . aureus TSA +a + + + + + + + + mti Pase + BHI-agar + + + + + + + + + - D-CS + Synthetic

S . aureus TSA + + + + + + + + + BHI-agar + + + + + + + + + D-CS + Synthetic

M. smegmatis TSA + + + + + + + + + + + BHI-agar + + + + + + + + + + + D-CS + + + + + + + + + + + Synthetic

a+ = well isolated colonies following incubation of 24 hours for S. aureus variants and 48 hours for the mycobacterial strain k- = no visible growth following incubation for 72 hours 71 turbidimetrially (as per cent transmission). These results are illustrated in Fig, 2. For each experiment, an overnight broth culture was diluted with fresh medium so that each culture tube contained approximately 10,000 bacteria per ml of final medium.

Resistance to D-cycloserine was demonstrated by the mycobacterial strain when grown in synthetic medium devoid of L-alanine, as well as the other two media employed. The resistance to D-cycloserine demonstrated by the myco­ bacterial strain occurred in all three broth cultures in the presence of D-cycloserine, The resistance to this antibiotic did not seem to be altered by the various cultural conditions employed and therefore it was felt that the strain was in fact resistant to the antibiotic D- cycloserine.

The graph (Fig, 2) illustrates that the inhibitory action of D-cycloserine was antagonized in TS-broth and

BHI allowing for growth of the S. aureus variants. From the results of these experiments, it seemed probable that the apparent resistance to D-cycloserine, initially encountered when the penicillin resistant and sensitive variants of strain U9W of S. aureus were tested using conventional cultural media (TSA and BHI-agar) in this study, was actually a reflection of the presence of

D-alanine in these media. Growth of these two variants 72

Figure 2. The effect: of various broth media on

the degree of resistance to D-

cycloserine demonstrated by S, aureus

(penicillin resistant), S. aureus

(penicillin sensitive), and H, smegmatis

607 B. /00 *3 f*3

9 0 *1“*—T Uninoculated Media 8 0 1*2

TO

. 'N 6 0 V

SO

4 0

_1 TS-broth 30 2 BHI J3 D-C3 Synthetic 20 medium

/ 0 S. aureus U9W ( P ;S. aureus U9W (P+ ) S. aureus U9W ( P ;S. M. smegmatis 607 B

0 .S' / S 90 20 30 40 SO 60 /CO 0 .5 / S /O 30 30 40 SO 60 /00 0 .5 / S /0 20 30 40 SO 60 /OO D-Cycloserine (ug/ml) 74 did not occur when employing synthetic medium with the antibiotic. Synthetic medium supplemented with L-alanine, prior to autoclave sterilization, inhibited the anti­ bacterial activity of D-cycloserine on the two variants of

staphylococci employed (Fig. 3).

In three series of experiments, only single step low grade resistance to D-cycloserine developed in 19 colonies of the penicillin resistant variant of S. aureus U9V/. The highest concentration to which resistance was demonstrated

to the antibiotic was 10 ug per ml of D-cycloserine. No

single step mutations to high level resistance ware

encountered. These mutants occurred at a mean mutation

rate of 6 X 10“^,

The mutants were maintained on T5A-slants without any

selective agent and were transferred bi-monthly. A com­

parison of resistance properties of the original 19 mutants

was carried out after four transfers. Only seven of the

original 19 demonstrated the same degree of resistance as

they exhibited when originally isolated and appeared stable.

Based on these findings, these seven mutants were retested

at two week intervals for the duration of the study and were

found to be stable. As concentrations of D-cycloserine

were increased above 10 ug per ml, colonies became mucoid.

Lack of Reversal of D-cycloserine Inhibition by

Mycobactin. a Growth Factor for Specific Mycobacteria.

Reversal of the growth inhibitory effect of D-cycloserine 75

Figure 3. Antagonism of D-cycloserine inhibition

aureus growth by heated prepa­

rations containing L-alanine. Time in Hours 24 16 12 0 8 4 KEY: o o o O' Heat D-cycloserine L-alanine 0 uM/ml 60 5 uM/ml 25

o o

Transmission Turbidity VO % A, 0 0 0 0 0 B. 0 G_ 2 G/15* 121 D CM o o r-1 o has further been reported for the compound Mycobactin, an essential growth factor for Mycobacterium paratuberculosis«

Limited studies have been reported in Mycobactin reversal 99 of D-cycloserine inhibition in M. paratuberculosis but no such activity has been observed with M. phlei or 118 butricum. Inasmuch as no other related studies have been reported, the reversal activity of Mycobactin demon­ strated to D-cycloserine was tested on sensitive cells of

S . aureus.

In contrast to the results obtained with heated

L-alanine, Mycobactin was found to possess no D-cycloserine reversal activity, in strain U9W P D-CS of S. aureus.

Preliminary studies indicated that supplementation of

D-cycloserine synthetic medium containing D-cycloserine

(1, 5, and 10 ug per ml) with 1 ug per ml of Mycobactin, dissolved in methanol, did not reverse the inhibitory activity of D-cycloserine. This was shown by the failure of growth on the supplemented plates. The addition of increased levels of Mycobactin (5 and 10 ug per ml) failed to reverse the inhibition of growth by D-cycloserine.

However, growth of isolated colonies was evident on plates supplemented with Mycobactin, but to which no antibiotic was added, suggesting that neither the Mycobactin or the diluent (methanol) was inhibitory to the staphylococci.

Reversal of D-cycloserine inhibition by Mycobactin could not be tested in M. smegmatis 607 B since no sensitive mutants 78 to the antibiotic have been obtained.

Spontaneous Mutation Rates. No spontaneously arising mutants sensitive to penicillin and/or D-cycloserine in

M. smegmatis 607 B were observed. Studies with resistant mutant #7 recovered from strain U9W of S. aureus (peni­ cillin resistant) resulted in the recovery of three spontaneous revertants to D-cycloserine sensitivity. No sensitive cells were recovered from resistant mutants numbers eight or nine. Mutants number one, two, three, and sixteen were not tested. Loss of the penicillinase marker was not studied in the resistant mutants of S. aureus^ however the three D-cycloserine sensitive colonies recovered remained penicillin resistant, A total of 20,971 colonies of M. smegmatis 607 B and 21,163 total colonies of re­ sistant mutants seven, eight, and nine of S. aureus, U9V/ were tested (Table 7). These rates were obtained using cloned cells of each strain.

The Effect of Acridine Orange and Elevated Temperature on Penicillin and D-cycloserine Resistant Cells. The selective loss of extrachromosoma1 particles following treatment with acridine dyes and elevated temperature have 33 105 been reported. * Various drug resistance factors m

aureus have been "cured" following treatment with these 32 69 selective mutagens * although similar studies have not been reported for the mycobacteria. D-cycloserine and penicillin resistance were selected for study as TABLE 7

SPONTANEOUS MUTATION RATE TO DRUG SENSITIVITY OF D-CYCLOSERINE RESISTANT CLONED CELLS OF S. AUREUS U9W and M. SMEGMATIS 607 B

Total Number of Number of Per Cent of Organism Cells Tested Sensitive Cells Sensitive Cells

M. smegmatis 20,971 0 0.00 £07 B

S . aureus 5,976 3 0.05 U9W 4 7

S . aureus 6,742 0 0.00 U9W # 8

S . aureus 8,444 0 0.00 U9W $ $ 80 no mutagenic work to establish their extrachromosoma 1 nature has been undertaken with this or other drug

resistant factors in M. smegmatis 607 B.

§.• aur&us U9VJ. When strain U9W (Pase+ D-CSr) was

treated with acridine orange at a concentration of 10 ug per ml in light, it was observed that between 80-90 per

cent of the treated cells tested lost resistance to the

antibiotic D-cycloserine. This was characteristic of the

three resistant mutants tested, i.e. seven, eight, and nine. Resistant mutants one, two, three, and sixteen were not tested. As shown in Table 8 the frequency of loss of

resistance in strain U9W did not seem to be affected by

changes in pH of the acridine medium. Studies did not

indicate that a combina trion' of acridine orange and elevated

temperature increased the rate of D-cycloserine sensi­

tivity. However sensitive variants were noted when cells were exposed to elevated temperature (44 C) alone. There

does seem to be a difference in the frequency of loss of

the D-cycloserine resistant determinant between the two mutagens employed. Although loss of the penicillinase marker was not studied, limited studies revealed that

approximately 2 per cent of the D-cycloserine sensitive

cells were also sensitive to penicillin following acridine

orange and elevated temperature treatment. More definitive

studies are needed before such a conclusion can be made however, TABLE 8

THE EFFECT OF MUTAGENIC AGENTS UPON LOSS OF PENICILLIN AND D-CYCLOSERINE RESISTANCE IN STRAINS S. AUREUS U9W AND M. SMEGMATIS 607 B

Concentration . Frequency of Total of Total number Strain Temperature of AO (ug/ml) pH Sensitive Cells Sensitives Cells Tested (per cent) Pase D-CS

U9W D-CS+ 37 10 7.3 NTC 84.1 1906 2258 # 1 37 10 7.6 NT 78.6 1974 25 L0 44 0 7.3 NT 11.1 456 3097 44 10 7.3 NT 83.8 2032 2422 44 10 7.6 NT 80.9 1394 1720

U9W D-CS+ 37 10 7.3 NT 81.3 1782 2190 # 2 37 10 7.6 NT 80.5 2496 3100 44 0 7.3 NT 9.4 243 2486 44 10 7.3 NT 89.5 1946 2396 44 10 7.6 NT 86.5 2987 3451

U9W D-CS+ 37 10 7.3 NT 85.7 2382 2780 # 3 37 10 7.6 NT 81.8 1934 2424 44 0 7.3 NT 10.1 257 2555 44 10 7.3 NT 90.9 2332 2564 44 10 7.6 NT 89.9 1996 2458

607 B 37 10 7.3 00.0 00.0 87667 P+ D-CS+ 37 10 7.6 00.0 00.0 42543 44 0 7.3 00.0 00.0 m w m mm 24096 44 10 7.3 00.0 00.0 11049 44 10 7.6 00.0 00.0 9123

temperature during mutagenesis bpH of medium during mutagenesis cNT=not tested 82

Inasmuch as the mutation rate to penicillin sensi­

tivity is approximately 2 per cent in strain U9U, this would tend to indicate that the mutagens were acting

independently and that there is no close association of

these determinants. Therefore more definitive studies must be carried out before it can be stated with certainty

that there is any co-elimination of the penicillinase

and D-cycloserine determinants with these mutagens.

M. smegmatis 607 B. Large populations of cells were

tested for loss of the penicillinase and/or D-cycloserine

determinant following exposure to acridine orange and

elevated temperature. A total of 130,210 cells were tested

for loss of D-cycloserine and penicillin resistance with

acridine orange and 24,096 cells at elevated temperatures

(Table 10). No mutants were recovered in any of these

experiments.

The Mutagenic Effect of Ultraviolet Radiation on

Penicillin and D-cycloscrine Resistant Cells. The lethal

effect of ultraviolet radiation on M, smegmatis 607 B was

studied by taking samples at 20 second intervals for a

total of approximately three minutes, diluted and plated

onto media. Survival of the cells plotted against time of

ultraviolet treatment is shown in Fig. 4. It can be seen

that the effect of ultraviolet irradiation is time de­

pendent. The dose yielding approximately 5 per cent

survival (2 minutes) was used to induce drug sensitive 83

Figure 4, Survival curve of M. smegmatis 607 B

after irradiation with ultraviolet rays. Surviving cells (%) 100- 80 90- 60- 40- 30 20 - ie (sec) Time 4 10 180 160 140 84 85 mutants. A total of 24,749 cells of M, smegmatis 607 B were tested. Of this total 14,627 cells were initially

tested for mutants sensitive to the antibiotic penicillin and 10,122 were initially tested for sensitivity to

D-cycloserine. The plates then were replicated onto the alternate antibiotic medium to detect mutants for the

second antibiotic not originally tested for. Of the total

cells tested, no sensitive mutants were found for either of the two antibiotics, penicillin or D-cycloserine.

Transduction of Penicillin and D-cycloserlne Re­ sistance. D-cycloserine resistant mutants seven, eight, and nine of strain U9VJ, which were also resistant to penicillin, were employed as various donors of these genetic determinants to the penicillin and D-cycloserine

sensitive variant of S. aureus being the recipient. In addition, several D-cycloserine sensitive mutants obtained

from treatment with acridine orange also were used as recipients of the determinant for D-cycloserine, As shown by the results in Table 9, all mutants tested, as well as

the penicillin and D-cycloserine sensitive variant of

strain U9W, were competent recipients of either the penicillinase or D-cycloserine determinant. The sensitive strains obtained by acridine orange treatment did not have a lower ability to act as recipients of these markers. Co­ transduction of the penicillinase and D-cycloserine determi­ nants was not observed. Limited studies failed to reveal TABLE 9

TRANSDUCTION OF THE PENICILLINASE AND D-CYCLOSERINE DETERMINANTS TO PENICILLIN AND D-CYCLOSERINE SENSITIVE CELLS OF STRAIN U9W OF S. AUREUS

Donor Recipient Pase+ D-CS + Pase+ and D-CS+

80/U9W 7 U9W p "d -cs 4,200c 4,980 0 U9VJ p “d -c s “ i ° 2,940 6,200 0 U9W P“D-CS“8D 2,420 3,460 0

80/U9W 8 U9W p "d -c s “ 5,120 6,240 0 U9W P ’D-CS‘ 1 5,780 5,460 0 U9W P~D-CS”8 4,480 6,080 0

80/U9W 9 U9W p ”d -c s “ 3,340 3,780 0 U9W p "d - c s “ i 2,880 3,240 0 U9W P-D-CS“8 3,420 4,660 0

aStock penicillin and D-cycloserine sensitive variant of strain U9VJ of S . aureus Random D-cycloserine sensitive mutants selected following treatment with acridine orange, 10 ug per ml cFrequency of transduction. Expressed number of transductants recovered per 10^0 phage particles in the transduction mixtures. 87

D-cycloserine resistant recombinants when various D-

cycloserine sensitive mutants were used as alternate donors 8 and recipients, in populations of 10 cells.

Extraction of DNA. To extract DNA from M. smegmatis

607 B and S, aureus U9W, prolonged incubation with lysozyme

(10 mg per ml) continued for 18 hours, at the end of which time lysis v/as evident by the presence of a highly viscous

suspension. The detergent sodium lauryl sulphage then was

added for an additional 3-6 hours, however the suspension did not become noticably transparent as is the usual case.

This method of disruption seemed to afford release of

sufficient amounts of DNA for these experimental pro­

cedures, therefore it was not necessary to increase the

amount of cells used for extraction. Approximately 1 mg

of DNA was extracted from two to three grams of wet

packed cells. Mo apparent difference in the amount of DNA

extracted from either M. smegmatis 607 B or S. aureus U9W

was noted, when equivalent amounts of cells were used for

an extraction. The ratio of absorptions at 260 and 280

( ^ ^ / ^ 2gg) £ori various test samples were as follows:

£!• smegmatis 607 B: 0.50, 0.52, 0.52 and 0.53 S. aureus:

0.50, 0.50, 0.52, and 0.55, The absorption ratios for the

standard sperm DNA were: 0.53, 0.53, 0.53, 0.53, 0.53,

0.53, 0.54, and 0.54.

Determination of DNA Base Composition. It has been

shown that the acquisition of multiple drug resistance by 88 conjugation may be correlated with the addition of a physically recognizable high molecular weight satellite 0 & DNA fraction. Studies have shown that the extrachromo- somal determinants reside in this satellite band of DNA.

DNA analysis by means of analytical ultracentrifugation was employed in an attempt to disclose satellite bands of

DNA in the test organisms.

The buoyant densities of each isolated DNA were determined by equilibrum density gradient centrifugation in cesium chloride, with a density of 1,7 g per ml, by 90 the method of Schildkraut et al. The content of guanine plus cytosine (GC per cent) of the DNA was calculated from the values obtained for buoyant density (See Materials and

Methods). The resulting ultraviolet absorption films were traced with a microdensitometer (Beckman model RB Anal- ytrol film densitomiter, slit width of 100 u). These densitomiter tracings were quite reproducible when retraced and reasonably independent of small alterations in speed of scanning etc.

The buoyant densities of the mycobacterial main DNA preparations studied ranged from 1.723 to 1.724 g/cc.

Repetition (three times) indicated that the method permitted highly reproducible results. The buoyant densities were calculated by the position of an internal standard of E. coli K 12 as reference with the density taken to be 1.710 g/cc. This was sufficiently different from byoyant density values recorded elsewhere for M. tuberculosis and M. phlei in addition to S, aureus such that no overlapping of DNA bands would occur due to close similarities in buoyant densities. All DNA prepa­ rations of M. smegmatis 607 B showed two symmetrical bands in the cesium chloride density gradient. The buoyant density of the extra satellite band was calculated to be

1.670. The mole per cent guanine plus cytosine (per cent GC) of the main band of DNA was calculated to be 64.3 per cent for a buoyant density of 1.723; the G-C value for the extra band was found to be 10.2 per cent where the buoyant density was 1.670.

Plate VIII represents the ultraviolet absorption picture of the cesium chloride density gradient equilibrium run as well as the corresponding densitometer trace for a sample of DNA from M. smegmatis 607 B. Photographs taken at intervals during a run indicated that equilibrium was attained in 16-18 hours after full speed was reached, as illustrated in photo series plates IX and X.

The buoyant densities of the staphylococcal DNA preparations examined were in the range of 1.691 to 1.692 g/cc. All preparations showed only a single symmetrical band where the per cent G-G of the DNA was determined to be 30.6 per cent for a buoyant density of 1.691. No extra or satellite bands were found in any of four consecutive equilibrium runs. Each run was comprised of DNA from a 90

Plate VIII. Densitometer trace and corresponding

ultraviolet photograph of DNA from

M. smegmatis 607 B. (a) satellite

band of DNA (b) reference band of

DNA (E. coli K 12) (c) main band

of DNA 91

1.723 1.710

670 r 92

Plate IX. Ultraviolet photographs of mycobacterial

DNA coming to equilibrium during a 24

hour run. (a) zero time picture

(b) plus 15 minutes (c) plus 1 hour

20 minutes (d) plus 3 hours 28 minutes

(e) plus 5 hours 36 minutes (f) plus

7 hours 44 minutes (g) 9 hours 52

minutes (h) plus 12 hours. B

C

H 94

Plate X. Ultraviolet photographs of mycobacterial

DNA coming to equilibrium during a 24

run. (i) plus 14 hours 8 minutes

(j) plus 16 hours 16 minutes (k) plus

18 hours 24 minutes (1) plus 20 hours

32 minutes (m) plus 22 hours 40 minutes

(n) plus 24 hours 48 minutes (o-p)

duplicate pictures taken at 24 hours

48 minutes. I

J

K

L

M

N

O fresh extraction where the sample was not held longer than

18 hours prior to the run.

The densitometer trace and ultraviolet photograh of staphylococcal DNA at equilbrium are illustrated in Plate

XI. Interval pictures during a run indicated that equi­ librium was reached around 23-24 hours (Plates XII and

XIII). This is in contrast to the mycobacterial DNA which reached equilibrium at approximately 16-18 hours. 97

Plate XI. Densitometer trace and corresponding

ultraviolet photograph of DNA from

_S, auretis U9W, Pase+ D-CSr .

(a) staphylococcal DNA (b) refer­

ence band of DNA (E. coli K 12). 98

710 1.691 99

Plate XII. Ultraviolet photographs of staphy­

lococcal DNA coming to equilibrium

during a 24 hour run. (a) zero

time picture (b) plus 15 minutes

(c) plus 1 hour 20 minutes (d) plus

3 hours 28 minutes (e) plus 5 hours

36 minutes (f) plus 7 hours 44

minutes (g) plus 9 hours 52 minutes

(h) plus 12 hours H 101

Plate XIII, Ultraviolet photographs of staphy­

lococcal DNA coming to equilibrium

during a 24 hour run, (i) plus 14

hours 8 minutes (j) plus 16 hours

16 minutes (k) plus 18 hours 24

minutes (1) plus 20 hours 32

minutes (m) plus 22 hours 40

minutes (n) plus 24 hours 48

minutes (o-p) duplicate pictures at

24 hours 48 minutes.

DISCUSSION

Experiments in the early part of this investigation established certain baselines necessary for the mutagenic studies and DNA analysis of the two organisms in this latter part of the study. The elimination of pellicle formation by M. smegmatis 607 B, without the aid of surfactants, was important in the determination of drug resistance in this organism and for phage propagation techniques. The Tween has been reported to exaggerate the effect of various antibiotics such that seemingly sensitive cells are actually resistant. ^ In addition, surfactants such as Tween 80 exert an inhibitory effect on phage propagation.

The use of synthetic medium devoid of L-alanine was found to be necessary in establishing D-cycloserine resistance in S. aureus as commercial media negated the inhibitory effect of this antibiotic, inferred to be by racemization of L-alanine to D-alanine upon autoclave sterilization. The basis for penicillin resistance was determined by tests employed to detect the presence of the penicillin destroying enzyme penicillinase.

It was necessary to elucidate the inhibitory action

103 104 of diluents on various mycobacteriophages lytic for

M . smegmatis so any change to phage susceptibility demon­ strated by variants obtained could be attributed to mutation and not due to external influences on the phage. In a similar manner, the host range of the mycobacterial species was established to identify mutants as being derivatives of the original parent strain and not contaminants. Any change in plaque formation might also have been employed to denote a new characteristic of any mutants obtained.

Inasmuch as variants in colonial morphology have been reported to occur at low frequencies in M. smegmatis. studies were undertaken to ascertain whether variants were present in this strain and at what rates these occurred in order to distinguish these variants from those which may have been induced by the mutagens employed in the study.

Attempts were made in the second part of the study to identify the drug resistant determinants for penicillin and D-cycloserine as being extrachromosomal in nature by the use of various mutagens such as acridine orange, elevated temperature, and ultraviolet irradiation. The mutagens have been reported to elicit a higher rate of change for nonchromosomal genetic material. These results were then to be correlated with the identi'"cation of satellite bands of DNA wherein extrachromosomal detenni- nants have been shown to reside.

The analysis of nonchromosomal inheritance has made 105 rapid progress in the past ten years especially, since the 88 first report of a non-Mendelian gene in 1909. Much work has been reported on the extra-chromosomal nature of drug 72 resistance in numerous bacteria. An early study by 37 Hirota reported the selective ,,curing,, of the fertility factor of E. coli in the autonomous state (nonchromo- somal) by acridine dyes. Numerous confirmations of these and similar findings have been reported such that the elimination or "curing" of a genetic determinant by acridine dyes is now accepted as indirect evidence of its extra-chromosomal nature. The work in this study was extended to include other mutagens such as elevated temperatures^ and ultraviolet radiation under certain circumstances'^*^ for the select elimination of extra­ chromosomal genetic material.

The genetic determinant governing D-cycloserine resistance in strain U9W of S. aureus was shown to be lost at a very high frequency following treatment with acridine orange or at a some what lower frequency following ex­ posure to elevated temperature in this study (Table 8 ),

A combination of acridine orange and elevated temperature did not show an additive effect on the rate of elimination to drug sensitivity. This was probably due to the inhibitory action high temperature exerts on acridine thus 2 lowering their effectiveness. The results of the present study would tend to indicate that the determinant(s) for 106

D-cycloserine resistance is extrachromosomal in nature in

S . aureus U9W. Since the spontaneous mutation rate to drug sensitivity (Table 7) was much lower than the induced rate

(Table 8 ) it seems plausible that the resistant mutants were indeed stable and any loss of the determinant(s) demonstrated the effectiveness of the mutagens. Such findings are compatible with those of other investi- on oo oo gators. * * Similar studies with regard to the 70 penicillinase marker in this strain have been reported.

Transduction of tho penicillinase marker among the pathogenic staphylococci v;as first observed by Ritz and 84 Baldwin. This marker has been shown previously to be 70 transduced in strain U9V/ of S. aureus and again in the experiment reported here. Transduction of the determi­ nant^) governing resistance to D-cycloserine was further reported in this investigation. No difference in competency of recipients was observed in any of the trans­ duction experiments, although differences have been 82 reported by other investigators. Transduction of

D-cycloserine resistance has been reported for derivatives of E. coli K 12.*^ The strain from which the D-cycloserine resistance property was eliminated in the aforementioned

E. coli strain and in strain U9W of S, aureus of this study have not so far been observed to revert to D-cycloserine resistance, an observation which would tend to indicate the irreversible elimination of this determinant. Various 107

D-cycloserine sensitive cells were used as alternate donors and recipients in transduction experiments in attempts to recover resistant recombinants derived from sensitive cells.

These transduction experiments failed to yield any D- cycloserine resistant recombinants in this stud}'. Similar suggestions of irreversible elimination have been made in cc oo other reports. »

Penicillinase producing transduetants are charac­ teristically distinguishable by the presence of satellite colonies surrounding the original penicillin resistant 84 recombinant. The enzyme, penicillinase, which is produced by the recombinant following transduction, destroys the penicillin in the transduction medium and allows non­ recombinants, sensitive to penicillin, to grow. Satellite colonies were not noted surrounding any of the D-cycloserine resistant transduetants which might tend to rule out the presence of an extracellular enzyme that would account for

D-cycloserine drug resistance.

Although co-transduction of closely linked determi- 71 83 nants has been found to occur in S, aureus * co­ transduction of the penicillinase and D-cycloserine resistant markers was not observed in this study. This 35 might imply that the markers are not as closely linked as was hypothesized.

The probability that distant markers are co-transduced is small, about 10” , In transduction, approximately 10" 108 -7 to 10 of a total population of phage particles are able 3 to yield a transductant. If one in 10 particles carries a particular donor marker and each recipient bacterium can absorb and be infected by ten particles at high multi­ plicities of infection, the probability that a double transductant for two unlinked markers will arise is about 10-8.35

A second alternative is that the determinant(s) of resistance to D-cycloserine is located on a separate plasmid, independent of any association with those governing penicillinase production. Multiple plasmids have 82 been shown to exist in the staphylococci and this has been recently suggested for the determinant of resistance to macrolide antibiotics in a strain of Staphylococcus where this determinant docs not show any association with 55 the penicillinase determinant. It will be recalled, however, that both penicillinase and D-cycloserine resistant determinants were lost following acridine and elevated temperature treatment although at a lower frequency (2 per cent).

The concept of extrachromosomal genetic material

(plasmids) is new and relatively unstudied among the mycobacteria (Juhasz, personal communication). One refer­ ence to the possibility of plasmids existing in the mycobacteria was made by Jones and White in 1968. That the determinants governing penicillin and/or D-cycloserine 109 resistance in M. smegmatis 607 B were extrachromosomal in nature could not be concluded due to the inability to obtain any mutants sensitive to these drugs following treatment with acridine orange, elevated temperature, or with ultraviolet radiation. Reports have indicated, however that the ease of removing plasmids from cells is not 82 equally easy therefore it is impossible to rule out that the determinants were not extrachromosomal based solely on their inability to be eliminated.

The failure to obtain penicillin and/or D-cycloserine sensitive variants could be based on the state in which the genetic determinants are found within the cells of 607 B. 43 Jacob et al, in describing the fertility factor in

E. coli. reported loss of the factor in the autonomous (F+) or extrachromosomal state with acridines or elevated temperature. The fertility factor, when integrated with the chromosome (Hfr) was not "cured" following treatment with these mutagens. The drug resistant determinant in the mycobacterial strain could therefore be integrated with the chromosome and would not be "cured", or if so, only at a very low frequency.

Similar findings have been reported for the penicill- 82 inase determinant in certain strains of S, aureus.

Tetracycline and streptomycin resistance similarly have not 49 been "cured" from strains of S. aureus therefore the investigators indicated that the inability to "cure" these 110 determinants from cells could have been due to their chromosomal attachment.

A single chemical mutagen and two physical mutagens were employed in this study such that impermeability of chemicals could not be the exclusive reason for the inability to obtain mutants. If the chemical mutagen was ineffective due to impermeability, then the two physical mutagen3 should still have been effective in the elimi­ nation of the drug resistant determinants if the determi­ nants were indeed extrachromosomal in nature.

The mycobacteria and staphylococci in general are 2 highly sensitive to acridine dyes. Information is not available as to why some mutagens are unable to eliminate certain markers even though doses sufficient to kill are 64 employed. A correlation between mutability and sensi- 115 tivity to mutagenic agents is not always direct.

Acridine dyes, under the influence of light, cause ex- 2 91 tensive destruction of guanine. » In the analysis of mycobacterial DNA, an extra or satellite band was found.

The per cent G+C however was extremely low, 10,2 per cent.

If indeed the determinants governing resistance to penicillin and/or D-cycloserine are located on this band, the low G+C content might account for the inefficiency of elimination by acridine orange which acts specifically on guanine. Inasmuch as a satellite band high in per cent

G+C was not found in the penicillin and D-cycloserine Ill

resistant S. aureus strain, the ease with which these

determinants were eliminated is difficult to explain.

The lethal damage of ultraviolet radiation and certain

chemicals (acridines) to bacteria such as E, coll are to

lid some extent reversible. It is not implied that the

repair mechanisms to certain chemicals is similar to that

which accounts for repair of radiation induced damage,

however the rate of mutation by an acridine has been shov;n

to be affected by repair mechanisms which exist in

radiation resistant cells,Cross resistance with

ultraviolet radiation and certain acridine dyes (acridine

orange and acriflavine) has been reported in a high

proportion of cells in a strain of E. coll. Although this

phenomenon in cells of M. smegmatis 607 B was not directly

studied in this investigation, the possibility that some mechanism of repair of damage to DNA induced by ultraviolet

radiation and/or acridine orange may be occurring, cannot

be excluded, A marker resistant to ultraviolet radiation

would tend to have a higher G+C content as ultraviolet

radiation causes thymine dimers.^ If any such resistance

is being demonstrated by 607 B, this could not be accounted

for because of high A-T content demonstrated by the extra

(satellite) band of DNA, assuming the genetic determinants

are indeed located on this band.

The inducability of certain mutations by ultraviolet

radiation may vary with the various types of mutations 112

(auxotrophic mutants and certain drug sensitivities) being 103 induced. This may occur in spite of the fact that most mutations may be photoreactivated. No explanation for thi3

insensitivity has been given. Certain post-irradiation conditions have been shown to modify the extent of ultra­ violet radiation damage such as constitution of plating medium and temperature of incubation. post-irradiation treatment has been shown to influence recovery of certain 54 mutants such as drug resistant mutants. Konicek and

Malek found that various post-irradiation operations can exert a positive or a negative influence on induced mutants.

A recovery medium rich in amino acids was used during the

sensitive post-irradiation period in the aforementioned report. This was found to bring about an increase in the

frequency of recovery of induced mutants'^ based on the

significance of protein synthesis occurring during this

time. However Witkin and Thail shovied, studying similar mutants, that po3t-irradiation incubation in a medium poor in amino acids did not decrease the number of induced mutants. It is felt that post-irradiation conditions

could influence the recovery of drug sensitive mutants and may serve as some explanation in the inability to recover

these mutants in this study.

The data from this study would not tend to support

the contention that the determinants governing penicillin

and/or D-cyclosorine resistance in M. smegmatis 607 B are 113 extrachromosomal in nature. However it further cannot be stated unequivocally that the location is chromosomal.

Rather the findings are inconclusive at this time and must await further investigation.

The results of this study indicated (Table 8 ) that the determinant of D-cycloserine resistance in S. aureus was extrachromosomal in nature and possibly in M. smegmatis that the D-cycloserine and penicillinase determinants were 26 chromosomal in nature, A report by Falkow et ad., indicated that the extrachrcmoaomal nature of R-factors

(resistance factor) of the enterics was correlated with the addition of one or more physically recognizable satellite DNA fractions. The satellite band of DNA 25 disappeared upon acridine "curing,,» Upon re-infection with the R-factor, satellite fractions were recovered again thus showing this to be the genetic material of the

R-factor,

There is so little in the literature on mycobacterial

DNA and it is doubtful that any published work exists regarding satellite bands (Wayne, personal communication).

Further, such extra bands have not been reported in the staphylococci.

In this investigation an extra or satellite band of

DNA from the penicillin and D-cycloserine resistant variant of S, aureus U9W was not revealed (Plate XI),

The buoyant density of the single band was 1,691, with 114 per cent G->'-C being 30.6, These are well within the ranges reported by DeLay^ and Hill?^ If extrachromosomal genetic material can indeed be identified by the presence of satellite bands in this strain of S. aureus and if these genetic determinants do reside in the extra bands, then these data would tend to conflict with the results of the high frequency loss of these determinants by mutagens purported to be selective for extrachromosomal genetic material. It is possible that sensitivity to acridine dyes may not be an obligate consequence of the extrachromo- 82 somal state after all.

It cannot be excluded that the extra (satellite) band had the same or very similar D M composition as the chromo­ somal band. The sensitivity of the densitometer is however of such a magnitude that if satellite DNA was similar in base composition as the main band, a shoulder would have been demonstrated on the peak of the main band. This was not evident as illustrated in Plate XI, An extra band of the same composition would not have been detected.

A fragment equalling approximately 0,5 to 1,0 per cent of the bacterial genome may still be detected as a major density peak in cesium chloride. This same fragment as part of a randomly fragnenting bacterial "chromosome" might be broken into two pieces, most of the time, and 2fi would therefore not be detected. No other studies on staphylococcal satellite DNA could be found, therefore no 115 approximation regarding the per cent of the total DNA that a satellite fraction could be, was made. That such a small fragment exists in this strain of Staphylococcus and is being broken thus making it undetectable must remain only an attractive hypothesis at this time.

Examination of the mycobacterial DNA did however reveal an extra band (Plate VIII), In a personal com­ munication from Sellers, an extra band has been observed in M . smegmatis 607, however these results were not reproducible. It was felt that the technique for cell rupture (sonication) may have been a basis for this.

However, the results described here were quite reproducible and a less harsh treatment for cell rupture was used

(lysozyme treatment) thus minimizing breakage, denatur-

ization and other related problems with the DNA,

Although it cannot be stated with certainty what this

band actaully represents, certain possibilities can be ruled out. The material would not likely be protein or RNA

as the former exhibits a buoyant density in cesium chloride around 1,3 g per ml and 1,90 g per ml for the latter,

In addition the extraction procedure included treatment with RNase.

The buoyant density of the major band of myco­

bacterial DNA was 1,723 and the per cent G+G being 64,3,

phlei. a saprophytic Mycobacterium, has been reported 19 to have a buoyant density of 1,732 with per cent G+C 116 O £ being 66-70. The buoyant density for pathogenic mj'co- bacteria, M. tuberculosis. has been reported to be 1.724 which seems to be clearly distinguishable from the saprophytic mycobacteria. Although M. smegmatis 607 B is considered to be saprophytic now, its buoyant density of

1,723 closely approximates that recorded for M. tuber­ culosis , It will be recalled that the history of this organism reveals its purported derivation from M. tuber­ culosis horninis. The satellite band demonstrated a buoyant density of 1.670 with per cent G+C being 10.2.

Single stranded DNA, including that obtained from de- naturization, could possibly further be ruled out as it has a somewhat higher density than the double helical DNA, due to greater binding by the heavy cesium ions,^”*

The extra band present could be attributed to one of several possibilities. The satellite band could indeed correspond to extrachromosomal DNA even though the per cent G+C is very low (10.2). A report by Bernardi^ indicated that the per cent of A+T in mitochondrial

(cytoplasmic) DNA from several strains of Yeast was from

83-87. The per cent A+T in the extra mycobacterial band was approximately 89,8 per cent. Further an extra band of DNA need not always have a buoyant density higher then the main band."** Extra bands with densities both higher and lower have been reported in mouse and guinea pig DNA respectfully. If this strain is lysogenized 117 with a phage, then another alternative exists wherein the extra band noted could corresond to the viral DNA if attachment to the chromosome became unstable during ex­ traction procedures. Buoyant densities as low as 1,50 104 have been reported for some DNA viruses. An RNA virus could be ruled out, as RNase was employed in the extraction procedure, A single report by Tokunaga and Sellers of the buoyant density and per cent G+C of rnycobacterio- phage D29 indicated this to be 1.72.2 and 63.77 respectfully.

If all buoyant densities of mycobacteriophage were in the general area of 1.72, this alternative would tend to be excluded although such a decision must await more detailed investigations.

Three other alternatives exist. Unusual bands not containing DNA, based on the ineffectiveness of DNase, have been found which band near the DNA region. Com­ mercially prepared glycogen produces a band with a density around 1.675 g per m l . ^ Preparations of fungal DNA contain a polysaccharide in the region corresponding to a density of 1.680-1.685 g per ml.*^ Teichoic acids have also been found to band in cesium chloride gradients in 114 the region of DNA although no specific buoyant density was reported. The use of schlieren optics was employed in the aforementioned studies whereas absorption optics

(ultraviolet) were utilized in this investigation which tend to be specific for nucleic acids. The extra band in 118 this study exhibited a buoyant density of 1,670 which approximates that reported for glycogen. Certainly however no conclusions can be drawn until further studies are undertaken to ascertain the exact chemical nature of the extra (satellite) band in this mycobacterial strain.

Because absorption optics were employed in this study, material other than nucleic acid however would tend to be ruled out.

Due to the inability to obtain mutants sensitive to

D-cycloserine and/or penicillin with M. sinegmatis 607 B, the elucidation of these genetic determinants could not be fully carried out with regard to mutagenesis. Some insight may have been gained however in the observance of an extra or satellite band accompanying the main DNA band.

This may be a possible site for extrachromosomal (plasmid) genetic material. It is possible that resistance to both these antibiotics are not plasmid associated character­ istics as was demonstrated to be the case in the staphylo­ cocci or that plasmids in this strain of mycobacteria do not behave in the same manner to mutagens as they did in

S, aureus U9W, SUMMARY

Drug resistance demonstrated to the antibiotics penicillin and D-cycloserine was studied in a Gram positive coccus, S. aureus U9W, and an acid fast bacillus,

M . smegmatis 607 B, The results of the study showed:

(1) D-cycloserlne resistance in S . aureus was

induced to D-cycloserine sensitivity at a high frequency

following exposure to acridine orange and a somewhat lower

frequency for elevated temperature. Use of both mutagens

simultaneously did not produce an additive effect in

eliciting a greater number of sensitive mutants. The pH

of the medium did not further affect the mutation rate.

(2) D-cycloserine or penicillin sensitivity in

smepmatls 607 B was not induced following exposure to

any of the mutagenic agents employed which included

acridine orange, elevated temperature, or ultraviolet

irradiation.

(3) The determinant(s) for D-cycloserine resistance was found to be transducible to D-cycloserine sensitive

cells in S. aureus U9W. No differences in the competency

of the recipients were noted. The determinants for D-

cycloserine and penicillin resistance were not co-trans-

ducible,

119 120

(4) The buoyant density of the parent band of staphylococcal DNA was 1,691, with per cent G+C being

30,6, No extra or satellite bands were found,

(5) The buoyant density of the mycobacterial parent band was 1,732 with per cent G+C 64,3, An extra or satel­

lite band was demonstrated. The buoyant density of this band was 1,670 with per cent G+C being 10,2,

(6) The inhibitory action of D-cycloserine was an- tagnoized, by the probable presence of D-alanine, in commercial laboratory media, L-alanine, following autoclave

sterilization, was found to reverse the anti-staphylococcal action of D-cycloserine inferred to be racemization of

L-alanine to D-alanine, Mycobactin did not seem to reverse the inhibitory action of D-cycloserine on sensitive cells

§.• aureuo, although it has been reported to do so in a mycobacterial species. BIBLIOGRAPHY

Abraham, E.P. and E.B. Chain. 1940. An enzyme from bacteria able to destroy penicillin. Nature (London). 146;837.

Albert, A. (ed.). 1966. The Acridines. Their Preparation, Physical, Chemical, and Biological Properties and Uses. St. Martin's Press, New York.

Arber, W, 1960. Transduction of chromosomal genes and episomes in Escherichia coli. Virology 11;273.

Asheshov, E.H, 1966. Chromosomal location of genetic elements controlling penicillinase production in a strain of Staphylococcus aureus. Nature ( London), 2107 804.

Asheshov, E.H. 1966. Loss of antibiotic resistance in Staphy 1ococcus aureus resulting from growth at high temperatures. J, Gen. Microbiol, 42;403.

Avers, C., C. Pfeffer and M. Rancourt. 1965. Acriflavine induction of different kinds of "petite" mitochondrial populations in Saccharo- myces cerevisiae, J. Bacteriol. 90:481-490.

Avery, R.C. and C.B. Ward. 1945. The inhibitory effect of atabrine on some acridine derivatives upon acid fast bacillin in vitro. J. Pharm. Exper, Ther, 85:258-264,

Balls, M.E. (ed.). 1968. Antagonists and Nucleic Acids. North-Holland Publishing Company, Amsterdam.

Barber, M. 1949. The incidence of penicillin sensi­ tive variant colonies in penicillinase producing strains of Staphylococcus pyogenes, J. Gen. Microbiol. 3:2T4. 122

10, Bernardi., G., M. Faures, G, Piperno, and P, Slominski. 1970, Mitochondrial DNA-s from respiratory sufficient and cytoplasmic respiratory- defficient mutant yeast, J, Mol, Biol, 48:23-42,

11, Bondi, A,, J, Kornblum and C, Forte, 1957, Inhibition of antibacterial activity of cycloserine by alpha-alanine, Soc, Exptl. Biol, Med, 96:270- 272. ”

12, Bowman, B.U,, Jr. 1958, Quantitative studies on some mycobacterial phage-host systems. Amer. Rev, Resp. Dis, 79;52-62.

13, Campbell, A.M. 1969, Episemes. H.O. HaIvorson, H.L. Roavnan, and Eugene Bell, eds. Harper and Rov;, Publishers, New York.

14, Carlberg, D, and G.J. Jann. 1963, DNA, protein, and inducible enzyme synthesis in phage infected M. smegmatio ATCC 607. 88:235-239.

15, Chain, E,B, and E.S. Duthie. 1945. Lancet. 1:652. In: Resistance of Bacteria to the Penicillins. A, de Reuck and M. Cameron, eds. Little, Brovin, and Company, Boston. 1962.

16, Cuckler, A,C., B.M, Frost, L. McClelland, and M, Solotorovsky. 1955. The antimicrobial evalu­ ation of oxsmycin (D-4-amino~3-iso>:aKolidone), a new broadspectrum antibiotic. Antibiot. and Chemother, 5:191.

17, Curtiss, R., III,, L.J, Charamella, C.M, Berg, and P.E, Harris. 1965. Kinetic and genetic analyses of D-cycloserine inhibition and resistance in Escherichia coli. J. Bacteriol. 90;1238-1250.

18, David, H.L., K. Takayama, and D.S. Goldman. 1969, Susceptibility of mycobacterial D-alanyl- D-alanine synthetase to D-cycloserine. Amer. Rev. Resp. Dis. 100;579-581.

19, DeLay, J. 1970. Reexamination of the association betweeen melting point, buoyant density, and chemical base composition of DNA. J. Bacteriol. 101:738-754. 123

20. Demerec, M. 1945, Production of staphylococcus strains resistant to various concentrations of penicillin, Proc. Natl, Acad. Sci. U.S. 31;16.

21. Dufour, A,P., R.A, Knight, and H.W, Harris. 1966. Mycobacterial penicillinase activity. Amer. Rev. Resp. Dis. 94;965-968.

22. Dyke, K.G.H. and M.H. Richmond. 1967, Occurrence of various types of penicillinase plasmids among hospital staphylococci. J. Clin. Path. 20;75.

23. Ephrussi, B. and H. Hottinguer. 1950. Direct demon­ stration of the mutagenic action of euflavine on baker's yeast. Nature (London). 166;956.

24. Fairbrother, R.W., L. Parker and B.R. Easton. 1954. The stability of penicillinase producing strains of Staphylococcus aureus. J. Gon, Microbiol. 10 ; W . ------

25. Falkow, S. and R.V. Citarella. 1965. Molecular homology of F-merogenote DNA. J. Mol. Biol. 12:138.

26. Falkow, S., R.V. Citarella, J.A. Wohlhieter, and T. V/atanabe. 1966. The molecular nature of R-factors. J. Mol. Biol. 17;102-116.

27. Gardner, A.D, 1940. The morphological effects of penicillin on bacteria. Nature (London). 146:837,

28. Gardner, G.M, and R.S.A. Weiser. 1947. A bacterio­ phage for Mycobacterium smegmatis. Proc. Soc. Exptl, Biol. Med. 66T2J55^ 2^ ]

29. Geronimus, L.H. and S. Cohen. 1957. Induction of staphylococcal penicillinase. J. Bacteriol. 73:28-34.

30. Gibor, A. and S, Granick. 1962. Bleaching phenomenon in Euglenia gracilis strain Z, J, Cell Biol. 15:5"99-6'0jC

31. Gunderson, W.B. 1963. New type of streptomycin resistance resulting from action of the episome- like mutator factor in E. coli, J. Bacteriol. 86:510-516. ~ 124

32. Hannon, S.A. and J.K. Baldwin. 1964. Nature of the determinant controlling penicillinase production in Staphylococcus aureus. J. Bacteriol. 87:593- 597.

33. Hashimoto, H., K. Kono and S. Mitsuhashi. 1964. Elimination of penicillin resistance of Staphy- locoecus aureus by treatment with acriflavine. J. Bacteriol. 88:261-264.

34. Hawkins, J. and V. McClcan. 1966. Effect of peni­ cillin G and D-cycloserine on mycobacteria in vitro and in vivo. Amer. Rev. Resp. Dis. 94: 118-120.

35. Hayes, W. 1968. The Genetics of Bacteria and Their Viruses. J, Wiley and Sons, Inc. New York.

36. Hill, L.R, 1966. An index to deoxyribonucleic acid base compos ition of bacterial species. J. Gen. Microbiol. 44;419-437.

37. Hirota, Y. 1960. The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Nat. Acad. Sci, 46:57-6*4.

38. Hoeprich, P.D. 1963. Alanine:cycloserine antagonism. III. Quantitative aspects and relation to heating of culture media. J. Lab. Clin. Med. 62:657-662.

39. Hoeprich, P.D. 1964. Alaninc-cycloserine antagonsim. Amer. Rev. Resp. Dis. 90:646-648.

40. Horvath, S. 1969. Transformation of phage resistance. Acta microbiol. Acad. Sci. hung. 16;77-83.

41. Howe, W.B., G.L. Melson, C. H. Meridith, J.R. Morrison, M.H. Platt and J.L. Strominger. 1964. Stepwise development of resistance to D- cycloserine in Staphylococcus aureus. J. Pharmacol. Expt’l. The rap. 143:2~82^2~84.

42. Hand, C.N. and S. Baines. 1949. The nature of mycobacterial penicillinase. J. Path. Bacteriol. 61:392.

43. Jacob, F., S. 3renner and F. Cuzin. 1963, On the regulation of DNA replication in bacteria. Cold Spring Harbor Quant. Biol. 28:334. 125

44. Jones, W. and White. 1968. Lysogeny in myco­ bacteria. I, Conversion of colony morphology, nitrate reductase activity, and Tween 80 hydrolysis of Mycobacterium sp. ATCC 607 associated with lysogeny. Can. J. Microbiol. 14:551-555.

45. Juhasz, S.E. 1960. Interspecific hybridization among mycobacteria. Nature (London). 185;265.

46. Juhasz, S.E., S. Gelbart and M. Harize. 1969. Phage- induced alteration of enzymic activity in lysogenic Mycobacterium smogmatis strains. J. Gen, Microbiol. 56:251-255.

47. Kapadia, R.T. and M. Srogl. 1969. Inactivation of B. subtills transforming DNA by mutagenic agents. FoT. Microbiol. 14:51-53.

48. Kasik, J.E. 1965. The nature of mycobacterial penicillinase. Amer. Rev. Resp. Dis. 91:117- 118.

49. Kasuga, T., H. Hashimoto, and S. Mitsuhashi. 1968. Drug resistance of staphylococci. J. Bacteriol. 95:1764-1766.

50. Kirby, W.M.M. 1944. Extraction of a highly potent penicillin inactivator from penicillin resistant staphylococci. Science. 99:452-453.

51. Kirby, K.S. 1964. Isolation and fractionation of nucleic acids. Chester Beatty Research Institute, of the Institute of Cancer Research, Royal Cancer Hospital. London, England.

52. Kirby, W.M.M. and R.J. Dubos. 1947. Effect of peni­ cillin on tubercle bacillus in vitro. Proc. Soc. Exptl. Biol. Med, 66;12’0.

53. Knight, V. and R. Tompsott. 1950. Relationship of type of growth of Mycobacterium tuberculosis to anti-tuberculosis activity of subtilin. Proc. Soc. Exptl. Biol. Med. 73:55.

54. Konicek, J. and I. Malek. 1968. Mutagenic effect of ultraviolet radiation on the non-acid fast strain of Mycobacterium phlejL. Fol. Microbiol. 13:282- 287. 126

55. Kono, M., K. Ogawa, and Susurau Mitsuhashi. 1968. Drug resistance of staphylococci. J. Bacteriol. 95:886-892.

56. Korman, J. and W. Beenaan. 1962. Gerietic transduction with staphylophage. J. Bacteriol. 84;228.

57. Kubica, G.P. and W.E. Dye. 1967. Laboratory methods for clinical and public health: mycobacteriology. Public Health Service Publication. No. 1547. U.S. Government Printing Office, Washington, D.C.

58. Lederberg, J. 1952, Cell genetics and hereditary symbiosis. Physiol, Rev. 32;403.

59. Lederberg, J. and J. St. Clair. 1958, Protoplasts and L-typc; growth of Es_c]ierichia. coli. J. Bacteriol. 75;143.

60. Lillick, L.R., R. Strang L.J. Boyd, M. Schwimmcr, and M.G. Mulinos. 1955/1956. Cycloserine in the treatment of nontuberculosis infections. Antibiotics Ann. p. 158.

61. Luria, S.E. and M. Delbruck. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics. 28;491.

62. Lutkenhues, A. and W. Ritzenfeld. 1965. Zur peni- cillin-virkung auf schnell wachsende Mycobakterieri. Naturwssensehaf ten. 52 : 170.

63. Lyman, H. H.T. Epstein, and J.A. Schiff. 1961. Studies of chloroplast development: in Euglena: I. Inactivation of green colony formation by ultraviolet light. Biochim. Biophys. Acta. 50:301-309.

64. McCalla, D.R. 1965. Chloroplast mutagenesis; effect of N-methyl-N1-nitro-N-nitrosoguanidine and some other agents on Euglena, Science. 148:497- 499.

65. Mahler, H.R. and E.H. Cordes. 1966. Biological Chemistry. Harper and Row, Publishers. New York.

66. Mandel, M., C. Sehildkraut and J. Marmur. 1966. Use of CsCl density gradient analysis from determining the guanine plus cytosine content of DNA. Meth. Nucl. Acids. Vol. II, pp. 1-21. 127

67. Marmur, J. 1961. A procedure for the Isolation of DNA from microorganisms. J. Mol. Biol. 3:208- 218.

68. Marmur, J., R. Rownd, S. Falkow, L.S. Baron, C. Schildkraut, and P. Doty. 1961. The nature of intergenic episomal infection. Proc. Nat. Acad. Sci, U.S. 47:972.

69. May, J.W., R.H, Houghton and C.J. Perret. 1964. The effect of growth at elevated temperatures on some heritable properties of Staphylococcus aureus. J. Gen, Microbiol. 37:157-lb9.

70. Miller, M.A. and S.A, Harmon. 1967. Genetic Associ­ ation of determinants controlling resistance to mercuric chloride, production of penicillinase and synthesis of methionine in Star;hy 1 ococcus aureus. Nature (London). 215:531-532.

71. Mitsuhashi, S., M. Mar Laura, K. Kono and H. Oshirna. 1963, Elimination of drug resistance of Staphylococcus aureus by treatment v;Lth acri- flavine. J. Bacteriol. 86;162-164.

72. Mitsuhashi, S. 1969. Review: the R-factors. J. Infect. Dis. 119;89-100.

73. Mora, J. and L #F. Bojalil, 1965. Antagonism of the D-alanine reversal of D-cycloserinc action by L-alanine in Mycobacterium acapulccnsis. Soc. Exptl. Biol. Med. 119 : 4 9 - 3 2~7

74. Neuhaus, F.C. 1967, D-cycloserine and O-carbamyl- D-serine. In: Antibiotics I: Meehanj.sm of Action. Springer"Verlag, New York.

75. Novick, R.P. 1963. Transduction of mutations affect­ ing analysis by penicillinase formation in Staphylococcus aureus. J. Gen Microbiol. 33: Hl-l36~.

76. Novick, R.P. 1967. Penicillinase plasmids of Staphylococcus aureus. Fed. Proc. 26:29.

77. Novick, R.P. and M.H. Richmond. 1965. Nature and interactions of the genetic elements governing penicillinase synthesis in Stanhylococcus aureus, J. Bacteriol. 90:467-480. 128

78. Park, J.T. 1952. Uridine-5-pyrophosphate derivatives. I. Isolation from S. aureus II. A structure common to three derivatives III. Araino-acid containing derivatives. J. Biol. Chcm. 194:877- 937 .

79. Perry, D., and H.D. Slade. 1964. Intraspecific and interspecific transformation in Streptococci. J. Bacteriol. 88:595.

80. Reitz, R., H.D. Slade, and F.C. Neuhaus. 1966. On the biochemical basis of D-cycloserine resistance. Fed. Proc. Abstr. 25:344.

81. Richmond, M.H. 1967. Associated diploids involving penicillinase plasmids in _S. aureus. J. Gen. Microbiol. 46;85.

82. Richmond, M.H. 1968, The plasmids of Staphylococcus aureus and their relati.on to other extra- chromosomal elements in bacteria. In; Advances in Microbia 1 Physiology. Vo lu re 2, p. 43.

83. Richmond, M.H. and M. John. 1964. Co-transduction by a staphylococcal phage of the genes responsible for penicillinase synthesis and resistance to mercury salts. Nature (London). 205:1.360-1361.

84. Ritz, II. L. and J.N. 3nldv;in. 1958. Induction of penicillinase production in staphylococci by bacteriophage. Bacteriol. Proc. 40.

85. Russell, R.L., G.J. Jann and S. Fro.nan. 1960. Lysogeny in the mycobacteria. I. The establish­ ment of lysogeny. Amer. Rev. Resp. Dis. 82: 384-393.

86. Russell, R.L., G.J. Jann and 3. Froman. 1963. Lysog eny in the mycobacteria. II. Same phage- host relationships of lysogenic mycobacteria. Airier. Rev. Resp, Dis. 87:528-538.

87. Sagan, L. and S. Scher. 1961, Evidence of cytoplasmic DNA in E. gracilis. J, Protozool. 8(suppl.):8.

88. Sager, R. and Z. Ramanis, 1963. The particulate nature of non chromosomal genes in Chlamydo.nonas. Proc. Nat. Acad. Sci. U.S. 50:260-267. 129

89. Salov'ev, V.N., V.S. Zueva, and E.P. Pavlov. 1969. Increase in the antimicrobial activity of tuberculostatic drugs under the effect of acridine derivatives. Probl.. Tuberk. 47(1): 83-87.

90. Schildkraut, C.L., J, Marmur, and P. Doty. 1962. Determination of base composition of DNA from buoyant' density in cesium chloride, J. Mol, Biol. 4:430-443.

91. Singer, G. and H. Fraenkel-Conrat. 1966. Dye- catalyzed photoinactivation of tobacco mosaic virus RNA. Biochcm. 5>:2446.

92. Soltys, M.A. 1952, Cited in: J.E. Kasik, 1965. The nature of mycobacterial penicillinase. Amer. Rev. Resp. Dis. 91.: 117-118.

93. Sonea, S., R. Lawlor and J. DeRepentigny. 1966. High yields of avirulent Staphy1ococcns aureus mutants obtained with acridine orange. Bacteriol. Proc. 4_8.

94. Stocker, B, S. Smith and H. Ozcki. 1963. High in- fectivity of S, typhimurlum newly infected by the col I factor. J. Gen, Microbiol. 30:201.

95. Strange, R.E. and F.A. Dark. 1956. Drug resistance: an unidentified amino-sugar present in cell walls and spores of various bacteria. Nature (London). 177:186.

96. Strominger, J. 1962. Biosynthesis. In: The Bacteria, Volume III. Academic Press, Inc., New York, p. 413.

97. Strominger, J. 1968. Enzymatic reactions in bacterial cell wall synthesis sensitive to penicillins and other antibacterial substances. In: Micro­ bial Protopi as ts, Spheroplasts, and L-Forms. L.B. Cuze, ed. WilTiams arid Wilkins, Co, pp. 55- 60.

98. Sunahara, S. 1968. Comparison of single, double, and triple drug regimens in the re-treatment of drug-resistant pulmonary tuberculosis, Kekkaku. 43:199-207. 130

99. Sutton, VJ.B. and L. Stanfield. 1955. The reversal of cycloserine inhibition by Mycobactin, a growth factor for mycobacteria. Antibiot. Chemother. 5:582-584.

10 Tokunaga, T., Y. Mizuguchi, and T. Murohashi. 1964. Phage induction of lysogenic mycobacteria. Amer. Rev. Resp. Dis. 90: 431-436. 101. Tokunaga, T. and M.l. Sellers. 1964. Infection of Mycobacterium smegmatis with D29 phage DNA. J. Exptl.'Med. 1197139^149.

102. Tsukantura, M. and M. Hashimoto. 1960. Transfor­ mation of isonizaid and streptomycin resistance in M. avium by the DNA derived from isoniazid and streptomycin double resistant cultures. Amer. Rev. Resp. Dis. 81:403-406.

103. Vcchet, B. 1968, The enhancement of the mutagenic effect of ultraviolet radiation in Escherichia coli by caffeine and acriflavine, Fol. Microbiol. 73:380-390.

104. Vinograd, J. and J.E. Hearst. 1962. Equilibrium sedimentation of macrcmolecules and viruses in a density gradient. In: Progress in the Chemistry of Organic Natural Products XX. Springor-Verlag. New York, pp. 372-422.

105. V/atanabe, T, 1963. Infective heredity of multiple drug resistance in bacteria, Bacteriol. Revs. 27:87-115.

106. V/e aver, J. and P. Pat tee. 1964, Induced resistance to erythromycin in St:aphylococcus aureus . J, Bacteriol. 88:574-580.

107. Weinberger, H, 1965. In vivo studies on staphylococcal penicillinase. J. Bacteriol. 90:589-593.

108. White, A,, F. Foster and L. Lyons. 1961. Alteration of colonial morphology of mycobacteria associ­ ated lysogeny. Amer. Rev. Resp. Dis, 83:118.

109. White, A., F, Foster, and L. Lyons. 1962. Alteration of colonial morphology of mycobacteria associ­ ated lysogeny. J. Bacteriol. 84:815-818. 131

110. White, A, and V. Knight. 1958. Effect of Tween 80 and serum on the interaction of mycobacterio- phage D-29 with certain mycobacterial species. Amer. Rev. Resp. Dis. _7_7 : 134-145.

111. Witkin, E.M. and E.C. Theil. 1960. The effect of post-treatment with chloramphenicol on various ultraviolet-induced mutations in Escherichia coll. Proc. Nat. Acad. Sci. U.S. 4bl2*26

112. Woodruff, H.C. and J.W. Foster. 1945. Microbiologic aspects of penicillin. VII. Bacterial peni­ cillinase. J. Bacteriol. 49:7.

113. Yasuda, S. and M, Sekiguchi. 1970. Mechanism of repair of DNA in bacteriophage. II. Inability of ultra- violet-sensitive strains of bacteriophage in inducing an enzyme activity to excise pyrimidine dimers. J. Mol. Biol. 47:243-255.

114. Young, F. and A.P. Jackson, 1966. Cited in: M. Mandel C. Schildkraut and J. Marmur. Use of CsCl density gradient analysis from determing the guanine plus cytosine content of DNA. Meth. Nucl. Acids. Vol. II. 1966. pp. 1-21.

115. Zampieri, A. 1967. Mutagenic Action of Proflavine on E* C °-IA K 12, E. co 1JL B and radioresistant mutants. Giornale di Microbiol. 15:131.-136.

116. Zampieri, A. and J. Grecnsburg. 1965. Acridine orange and crystal violet as rad 5 o iiimetic agents. Giornale di Microbiol. 13;177-190.

117. Zinder, N.D. and J, Lederberg. 1952. Genetic exchange in Salmonella. J. Bacteriol. 64:679-699.

118. Zygmunt, W.A. 1963, Antagonism of D-cycloserine inhibition of mycobacterial growth by D-alanine. J. Bacteriol. 85:1217-1220.