Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroidest

The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyli under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 817 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyli molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides. bically in the dark, the bacteriochlorophyll concentration can be varied by the degree of aeration or by altering the light intensity, and both types of approach have shown the parallel between pigment and membrane content. This raises the question of the role of the bacteriochlorophyll molecule in the formation and structure of the membranes and whether synthesis of the entire molecule is needed for their development. In an attempt to answer some of these questions we have examined the ultrastructure of mutant strains of Rhodopseudomonas spheroides with blocks at various stages in bacteriochlorophyll synthesis. Some of these form magnesium-containing precursors when grown under low aeration and presumably fail at steps on the chlorophyll branch of the biosynthetic path (13). Mutant TA-R has also been examined: unlike the wild type this strain makes abundant bacteriochlorophyll when grown with high aeration (10). The characteristic intracytoplasmic membranes seen by electron microscopy of photosynthetic bacteria are presumed to contain chlorophyll and other components of the photosynMATERIALS AND METHODS Organisms. The wild type strain of R. spheroides and the various mutants derived from it have been described previously (8, 10, 13). Their characteristics are summarized in Table I. Stock cultures were maintained on yeast extract-malateglutamate agar (8), either as stab cultures grown in the light Table I. Summary of Characteristics of Wild Type and Mutanits Strain Growth' Condition Chlorophyll Precursors Accumulated Bacteriochlorophyll Carotenoids Chromatophore Formation nmoles/mg dry wt. of cells Wild type Low aeration Traces 4.8 4.0 + High aeration Nil <0.1 <0.1 L-57 Low aeration Nil ND2 ND 8-29 Low aeration 2-Devinyl-2-a-hydroxyethyl ND 10.0 chlorophyllide a (P-662) 8-17 Low aeration Bacteriochlorophyllide (P-770) ND 1.0 TA-R High aeration Nil 1.5 1.8 1 All cultures were grown in the dark. 2 Not detectable. thetic apparatus (2, 9). Examination of cells grown under various conditions has shown that the concentration of bacteriochlorophyll is correlated with the amount of these membrane structures. In the case of Athiorhodaceae which can grow aeroI This work was supported by grants from the National Science Foundation (GB 14967) and the National Institutes of Health (5 ROI AM 11148). 2 Predoctoral trainee of the National Institutes of Health Training Grant GM-1297. Present address: Department of Microbiology, University of Indiana Medical School, Indianapolis, Ind. (wild type and strain TA-R) or as slants incubated in the dark for those strains which did not make bacteriochlorophyll. Growth. Cells were grown in malate-glutamate medium supplemented with 0.2% (w/v) Difco yeast extract. For growth with low aeration, Erlenmeyer flasks were filled to 80% capacity with growth medium, and shaken on a rotary shaker at 200 rpm in the dark; these conditions promote membrane and chlorophyll synthesis in the wild type (Table I). For growth with high aeration, cells were incubated in 500 ml volumes in 2-liter Fernbach flasks on a reciprocal shaker operating at 55 oscillations/min. Such conditions repress pigment synthesis in