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Biochemical and Genetic Analysis of Leucine-, Isoleucine- and Valine-Dependent Mutants of Staphylococcus Aureus Creed Delaney Smith Iowa State University

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Biochemical and Genetic Analysis of Leucine-, Isoleucine- and Valine-Dependent Mutants of Staphylococcus Aureus Creed Delaney Smith Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1966 Biochemical and genetic analysis of leucine-, isoleucine- and valine-dependent mutants of Staphylococcus aureus Creed DeLaney Smith Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons Recommended Citation Smith, Creed DeLaney, "Biochemical and genetic analysis of leucine-, isoleucine- and valine-dependent mutants of Staphylococcus aureus " (1966). Retrospective Theses and Dissertations. 5336. https://lib.dr.iastate.edu/rtd/5336 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. This dissertation, has been microfihned exactly as received 67-2094 SMITH, Creed DeLaney, 1928- BIOCHEMICAL AND GENETIC ANALYSIS OF LEUCINE-, ISOLEUCINE- AND VALINE-DEPENDENT MUTANTS OF STAPHYLOCOCCUS AUREUS. Iowa State University of Science and Technology, Ph.D„ 1966 Bacteriology University Microfilms, Inc., Ann Arbor, Michigan BIOCHEMICAL AND GENETIC ANALYSIS OP LEUCINE-, ISOLEUCINE- AND VALINE-DEPENDENT MUTANTS OP STAPHYLOCOCCUS AUREUS by Creed DeLaney Smith A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OP PHILOSOPHY Major Subject: Bacteriology ) Approved: Signature was redacted for privacy. In „or Work Signature was redacted for privacy. Heaa or Major Department Signature was redacted for privacy. uate College Iowa State University Of Science and Technology Ames, Iowa 1966 il TABLE OP CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 MATERIALS AND METHODS 19 Media 19 Bacterial Strains 19 Bacteriophages 21 Isolation of Mutants 23 Biochemical Studies Zk Transduction 28 RESULTS 30 Growth Requirements of Strain 655 30 Selection of Mutants 33 Biochemical Studies 33 Genetic Studies kZ DISCUSSION 53 SUMMARY 63 ACKNOWLEDGEMENTS 65 LITERATURE CITED 66 1 INTSOEUCTION Methods of biochemical and genetic analyses were long believed to be Inapplicable to microorganisms in general, and to bacteria in particular. The small size of bacteria made them unsuitable for cytological studies. Because of their low degree of organization, no distinction between genotype and phenotype could be established. Consequently, it was long believed that the differences, both genetic and physiological, between higher organisms and microorganisms were of a funda­ mental nature, and that the laws valid for the former could not be applied to the latter. Because of their small size and rapid growth, bacteria quickly produce an enormous population in a very small volume of culture medium. These conditions are highly favorable for the appearance of variant forms and their study. Therefore, the very characters that originally seemed to exclude biochemical and genetic analyses of bacteria, in fact, make them the objects of choice for the study of variation, and it was from such studies that bacterial genetics was born. It has been shown that biochemical mutants deficient in their ability to synthesize essential metabolites have each lost a function under the control of a specific genetic determinant. The Interruption, at a definite point, of the chain of reactions leading to the biosynthesis of a specific essential metabolite is related to the absence of a specific 2 enzyme. Consequently, there is a olose relationship between genes and enzymes, eaoh gene determining the amino acid sequence of a particular enzyme. Problems of genetic control of enzyme synthesis are not, of course, confined to micro­ organisms. There is no reason to believe that most of the basic mechanisms Involved do not apply universally to living systems. However, because of their ease of manipulation, and, particularly, because of their suitability for fine structure genetic analysis, microorganisms, at present, almost monopolize research in this area of biochemical genetics. This study is concerned with the genetics and biosyn­ thesis of isoleucine, valine and leucine, in Staphylococcus aureus. aureus was chosen for this study because it is a pathogen of man and animals, unlike most previous experimental material used in the study of biochemical genetics. Further­ more, the necessary techniques for biochemical and genetic studies in this bacterium were available. Initially, the bio­ synthesis of isoleucine and valine, and the genetic control of the process was chosen for study. However, because of the close relationship of these pathways to the leucine biosyn- thetic pathway, it became necessary to broaden the scope of this study to include the effect of this amino acid as well. 3 LITERATURE REVIEW One of the basio questions that investigators in the field of genetics seek to answer is that of gene function, i.e.; how do genes control the properties of living cells and of complex, multicellular organsims? Prior to 1941, most investigators in the field of genetics concerned them­ selves with the genetic control of rather gross properties, such as wing shape in Drosophila, or the formation of conidia by Ascomycetes, Phenotypic traits were regarded simply as convenient genetic markers, because these investigators were primarily interested in studying genes as units of mutation and of recombination. However, a few investigators did exam­ ine the effect of gene mutation on discrete chemical activi­ ties of the cell and recognized that some genes have as their functions the governing of the activity of specific enzymes. For example, the earliest clear formulation of the concepts of biochemical genetics was made by the British physician. Sir Archibald Garrod, In 19O8, as a result of his studies on cer­ tain hereditary errors of human metabolism (Hayes, 1964). He pointed out that in man the enzymes concerned with the degra­ dation of the aromatic amino acids tyrosine and phenylalanine are under genetic control and that a gene mutation is re­ sponsible for the failure of one of these enzymes in the con­ genital diseases alkaptonuria and phenylketonuria (the cause of mental retardation in children). In subsequent years the 4 relationship between genetics and biochemistry became In­ creasingly apparent. Another early example of the effect of gene mutation on discrete chemical activities of the cell resulted from the studies of Beadle and Taturn (1941). In their studies with Drosophila, they succeeded in relating eye-color changes to mutationally produced blocks in the biosynthesis of eye pig­ ments, Though their experimental system was limited, from the results of their studies they hypothesized that genes control phenotypic properties by governing the formation of the enzymes of the metabolic pathways of eye pigments. They proposed that each of the constituents of the cell is synthesized by a series of biosynthetlc reactions and that each enzyme in the series is under the control of a single gene. If this were true, they reasoned, it should be possible to induce mutations, any one of which result in the failure of production of certain enzymes, thereby blocking that particular biosynthetlc pathway. In the case of microorganisms, if the end product of the blocked pathway could be supplied in the growth medium, then the mutant would be able to grow but would have a growth-factor requirement not shared by the wild-type. To prove this hypo­ thesis, Beadle and Tatum (1941) performed a series of experi­ ments on the biosynthetlc pathway of the amino acid arglnine in Neurospora erassa. They chose Neurospora as their new experi­ mental organism for two reasons: first. Its life cycle and genetics had been sufficiently studied so that any mutations 5 produced in it could be readily mapped on the chromosomes, and second; Neurospora was known to grow on a simple medium con­ taining a few inorganic salts together with a sugar as the carbon source. Following treatment of wild-type Neurospora with various mutagens, several thousand isolates were tested for their ability to grow on a nutritionally complete medium but not on a synthetic minimal medium adjusted, for growth of wild-type Neurospora, Large"numbers of mutants were isolated which were found to require one or another of almost every naturally occurring amino acid, vitamin, purine, and pyrimidine, as well as several new compounds previously not suspected to be essential for growth. Genetic analysis revealed in every case that a single gene had mutated, and in every case the nutritional deficiency could be ascribed to a specific block in a biosynthetic pathway. After determining the nature of the blocked reaction in a mutant, it became possible to compare mutant and wild-type for the presence of a particular enzyme. In every mutant a single enzyme present in the wild-type was found to be either lost or altered as a result of the single gene mutation. The work of Beadle and Tatum was extended to other microorganisms, particularly bacteria, and in a broad sense the "one gene-one enzyme" concept was substantiated. This relationship is not restricted to enzymes of biosyn­ thetic pathways. In the early studies of Garrod In I908 (Hayes, 1964) and the early studies of Beadle and Tatum (19^1), it is pointed out that it should also be possible to screen 6 for mutants blocked in degradatlve pathways. Most of the nutritional mutants
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