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The Isolation and Partial Characterization of the Lipopolysaccharides from Rhizobium Leguminosarum Biovar Trifolii Strains TB4, TB104, and TB112 (Llne)

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The Isolation and Partial Characterization of the Lipopolysaccharides from Rhizobium Leguminosarum Biovar Trifolii Strains TB4, TB104, and TB112 (Llne) Eastern Illinois University The Keep Masters Theses Student Theses & Publications 1990 The solI ation and Partial Characterization of the Lipopolysaccharides from Rhizobium leguminosarum biovar trifolii strains TB4, TB104, and TB112 Jill K. Miller Eastern Illinois University This research is a product of the graduate program in Zoology at Eastern Illinois University. Find out more about the program. Recommended Citation Miller, Jill K., "The sI olation and Partial Characterization of the Lipopolysaccharides from Rhizobium leguminosarum biovar trifolii strains TB4, TB104, and TB112" (1990). Masters Theses. 2302. https://thekeep.eiu.edu/theses/2302 This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. THESIS REPRODUCTION CERTIFICATE TO: Graduate Degree Candidates who have written formal theses. SUBJECT: Permission to reproduce theses. The University Library is receiving a number of requests from other institutions asking permission to reproduce dissertations for inclusion in their library holdings. Although no copyright laws are involved, we feel that professional courtesy demands that permission be obtained from the author before we allow theses to be copied. Please sign one of the following statements: Booth Library of Eastern Illinois University has my permission to lend my thesis to a reputable college or university for the purpose of copying it for inclusion in that institution's library or research holdings. l f ttJ9o I Date I respectfully request Booth Library of Eastern Illinois University not allow my thesis be reproduced because -------------- Date Author m The Isolation and Partial Characterization of the Lipopolysaccharides from Rhizobium leguminosarum biovar trifolii strains TB4, TB104, and TB112 (llnE) BY Jill K. Mill er THESIS SUBMIITED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 1990 YEAR I HEREBY RECOMMEND THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE lz '7i:R 199o DATE /8 ~ \l\'\,O DATE ABSTRACT The lipopolysaccharides (LPSs) from Rhizobium leguminosarum biovar trifolii TB4 and two transposon (Tn5) symbiotic mutants, R· leguminosarum bv. trifolii TB104 and TB112, were isolated and partially characterized. Bv. trifolii TB4 is a derivative of R· leguminosarum bv. trifolii ANU843. The two mutant strains elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. The LPSs were extracted using the hot phenol-water method and purified with gel filtration column chromatography using Sepharose 4B with an elution buffer consisting of EDTA and triethylamine. The hexose compositions were determined by gas chromatography of the alditol acetate derivatives. LPS components were quan­ titated by colorimetric assays. The LPSs of both mutants lacked 0-antigen sugars and some sugar residues of the LPS core oligosaccharides . Therefore, R. leguminosarum bv. trifolii TB104 and TB112 are missing LPS I, the complete form of LPS containing the 0-antigen found in t h e parent strain TB4. Polyacrylamide gel electrophoresis analysis verifies that the mutants. are missing the 0-antigen through the lack of bands in the LPS I higher molecular weight region. An immunoblot procedure using bv. trifolii ANU843 antisera confirmed these results. Thus, while the parent strain contains both LPS I (0-antigen complete) and LPS II (0-antigen incomplete), the mutants contain only LPS II. i DEDICATION to Blair and Erin ACKNOWLEDGEMENTS I thank Dr. Russell W. Carlson for his guidance, assist­ ance, and patience throughout the course of this work . Also , I thank Dr . K. Dale Noel for providing the bacterial strains used in this study. iii TABLE OF CONTENTS PAGE Abs tract . i Dedic atio n. ii Acknowledgeme nts . .. ............. ........... ........... iii Li st of Figures..... ......... .... v List of Tables .. ...... .. .... ...... .. .. ....... .. ... vi List of Appe ndic es .. ... ... ........ .. ...... ............ vii Introduction . 1 Materials and Me thods................................... 13 Results.. ........ ..... ............................... 21 Discussion...... .... ... .......................... 33 Appendices.......... .. ............................ 37 Literature Cited . .. .. .................... ............. 59 i v LIST OF FIGURES FIGURE PAGE 1. The general structural arrangement of the LPS components for £. typhimurium 8 2. A proposed structural arrangement of the LPS components for R· leguminosarum biovar legumino­ sarum, biovar trifolii, and biovar phaseoli 10 3 . Sepharose 4B EDTA-TEA gel filtration column of bv. trifolii TB4 LPS 23 4. Sepharose 4B EDTA-TEA gel filtration column of bv. trifolii TB104 LPS, water layer (a) and phenol layer (b) 24 ,... 0. Sepharose 4B EDTA-TEA gel filtration column of bv. trifolii TB112 LPS, water layer (a) and phenol layer (b) 25 6. 16 cm, 15% acrylamide DOC-PAGE gel 26 7. Gas chromatograms of bv. trifolii TB4 (a) and carboxyl reduced bv. trifolii TB4 (b) hexose alditol acetate derivatives 30 8. Gas chromatograms of bv. trifolii TB104 (a) and carboxyl reduced bv . trifolii TB104 (bl hexose alditol acetate derivatives 31 9. Gas chromatograms of bv. trifolii TB112 (a) and carboxyl reduced bv. tri folii TB112 (b) hexose alditol acetate derivatives 32 v LIST OF TABLES TABLE PAGE 1. Bacterial strains 12 2 . Tryptone yeast medium 1 3 3 . The nuclease treatment 16 4 . LPS yields of bv. trifolii strains 21 5. LPS compositions of bv . trifolii parent and mutants 28 vi LIST OF APPENDICES APPENDIX PAGE I . LPS extraction (hot phenol/water method) 37 II. Hexose assay 39 III. KDO assay 40 IV. Acetylation procedure 41 V. Carboxyl reduction procedure 43 VI. Uronic acid assay 45 VII. Assay for acetyl groups 46 VIII. Phosphate assay 48 IX . Pyruvic acid assay 49 X. Discontinuous polyacrylamide gel electrophoresis 51 XI. Silver staining procedure for lipopolysaccharides 55 XII. Immunoblot procedure 57 vii INTRODUCTION Nitrogen fixation is important both ecological ly and economically. In the developed countries of the world, the use of nitrogen fertilizers is both widespread and essential in meeting the agricultural needs of the population . In the underd~veloped countries of the world, the expense and unavailability of industrially produced nitrogen fertilizers make their use prohibitive. Therefore, nitrogen is a limiting factor of agricultural production throughout the world (1). The Gram-negative bacteria of the genera Rhizobi um and Bradyrhizobium are able to form nitrogen-fixing symbiotic relationships with members of the family Leguminoseae (beans, soybeans, clover, etc.). This mutually beneficial relationship provides nitrogen to the plant in the usable form of ammonia and carbon to the bacteria through the products of photosynthesis. Two benefits to be gained by using legumes as crop or pasture plants are the plant's independenc e of soil nitrogen and the potentially improved nitrogen-status of the soil resulting from the use of the legume (2). The host legume root nodule is the site of biological nitrogen fixation by the Rhizobium bacteria. Effective nodule de velopment, and consequently nitrog e n fixation, 1 occurs through a series of intricate interactions between the symbionts. First, rhizobia, present in the soi l, specifically bind to new host root hairs and elicit root hair curling. Generally, the root hairs that are invaded have not yet emerged from the developing root at the time of first bacterial contact. The presence of the bacteria causes the root hairs to curl as they develop. Secondly, the host cell wall is degraded in a pocket formed by the curled root hair and the bacteria penetrate the root hair cell wall . A tubular wall is deposited by the host between the invading rhizobia and the involuted root hair cel l wal l, forming an infection thread. This infection thread grows through the root hair cell wall into the root cortex . Thirdly, active cell division occurs in the root cortex forming a nodul e. Then, the bacteria are released from the infection thread into the cytoplasm of these nodule cells. The bacteria remain separate within the cytoplasm by peri­ bacteroid membranes which are derived from the infection thread. Fourthly, the bacteria differentiate into bacte- roids which produce the enzyme nitrogenase . The nodules are the site where atmospheric dinitrogen is converted into ammonia (3,4,5) . The expression of both host legume and microsymbiont rhizobial genes plays a role in nodule initiation and development. In both symbionts a portion of the genome is expressed only in the symbiotic state. Genome variation may 2 affect the sequence of nodule development. The legume produces nodulins, nodule-specific proteins. In the rhizo- bia, genes located on plasmids and on the chromosome are involved in the nitrogen-fixing symbiosis. Many genes for symbiotic function, including nodulation (nod genes) and nitrogenase activity (nif genes) occur on large Sym plasmids ( 3 , 6 , 7 , 8 ) • Early symbiotic functions encoded by the Sym plasmid includ.e bacterial binding to the root hairs, hair curling, and host specificity. Sym plasmid genes expressed during nodule development include genes for nitrogenase polypeptides and a regulatory g ene of nitrogenase activity ( 3 ) . Using transposon Tn5 mutagenesis,
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