The Morphology of Azotobacter Vinelandii
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All THE MORPHOLOGY OF AZOTOBACTER VINELANDII GROWN IN DIALYZED SOIL MEDIUM THESIS Presented to the Graduate Council of the University of North Texas in Partial Fulfillment of the Requirements For the degree of MASTER OF SCIENCE By Hoda A. Jradi, B.A. Denton, Texas August, 1992 Hoda, Jradi A. , The Morphology of Azotobacter vinelandii Grown in Dialyzed Soil Medium. Master of Science (Biology), August, 1992, 57 pp, 13 Illustrations, List of references, 6 Titles. This research describes the changes in cell morphology of Azotobacter vinelandii cells cultured in dialyzed soil medium. This particular culture medium was assumed to provide the bacteria with an environment similar to their natural habitat, the soil. Cells were grown in the medium for 4, 8 and 16 days and fixed with glutaraldehyde and osmium tetroxide. Sections were cut to a thickness of 60 to 90 nm. Observation of the cells was performed using electron microscopy. Electron micrographs of cells in young cultures showed morphological differences from cells grown in chemically-defined, nitrogen-free media. Electron micrographs of cells in older cultures revealed the presence of a cell form not previously described in the literature. These are cells approximately 0.5 pm in diameter surrounded by a thick, rigid membrane. TABLE OF CONTENTS Page LIST OF IllUSTRATIONS......................................iv INTRODUCTION................................................1i Morphology Pleomorphism and Monomorphism Cyst Formation Conclusion MATERIALS AND METHODS.....................................13 Cultures and Media Microscopic Observations Light Microscopy Electron Microscopy Negative Staining RESULTS.........................18 DISCUSSION...................................................48 LIST OF REFERENCES...........................................54 iii LIST OF ILLUSTRATIONS Figure Page 1. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing peritrichous flagellation....................22 2. Light micrograph of A. vinelandii grown in dialyzed soil medium for two days ...........24 3. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing double cell form and peritrichous flagellation-............................26 4. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing long rod shaped cells........................38 5. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing electron dense small cells, ghost cells and large spherical cells-............................30 6. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing rigid limiting membrane......................32 7. Electron micrograph of A.vinelandii grown in dialyzed soil medium for four days showing small cells with thick cell walls............ 34 8. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing multi-layered cell wall......................36 9. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days showing thick limiting edge of the cell, ghost cells and large rod shaped cells............... 38 10. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days Showing internal membrane of the cell ................ 40 iv LIST OF ILLUSTRATIONS--CONTINUED Figure Page 11. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days showing internal structure of the small cell.........42 12. Electron micrograph of A. vinelandii grown in dialyzed soil medium for 16 days showing small spherical cells, large oval cells and bizarre cells....................................44 13. Electron micrograph of A. vinelandii grown in dialyzed soil medium for 16 days showing separation of the cell wall..................46 V INTRODUCTION In 1890, Winogradsky's (48) concern with the nitrogen cycle, specifically the fixation of atmospheric nitrogen, together with his interest in the existence of oligonitrophiles, brought him success in isolating anaerobic, spore-forming bacteria capable of fixing atmospheric nitrogen which were placed in the genus Clostridium. The method that he employed depended on the removal of oxygen from the culture by aerobic organisms, making it possible for the development of anaerobic ones. His observations led him to believe that he had also encountered aerobic oligonitrophiles, but he was unable to obtain them in pure culture. In 1901, by applying the techniques used by Winogradsky in discovering the anaerobic nitrogen-fixing bacteria, Beijerinck (5) isolated pure cultures of aerobic nitrogen fixing bacteria that he called azotobacter. These came from the soils and canal waters of the city of Delft, Holland. He established the genus Azotobacter with two species, chroococcum and agile. Following this, the taxonomy and physiology of these bacteria became a popular subject of 1 2 intensive study that led to an extensive literature and several lasting controversies. Beijerinck (5) noted the similarity of characteristics between the two species of the genus Azotobacter and the blue-green alga which he had previously studied. Because of this resemblance, Beijerinck (5) named A. chroococcum after the cyanophytan of the family Chroococcacea. Jensen (19) and Kyle and Eisentark (23) disagreed with much of the work reported up to 1950 and considered Azotobacter a non-pigmented, blue-green alga. Imshenetski (18) also noted such similarities as nitrogen-fixation, cell dimensions and structures, division pattern, and capsule formation between Azotobacter and the blue-green alga, now cyanobacteria. By 1930, considerable disagreement surrounded the taxonomic position of these bacteria, their morphology, role in nature, ecology, and relationship to the plants. The description of the genus Azotobacter was surrounded by confusion as mentioned above, and the list of pleomorphic types was so lengthy that it was difficult to distinguish among different forms and also to understand existing terminology, especially when authors failed to publish photographic evidence for some of the descriptions (5, 14, 18, 19, 23). More than sixteen species of Azotobacter have been proposed by various authors, including Beijerinck's initial designations. This includes the following: Azotobacter 3 chroococcum, A. agile, A. vinelandii, A. woodstownii, A. svrmii, A. nigricans, A. araxi, A. lacticogenes, A. insigne, A. macrocytogenes, and A. paspali. Most of these species have been disregarded and Bergey's Manual of Systematic bacteriolocw (37) lists only: A. chroococcum, A. vinelandii, A. beierinckii, A. nigricans, A. armeniacus, and A. paspali. Green and Wilson (14) showed by biochemical analyses that major similarities between the two species chroococcum and beiierinckii existed. Moreover, he discovered that A. chroococcum and A. beilrinckii were practically identical, but differed significantly from A. vinelandii and A. agile. These results were confirmed by the work of De Ley and Parks (12), who studied deoxyribonucleic acid homologies and base ratio composition in the Azotobacter, and found antigenic similarities between the two species chroococcum and beiierinckii. Many differences between Azotobacter species have been made , sometimes on the basis of cultural characteristics (5, 17, 18, 20, 23). The description and morphological characteristics of Azotobacter are given in the of Bergey's Manual of Systematic Bacteriology (37) as follows: "Large ovoid cells 1.5-2.0 pm or more in diameter. Pleomorphic, ranging from rods to coccoid cells. Occur singly, in pairs or irregular clumps, and sometimes in chains of varying lengths. Do not produce endospores, but form cysts. Gram negative. Motile 4 by peritrichous flagella, or non-motile. Aerobic, but can also grow under decreased oxygen tensions. Water-soluble and water-insoluble pigments are produced by some strains of all species. Chemoorganotrophic, using sugars, alcohols and salts of organic acids for growth. Nitrogen-fixers; generally fix nonsymbiotically at least 10 mg of atmospheric nitrogen/g of carbohydrate (usually glucose) consumed. Molybdenum is required for nitrogen fixation but may be partially replaced by vanadium. Non-proteolytic. Can utilize nitrate and ammonium salts (all but one species) and certain amino acids as sources of nitrogen. Catalase positive. The pH range for growth in the presence of combined nitrogen is 4.8-8.5; the optimum pH for growth and nitrogen-fixation is 7.0-7.5. Occur in soil and water; one species occurs in association with plant roots. The mol% G + C of the DNA is 63.2-67.5 (Tm)-" Morphology A variety of morphological forms of the cells of bacteria in the genus Azotobacter have been reported by many investigators. This is a clear indication that there are many morphological variations in Azotobacter cells, and it is obvious that these were deemed of prime importance by several investigators including Lohnis and Smith (27) and Bisset and Hale (6). In 1913, Jones (20, 21) reported the presence of intracellular granules in the Azotobacter life- 5 cycle. He came to the conclusion that the filtrable inclusions represented reproductive bodies that were liberated from the mother cell and that these eventually gave rise to normal Azotobacter cells. Many authors (6, 16, 21, 26, 27, 28, 44, 49) considered the varied morphology of Azotobacter to be an expression of a complex life-cycle, with the variation in form representing stabilized stages of the cycle. The life-cycle they described