Subcellular Particulate Systems and the Photochemical Apparatus of Rhodospirillum Rubrum.

The photoactive pigments of the photosynthetic bacteria are believed to be localized in small spherical bodies called chromatophores.1 Biochemical studies2 have shown that chromatophore preparations catalyze a variety of light-dependent reactions (e.g., photophosphorylation of adenosine diphosphate) and they are, therefore, frequently considered to represent the functional equivalent of green plant chloroplasts. Electron micrographs3 of thin sections of mature light-grown photosynthetic bacteria indicate that chromatophores are densely packed throughout the cell, and it has been known for some time that these bodies can be readily obtained from extracts of bacteria disrupted by sonic oscillation or mechanical abrasion. These observations have led to the impression that chromatophores are discrete morphological entities dispersed throughout the cytoplasm. Sonic disruption of cells has been the most widely used method for preparing chromatophore suspensions. If sonication is extended beyond a critical time, however, photophosphorylation activity4' I and the ability to produce molecular hydrogen photochemically8 are lost. Continued sonic oscillation also leads to fragmentation of chromatophores.7 In view of these results, it seemed likely that various properties of subcellular particles obtained by sonication might be significantly altered from those of the native photochemical system in the intact cell. Studies were therefore initiated to produce fragile cell-forms, such as protoplasts, of Rhodospiiillum rubrum, from which the photochemical particulate system could be released by mild procedures.8 These preliminary experiments disclosed that virtually all of the pigmented material in "lysates" (made by osmotic shock) of Rhodospirillum protoplasts was readily sedimented at low centrifugal force. Since free chromatophores obtained by the usual procedures are sedimented only at much higher forces (e.g., 46,000 to 54,000 X g for 50 minutes2) it was suggested that (in iPvo) these bodies are associated with membranous components of the cell. The present experiments with several types of altered cell-forms of Rhodospirillum offer further evidence that the system responsible for photochemical generation of adenosine triphosphate is normally associated with the cytoplasmic membrane or a system of membranous extensions penetrating the cytoplasm. Materials and Methods. Rhodospirillum rubrum (strain SI) was grown photosynthetically at approximately 30'C in either the G3X or G5 media described by Kohlmiller and Gest.9 Both media contain L-glutamic and DL-malic acids as major components, but differ with respect to their content of complex supplements (yeast extract, peptone). Phase contrast examinations were made with the Leitz Ortholux microscope. Rates of photophosphorylation of adenosine diphosphate (ADP) were estimated by determining utilization of inorganic phosphate as outlined by Frenkel.4 Bacteriochlorophyll estimations were made using the methods of Cohen-Bazire et al.10