Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1989 Physiology of the wall-less Anaeroplasmataceae (Class Mollicutes) and related walled bacteria James P. Petzel Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons Recommended Citation Petzel, James P., "Physiology of the wall-less Anaeroplasmataceae (Class Mollicutes) and related walled bacteria " (1989). Retrospective Theses and Dissertations. 9077. https://lib.dr.iastate.edu/rtd/9077 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]. 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University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9008657 Physiology of the wall-less Anaeroplasmataceae (Class Mollicutes) and related walled bacteria Petzel, James P., Ph.D. Iowa State University, 1989 UMI SOON.ZeebRd. Ann Aibor, MI 48106 Physiology of the wall-less Anaeroplasmataceae (Class Holllcutes) and related walled bacteria by James P. Petzel A Dissertation Submitted to the Graduate Faculty In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major: Microbiology Approved: Members of the Committee: Signature was redacted for privacy. Signature was redacted for privacy. Charge of Major Work Signature was redacted for privacy. Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. F I Graduate College Iowa State University Ames, Iowa 1989 1i TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 Dissertation Format 2 LITERATURE REVIEW 4 The Class Mol Hautes 4 Physiology of the Molllcutes 17 Phylogeny of the Molllcutes 33 SECTION I. ENZYMIC ACTIVITIES OF CARBOHYDRATE, PURINE, AND PYRIMIDINE METABOLISM IN THE ANAEROPLASMATACEAE (CLASS MOLLICUTES) 41 Abstract 41 Introduction 42 Materials and Methods 44 Results 50 Discussion 54 References Cited 66 SECTION II. PYROPHOSPHATE-DEPENDENT ENZYMES IN WALLED BACTERIA PHYLOGENTICALLY RELATED TO THE WALL-LESS MOLLICUTES 72 Abstract 72 Introduction 73 Materials and Methods 75 Results and Discussion 78 References Cited 97 Ill SUMMARY AND DISCUSSION 102 Enzymic Activities of Strictly Anaerobic MolUcutes 102 Enzymologlcal Evidence Supportive of the Phylogenetic Relationships among MolUcutes and their Walled Relatives 107 REFERENCES CITED 116 ACKNOWLEDGMENTS 132 APPENDIX A: CULTURE MEDIA FOR ANAEROPLASMA SPP. AND ASTEROLEPLASHA ANAEROBIUM 135 APPENDIX B: ENZYMIC ACTIVITIES OF STRICTLY ANAEROBIC MOLLICUTES DETERMINED WITH API ZYM GALLERIES 138 APPENDIX C: STIMULATION OF GROWTH OF ANAEROPLASMA SPP. BY VARIOUS CARBON SOURCES 145 iv "We dance round in a ring and suppose But the secret sits in the middle and knows." The Secret Sits, by Robert Frost. 1 GENERAL INTRODUCTION The wall-less bacteria of the class Molîlcutes (formerly called mycoplasmas) are classified Into three orders: the Mycoplasmatales, which Include the sterol-requlring genera Mycoplasma, Ureaplasma, and Spiroplasma; the Acholeplasmatales, which Include the sterol-nonrequlring genus AcholepTasma; and the Anaeroplasmatales, which Include the sterol- requlring Anaeroplasma and the sterol-nonrequlring Asteroleplasma. The Mycoplasmatales and Acholeplasmatales have been studied for a number of years because of their Importance In human and animal medicine. In addition, Horowitz (1984) has proposed that because the molîlcutes are the smallest, free-living cells with small genomes and limited metabolic capabilities, they should serve as model organisms In an investigation of minimal requirements for independent cellular life. Many aspects of the carbohydrate, purine, pyrimidine, arginine, and urea metabolism and of the membrane physiology in the Mycoplasmatales-anà Acholeplasmatales have been elucidated. On the other hand, the first strictly anaerobic mo111cute was not described until 1973, and little is known about the intracellular metabolism of the Anaeroplasmatales. Therefore, I investigated some aspects of the physiology of the strictly anaerobic molîlcutes. In this dissertation, some enzymic activities associated with carbohydrate, purine, and pyrimidine metabolism were described for Anaeroplasma intermedium and Asteroleplasma anaeroblum. The most 2 distinctive feature noted in these organisms was the presence of several pyrophosphate-dependent (PPi-dependent) enzymes; some of these enzymes had been detected previously in other mollicutes by other investigators. These PPi-dependent enzymes occur infrequently among most bacteria. However, because these enzymes occur with greater frequency in the Mollicutes, six species of walled bacteria proposed to be phylogenetically related to the Mollicutes based on rRNA-sequence analyses were examined for PPi-dependent enzymes. The demonstration of one or more PPi-dependent enzymes in each of the walled bacteria is a general phenotypic indicator of the phylogenetic relatedness of these bacteria and the Mollicutes, and the distributions of the PPi-dependent enzymes among these bacteria correlated with phylogenetic subdivisions of this group based on 16S-rRNA analysis. Dissertation Format This dissertation is in the alternate thesis format; it includes two sections that are manuscripts. The manuscript that forms Section I has been accepted for publication in Archives of Microbiology. Section II is a manuscript that has been submitted to the International Journal of Systematic Bacteriology. A general literature review precedes the first manuscript, and a general summary and discussion follows the second manuscript. There are separate lists of references for each of the manuscripts as well as a separate list for the literature review and summary and discussion. 3 The doctoral candidate, James P. Petzel, was the principal investigator In these studies with the exception of the examination of the Embden-Meyerhof-Parnas pathway in Anaeroplasma Intermedium and pyrimidine metabolism in Asteroleplasma anaerobium. The data for these studies were collected by David DeSantis and Marshall V. Williams of the Ohio State University, respectively, using cells provided by the doctoral candidate. In addition, the doctoral candidate provided cells used in the investigations of McElwain et al. (1988) and Weisburg et al. (1989) and is co-author on those reports. 4 LITERATURE REVIEW The Class Molllcutes Introduction and history Prokaryotes that are devoid of cell walls are grouped in the Class Molllcutes. These organisms are very small (0.1 to 0.3 pm in diameter), and they can pass through membrane filters that retain other bacteria. Molllcutes are divided into three orders, the Mycoplasmatales (Freundt et al., 1984; Razin and Freundt, 1984), the Acholeplasmatales (Freundt et al., 1984), and the Anaeroplasmatales (Robinson and Freundt, 1987). Unlike these eubacteria, the remaining organism in the Class Molllcutes, Thermoplasma acidophilum, is an archeobacterium (Razin and Freundt, 1984; Woese et al., 1980)., and it will not be discussed further. The term "molllcutes" is currently used to refer to wall-less bacteria (Razin and Freundt, 1984). Previously, the term "mycoplasma" was used to refer to either any wall-less bacterium or specifically to members of the genus Mycoplasma, causing confusion. In the present discussion, the term "molllcutes" will be used, even when discussing publications that appeared before the term was invented. The names "mycoplasmas", "ureaplasmas", "spiroplasmas", and "acholeplasmas" will be used to refer to members of the respective genera. The term "strictly anaerobic molllcutes" will be used to refer to members of the family Anaeroplasmataceae. For clarity,