SOME PHYSIOLOGICAL STUDIES OF THE PHOTOSYNTHETIC AND DARK METABOLISM OF PURPLE SULFUR BACTERIA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of the Ohio State University by JOHN JACOB TAYLOR, B, Sc., M. Sc. The Ohio State University 1957 Approved byi -4 ^ {■ ■/[ CL-t'-H.'&C Adviser Department of Bacteriology ACKNOWLEDGMENT I wish to express my appreciation and gratitude to Dr. Chester I. Randles for his assistance and timely guidance throughout this investigation. ii TABLE OP CONTENTS INTRODUCTION ............................................... 1 LITERATURE R E V I E W ........................................ 4 MATERIALS AND METHODS O r g a n i s m s .............................. 11 Medium ................................... ...oil Cultures ..... ................ ...... 12 Sulfate Determination * ..........................14 Patty Acid Determination ........... 14 Determination of Sulfhydryl Compounds ..... 15 Determination of Carbon Dioxide Uptake .... 16 RESULTS Characterization Studies: Oxidation of Intracellular sulfur .... 19 Nitrogen-fixing cultures ............ ...23 Fatty acid analysis of filtrates ..... 23 Growth on sulfur compounds ........ 24 Carbon Dioxide Fixation (Illuminated) ..... 27 Carbon Dioxide Fixation (Non-Illuminated) . 33 DISCUSSION ................................................ 38 SUMMARY .................................................. 59 HI BLIOGRAPHY . ........................... 63 iii INTRODUCTION With the definition and elaboration of new methods and techniques for studying the physiology of micro­ organisms hes come an increasingly detailed characteri­ zation of the pnotosynthetic process. Before the begin­ ning of the pest decade, investigations of the photosyn­ thetic mechanisms had been more vigorous and perhaps more fruitful for the plant physiologist and the algolo- gist then for the bacterial physiologist. The plant physiologist hes at his disposal a clearly defined, photochemically active, and at present conveni­ ently isolated chloroplast which he may remove from the green plant cell and study quite independently of other cell constituents. The source of these chloroplasts is, with the exception of the fungi, as widespread and as diversified as the plant kingdom. But despite this diversity of source, all these chloroplasts contain identical photosynthetic pigments which are responsible for identical photo-autotrophic processes. On the contrary, the bacterial physiologist hes no such convenient means of isolating in an active form the chlorophylls from the photosynthetic bacteria since chloroplasts do not occur in these organisms. In addi- 1 tlon, all those bacteria known to possess a photosynthet­ ic mechanism may be classified, according to our present knowledge, in less than a dozen genera. Yet in this limited classification are included such divergent types of organisms as: strict autotrophs which must be cul­ tured anaerobically in the light; heterotrophs which may be cultured aerobically in the dark; and organisms with requirements between these two apparent extremes. Indeed, the photosynthetic bacteria may well provide a broad spectrum of assimilatory activities from the strict auto­ trophy of the green sulfur bacteria to the obligate het­ erotrophy found in the non-sulfur purple bacteria which, after repeated culture in the dark, have temporarily lost their ability to photosynthesize. Intermediate between the green and the non-sulfur purple bacteria are the purple sulfur bacteria. These organisms assimilate carbon dioxide in the light at the expense either of Incompletely oxidized inorganic sulfur compounds, or of certain simple organic acids. Their tolerance of oxygen varies among strains, although most are strict anaerobes. There has never been an unequiv­ ocal demonstration of growth In the dark, regardless of the composition of the medium or of the atmosphere above it. The non-sulfur purple bacteria, which are more readily isolated, grown, and retained in pure culture LITERATURE REVIEW Since the time of Lankester (1873) and Winogradsky (1889), accounts of the photosynthetic bacteria, and especially the purple bacteria, have contributed to conflicting ideas of the taxonomy and physiology of this group of organisms. The taxonomic status of the photo­ synthetic purple sulfur bacteria during the first quar­ ter of the twentieth century was based primarily on some­ what dubious reports of observations made of organisms morphologically different from those already recorded In the literature at the time. Many of the early descrip­ tions were the results of the chance appearance of an apparently new genus or new species in a sample of sul- fide-rieh lake water. Pure cultures of the organisms were often difficult if not impossible to prepare, and when success did attend the culture, the conditions, results, and the nature of the organisms obtained often varied considerably among investigators. The photosynthetic bacteria were even less clearly characterized physiologically. They were known to occur frequently, but not always, in environments in which sul­ fides were present. While some investigators were work­ ing with what appeared to them to be aerobic, autotrophic bacteria which evolved molecular oxygen during photosyn­ thesis, other workers studied anaerobic, heterotrophic 4 than the purple sulfur bacteria, have contributed much to our knowledge of bacterial photosynthesis. Eut their requirements for organic substrates and growth factors have made studies of their photosyntbetic metabolism somewhat tedious, even though the use of radioactive iso­ topes has gone far in overcoming the difficulties encoun­ tered in detecting the primary stages of carbon dioxide assimilation. In contrast, the purple sulfur bacteria will grow on strictly inorganic media. This ability has inclined many investigators to suggest these organisms as the more suitable subject for. a study of bacterial photosynthesis. Ebt during the past ten years, the purple sulfur bacteria have been largely neglected in such studies. The present investigation was initiated to deter­ mine the possible roles of various organic sulfur com­ pounds in the photosynthetic metabolism of the purple sulfur bacteria. The initial results obtained with sodium thioglycollate indicated that further work should be done with this compound alone. Therefore, an Inten­ sive study of the effects of thioglycollate on both the photosynthetic and the dark metabolism was undertaken. 5 bacteria which did not liberate molecular oxygen during illumination. This was the somewhat formidable view of the photo­ synthetic bacteria at the time that van J^iel (1931) pub­ lished his extensive review and monograph of the photo­ synthetic purple and green sulfur bacteria. Through his investigations and observations, van Niel qualified the contrasting reports of earlier workers by clearly delin­ eating the sulfur free the non-sulfur purple bacteria, and by defining the former group morphologically and physiologically. Three groups of purple sulfur bacteria were recog­ nized on the basis of morphology: the large cylindrical to ellipsoidal Chromatium-type. the spherical ThlocystiS' type, and the small bsciliary Pseudomonas-type. All types were either motile or non-motile, and all but the last type contained sulfur droplets within the cells if grown on media containing sulfide. The sizes and shapes of the three types varied considerably if the pH and/or sulfide concentration of the culture medium differed from those concentrations providing optimum growth at 25 C. In fact, the Pseudomonas-type appeared as the typical small bacillus, as a spiral of one-half to one turn, or as a coccus about one micron In diameter, each form depending on the pH and the amount of sulfide present. Through an anaerobic light-dependent process, the purple sulfur bacteria were shown to be capable of oxi­ dizing sulfide with concomitant incorporation of carbon dioxide into cellular substance according to the follow­ ing equations: HgS ♦ 2 H20 ♦ 2 C02 --*-2 (CHgO) ♦ H2S04 (1) Sulfur is available for storage as droplets following an incomplete oxidation of sulfide sulfur: 2 H g S ♦ C02 --- *(CH20) ♦ H20 ♦ 2 S (2) When the sulfide was depleted from the medium in which the cells were grown, intracellular sulfur droplets were further oxidized to sulfate: (3) 2 S ♦ 8 H 20 ♦ 3 C G 2 -- * 3 (CHgO) + 3 HgO ♦ 2 H 2S 0 4 Van Niel (1931) suggested the following scheme for the complete oxidation of sulfide: \ H SO, Although he found that sulfite and thiosulfate substi­ tuted for sulfide in the culture medium, sulfur droplets could never be found in cells grown under these condi­ tions. Neither was he able to demonstrate in his cul­ tures any of the Intermediates postulated in the above scheme. 7 In 1933, Muller reported that he was ahle to cul­ ture both the Chromatium- and the Thlocystls-type on acetic, propionic, lactic, pyruvic, succinic, fumaric, and malic acids anaerobically in the light. The Thio- cyatls-type also grew slightly on butyric acid, and a Chromatium-type "did not grow on glucose very well." The results of the carbon balance studies with these acids indicated that all of the added substrate was con­ verted into cell substance and a very small amount of carbon dioxide, an observation which seemingly contra­ dicted the usual anaerobic metabolism Involving an Incorporation of only about ten per cent of the sub­ strate into cell material. Van Kiel (1935) concluded that the simple organic acids which