in the Physiology of the Blue-Green Bacteria. (Algae): ..... R. A. Lewin (ed.), Physiology and biochemistry of algae. Academic Press Inc., New York. 6. Fogg, G. E. ...
Vol. 114, No. 2 Printed in U.SA.
JouRNAL OF BAcTRIoLOGY, May 1973, p. 701-706 Copyright 0 1973 American Society for Microbiology
Role of Reduced Exogenous Organic Compounds in the Physiology of the Blue-Green Bacteria (Algae): Photoheterotrophic Growth of an "Autotrophic" Blue-Green Bacterium L.
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INGRAM,' C. VAN BAALEN, AND J. A. CALDER
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, The University of Texas Marine Science Institute, Port Aransas, Texas 78373, and Department of Oceanography, Florida State University, Tallahassee, Florida 32306 Received for publication 24 November 1972
The unicellular blue-green bacterium Agmenellum quadruplicatum strain BG-1 was found to be capable of rapid photoheterotrophic growth but unable to grow in the dark on a variety of reduced organic substrates. The generation time on glycerol was 12 h, and on CO2, 3 h. Glycerol carbon was converted into cellular carbon with a very high efficiency. This high efficiency of carbon conversion, the action spectrum for growth on glycerol, cell pigmentation, gas exchange measurements, and immediate ability of photoheterotrophically grown cells to evolve 02 (upon the addition of C02) suggest the involvement of both photosystems I and H of photosynthesis during photoheterotrophic growth.
The blue-green bacteria have long been considered to be autotrophic. Enrichment procedures for this group, without exception, are based on their ability to grow at the expense of light, CO,, and minerals without organic supplements (1, 2, 29). After surveying a number of these organisms for their ability to grow heterotrophically in the dark, algal and microbial physiologists have largely concluded that blue-green bacteria are obligately autotrophic (8, 10, 15, 17, 28). Very few strains in this group have been found capable of dark heterotrophic growth (9, 16, 28), and most of these grow at an exceedingly slow rate and on a very limited number of substrates (glucose, fructose, and sucrose). Bulk CO2 fixation is coupled to photosynthesis during autotrophic growth of blue-green bacteria. During mixotrophic growth with both C02 and substrate under high light, C00 is still the preferred carbon source. However, by restricting the availability of C02 to the level found in air under high light, Pearce and Carr (21) demonstrated that it is possible to impair selectively the carbon side of photosynthesis and to force blue-green bacteria
to use reduced organic compounds as the major source of cellular carbon. This has been subsequently confirmed in Nostoc sp. strain Mac (13). Further, by omitting exogenous CO. completely, it is possible to force strain Mac to use a diversity of compounds in the light which do not support dark heterotrophic growth. This enhanced ability of a blue-green bacterium, capable of dark heterotrophic growth on only a limited number of substrates, to use reduced compounds as a sole source of carbon in the light suggests the possibility that other blue-green bacteria, previously regarded as obligate photoautotrophs, may exhibit versatile carbon metabolism in the light in the absence of
C02. In this paper, we describe the photoheterotrophic growth of the blue-green bacterium Agmenellum quadruplicatum strain BG-1 in the absence of CO2 with glycerol as the sole source of carbon. MATERIALS AND METHODS Organisms. A. quadruplicatum strain BG-1 was
originally isolated into axenic culture by C. Van Baalen (29). Strain BG-1 was grown batchwise in test-tube cultures at 39 C in medium ASP2 + B12 (24) as described previously (12, 18). Agitation was I Present address: Department of Microbiology, University provided in autotrophic cultures by continuous gassing with 1% CO,-enriched air. Photoheterotrophic of Florida, Gainesville, Fla. 32601. 701
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INGRAM, VAN BAALEN, AND CALDER
cultures were gassed continuously with tank N2 scrubbed through four successive CO2 traps (10% aqueous KOH). Illumination was provided by four 30-W deluxe cool white fluorescent lamps, two on each side of the bath, producing about 300 ft-c. Whole-cell spectra were obtained with a Bausch & Lomb Spectronic 505 colorimeter by use of the opal glass technique of Shibata et al. (27). Analyses. Growth was measured turbidimetrically with a Lumetron colorimeter and by dry weight after filtration on tared membrane filters (Millipore Corp., 0.45-ym pore size). Glycerol was determined according to the procedure of Burton (3). This reaction is nonspecific for all terminal vicinal hydroxyl groups. Total keto acids were determined colorimetrically (7). Nitrite was determined according to the method of Snell, as described by Nichols and Hason (19). Stable carbon isotope ratios were determined by use of the facilities of P. L. Parker, as described previously (4, 13). Gas exchange experiments were performed with a Clark 02 electrode (Gilson Medical Electronics, OX700) as described previously (13). Crude action spectra for photoheterotrophic growth were determined in a compartmented water bath by use of Bausch & Lomb second-order interference filters and appropriate Corning blocking filters. Incident light intensity was adjusted to 390 MW/cm2 in each case.
RESULTS No truly unicellular blue-green bacteria have been shown capable of dark heterotrophic growth. One of these bacteria, A. quadruplicatum strain BG-1, was selected as a model organism for the investigation of possible photoheterotrophic growth. PR-6, a morphologically identical organism with the same base ratio (Manley Mandell, personal communication), has been reported previously to be incapable of dark heterotrophic growth (28, 29). Figure 1 shows a typical growth curve for photoheterotrophic growth of BG-1 with glycerol as the sole source of carbon (mean generation time, 12 h). Glycerol disappearance parallels the increase in dry weight, suggesting a very high efficiency for carbon conversion. Table 1 shows a summary of five separate experiments conducted to determine the carbon efficiency of BG-1. These indicate that most of the glycerol carbon is converted into new cellular material. The difference in stable carbon isotope ratio relative to NBS20 of the glycerol carbon is -21.3 parts per thousand; that of BG-1 grown photoheterotrophically on glycerol is - 22.6 parts per thousand. This further substantiates that glycerol carbon serves as the major source of cellular carbon. Morphologically and ultrastructurally, photoheterotrophic cells are very similar to autotrophic cells. Abundant glycogen reserves
J. BACTERIOL.
are present in both. The pigmentation of BG-1 grown photoheterotrophically is very similar to that of autotrophically grown cells (Fig. 2). The relative amounts of chlorophyll a and phycocyanin are approximately the same. However, the total amounts of these two pigments are somewhat diminished with respect to the carotenoid levels and the cell concentration. A crude action spectrum was determined for photoheterotrophic growth of BG-1 (Fig. 3). This action spectrum closely resembles that of photosynthesis (14). Wavelengths absorbed by both photosynthetic pigments are most effective. Exponentially growing cultures were taken directly from photoheterotrophic or autotrophic growth and placed in a sealed 02 electrode chamber for gas exchange measurements (Fig. 4). Photoheterotrophic cultures contain a low initial level of dissolved 02 (5,uliters/ml), equal in magnitude to one-eighth air saturation. During the first few minutes, a short burst of 02 was evolved, after which a low steady rate of 02 evolution was rapidly achieved. This rate (10 Aliters/mg .h) appears to be constant and has been followed with the electrode for a period of more than 4 h. The low rate of net 02 evolution was accepted as being representative of normal photoheterotrophic growth. Upon addition of NaHCO8, the photoheterotrophic culture immediately resumed rapid photosynthetic activity at a rate (230 Aliters/mg of dry weight h) similar to that of autotrophic cultures. Dark respiratory rates for photoheterotrophic and autotrophic cultures (18 Aliters/mg of dry weight h) are identical either under high or low 02 concentration. Figure 4 shows a comparison of photosynthetic (bicarbonate added) and net phototoheterotrophic rates of 02 evolution with the rate of dark respiratory 02 consumption in photoheterotrophic cultures.
DISCUSSION Photoheterotrophic growth with a reduced organic substrate as the sole source of carbon does not appear to be an isolated phenomenon. Both strains Mac (13) and BG-1 will utilize reduced organic substrates in light in the absence of exogenously supplied CO2 which will not support their respective dark heterotrophic growth. As suggested by Rittenberg (26), the term "obligate autotroph" with respect to carbon metabolism should be abandoned. Photoheterotrophic growth effectively reduces the problem of obligate autotrophy to one of obligate phototrophy in the blue-green bacteria. There are a variety of possible mechanisms
VOL. 114,1973
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