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Smith, A. J., London, J. & Stanier, R. Y . (1967) J. Bacteriol. 94, 972-983 Smith, K. M., Brown, R. M., Jr., Goldstein,D. A. & Walne, P. L. (1966) ViroIogy28,580-591 Stanier, R. Y . (1964) in The Bacteria (Gunsalus,I. C . & Stanier, R. Y . ,eds.), vol. 5, pp. 445-464, Academic Press, London Stanier, R. Y . & van Niel, C. B. (1941) J. Bacteriol. 42, 437-466 Stanier, R. Y . & van Niel, C. B. (1962) Arch. Mikrobiol. 42, 17-35 Stewart, W. D. P. & Lex, M. (1970) Arch. Mikrobiol. 73, 250-260 Stitzenberger, E. (1860) in Dr. L . Rabenhorsts AlgenSachsensresp. Mittel Europa's Systematische Geordnet mit Zugrundlegung Eines Neuen Systems, Decaden I-C, Dresden Stewart, W. D. P., Rowell, P. & Tel-Or, E. (1975) Biochem. SOC.Trans. 3, 357-361 Sugiyama, T. & Akazawa, T. (1967) J. Biochem. (Tokyo) 63, 4 7 4 8 2 Tabita, R. F., Stevens, S. E. & Quijano, R. (1974) Biochem. Biophys. Res. Commun. 61,45-52 Taylor, B. F. & Hoare, D. S. (1969) J. Bacteriol. 100, 487-497 Taylor, M. M. & Storck, R. (1964) Proc. Nut. Acad. Sci. US.52, 958-965 Tolbert, N. E. (1971) Annu. Rev. Plant Physiol. 22, 45-74 Tolbert, N. E. (1973) Curr. Top. Cell. Regul. 7, 21-50 Van Baalen, C., Hoare, D. S. & Brandt, E. (1971) J. Bacferiol. 105, 685-689 Zelitch, I. (1964) Annu. Rev. Plant Physiol. 15, 121-139
The Utilization of Organic Substrates by Cyanobacteria ROGER Y . STANIER Service de Physiologie Microbienne, Institut Pasteur, Paris 7501 5 , France The dominant mode of energy-yielding metabolism in cyanobacteria is oxygenic photosynthesis, and the dominant nutritional mode of these organisms is photoautotrophy. Many cyanobacteria are obligate photoautotrophs, dependent on light as energy source and on CO, as principal carbon source. Other cyanobacteria are facultative photo- or chemo-heterotrophs. Facultative photoheterotrophs can use one or more organic compounds in place of CO, as a principal carbon source in the light; they do not, however, necessarily derive energy from the metabolism of the organic substrate, since light can in principle supply all energy requirements for growth. Facultative chemoheterotrophs can derive both carbon and energy from the dark metabolism of organic substrates. All facultatively chemoheterotrophic cyanobacteria have so far also proved to be facultative photoheterotrophs; however, the converse is not always true (Van Baalen et al., 1971 ; Rippka, 1972). The unambiguous demonstration of photoheterotrophy in cyanobacteria and in eukaryotes that perform oxygenic photosynthesis poses a methodological problem. I n this mode of photosynthesis, the reductive assimilation of CO, is intimately related to photochemical events, since reductant (NADPH) is generated through the complex sequence of reactions triggered by the photo-oxidation of water, known as non-cyclic photophosphorylation. To show that an organic compound can serve as the principal carbon source in the light, it is necessary to prevent or inhibit photosynthetic CO, assimilation. This has been achieved in several ways. The most direct means is to eliminate CO, from the culture medium; there is a risk, however, that minor but perhaps essential anaplerotic C0,-fixing reactions (e.g. synthesis of oxaloacetate from pyruvate or phosphoenolpyruvate) will also be prevented. Nevertheless, by the use of this technique Ingram et al. (1973) demonstrated photoheterotrophic growth of a cyanobacterium at the expense of glycerol. Photoheterotrophy in cyanobacteria has also been demonstrated in cultures exposed to very low light-intensities, insufficient to support measurable photoautotrophic growth. Van Baalen et al. (1971) observed that, under these conditions, the addition of glucose to the medium permitted growth. Presumably the lightflux permitted a rate of A W synthesis just sufficient to supply the maintenance energy 1975
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required under photoautotrophic conditions; growth on the addition of glucose is possible because the overall ATP requirement for biosynthesis from this carbon source is considerably less than that from COz. A third method of demonstrating photoheterotrophy, which has been developed and extensively applied in our laboratory (Rippka, 1972), involves the use of the herbicide 3-(3,4-dichlorophenyl)-l,l-dimethylurea. As shown by Bishop (1958), low concentrations of this compound inhibit Photosystem 2 activity, thereby blocking the photochemical generation of reducing power and the assimilation of COz, but still permit ATP synthesis by cyclic photophosphorylation. At a concentration of 1 0 , 3-(3,4-dichloro~ ~ pheny1)-1,l -dimethylurea completely arrests photoautotrophic growth of cyanobacteria. Reversal of the growth inhibition by glucose or other organic substrates constitutes prima facie evidence that these compounds can replace COz as the principal carbon source for photosynthetic growth. The growth rate at the expense of glucose in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea and in the light is much higher than the growth rate at the expense of glucose in the dark; indeed, some cyanobacteria that grow well under the former conditions appear to be incapable of chemoheterotrophic growth (Rippka, 1972). The phenomenon of obligate photoautotrophy, so prevalent in cyanobacteria, has long presented a challenge to the biochemist. Further, some aspects of the organic nutrition of cyanobacteria, summarized below, raise additional biochemical questions. (1) The range of substrates able to support heterotrophic growth is very limited. Glucose is the most widely used substrate, closely followed by fructose and sucrose. Other compounds that permitheterotrophicgrowth of a few strains are glycerol (Ingram et al., 1973), ribose and gluconate (R. Rippka, unpublished work). (2) Growth rates in the dark are very low, always far lower than the phototrophic growth rate of the same strain (Hoare et al., 1971; Rippka, 1972). (3) Even obligately photoautotrophic cyanobacteria readily assimilate amino acids and certain organic acids (notably pyruvate and acetate) in the light (Hoare & Moore, 1965; Hoare et al., 1967; Smith et al., 1967). Assimilation is strictly light-dependent, and none of these compounds can serve as a principal carbon source. Smith et al. (1967) found that acetate, the most rapidly assimilated of the compounds examined, contributed only about 10% to the carbon of newly synthesized cell compounds, the rest being derived from COz. (4) During photoautotrophic growth, assimilated carbon in excess of immediate biosynthetic requirements is stored as glycogen (Kindel & Gibbs, 1963). Glycogen also accumulates as the major assimilatory product during the light or dark metabolism of sugars (Cheung & Gibbs, 1966; Pelroy et al., 1972). Placed in the dark in the absence of a n exogenous substrate, all cyanobacteria perform an endogenous respiration, presumably at the expense of their glycogen reserve. This respiratory activity is coupled with ATP synthesis (Biggins, 1969). Endogenous respiration is sharply diminished by exposure to light, even of low intensity (Brown & Webster, 1953; Jones & Myers, 1963). Light has also been shown to inhibit severely the respiration of exogenous glucose, an effect almost completely abolished by 10p~-3-(3,4-dichlorophenyl)-1,l-dimethylurea (Pelroy et al., 1972). ( 5 ) The dark energy-yielding metabolism of many cyanobacteria appears to be strictly respiratory: several attempts to grow chemoheterotrophic strains anaerobically in the dark at the expense of sugars, or to demonstrate biosynthesis under these conditions, have given negative results (Pelroy et al., 1972; Pelroy & Bassham, 1973; R. Rippka, unpublished work). Hoare et al. (1971) reported very low rates of dark anaerobic growth at the expense of sugars by a Nostoc sp.; however, it is not certain that the cultures were rigorously 02-free. The possibility that certain cyanobacteria may possess weak fermentative capacity capable of supporting a very low rate of dark anaerobic growth merits further systematic exploration, particularly in view of the long-overlooked ability of some non-sulphur purple bacteria to grow anaerobically in the dark by fermentation (Uffen & Wolfe, 1970). VOL
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Recent biochemical studies make it possible to account for many of the physiological data outlined above in metabolic terms. The relevant biochemical findings are described below. Absence of a functional tricarboxylic acid cycle
Smith et al. (1967) observed that the photoassimilation of amino acids or organic acids by obligately photoautotrophic cyanobacteria results in entry of substrate carbon into a rather limited number of biosynthetic pathways. Further, the specific patterns of incorporation suggested that the tricarboxylic acid cycle was non-functional. This inference was confimed by enzymic analysis: in cell-free extracts of the three cyanobacteria examined, 2-oxoglutarate dehydrogenase activity was undetectable, and the activities of both malate dehydrogenase and succinate dehydrogenase were very low. There is also evidence for the absence of a functional tricarboxylic acid cycle in other cyanobacteria (Hoare et al., 1969; Van Baalen et al., 1971; Pelroy et al., 1972). This deficiency is probably characteristic of the group as a whole, and accounts for the inability of such compounds as acetate, pyruvate and dicarboxylic acids to serve as sole sources of carbon and energy for growth in the dark, even though they can be readily assimilated in the light. Respiratory metabolism of sugars
The pathway of glucose respiration has been studied in three facultatively heterotrophic cyanobacteria (Cheung & Gibbs, 1966; Pearce & Carr, 1969; Pelroy et al., 1972). The data of Pelroy et al. (1972) on a unicellular cyanobacterium, Aphanocapsu 6714, are the most complete. In all three cases the oxidative pentose phosphate pathway appears to be the major (and very probably the sole) route of glucose oxidation. Thus radiorespirometric experiments show that oxidation of glucose carbon atoms is highly asymmetric, COz arising preferentially from position 1.The cells always contain high activities of the two dehydrogenases operative in this pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). On the other hand the activities of enzymes specific to the Embden-Meyerhof pathway (phosphofructokinase) and the Entner-Doudoroff pathway (6-phospho-2-keto-3-deoxygluconatealdolase and 6-phosphogluconate dehydratase) are either very low or undetectable. Finally, in cells respiring glucose, glucose 6-phosphate and 6-phosphogluconate together account for over half the intracellular pool of phosphorylated intermediates. The two dehydrogenases of the oxidative pentose phosphate pathway are NADP+specific, in the cyanobacteria so far examined. This implies that respiratory electron transfer proceeds almost wholly through NADP+ rather than through NAD+. Interplay between the reductive and oxidative pentose phosphate cycles
Detailed kinetic studies on 14COzincorporation into components of the metabolic pool, conducted on four unicellular cyanobacteria, reveal that COz fixation proceeds viathe reductive pentose phosphate(Ca1vin-Benson) pathway(Pe1roy &Bassham, 1972). Typical data are shown in Fig. 1. The first stable product, and the only polymer formed in significant amounts during short-term fixation experiments, is glycogen. As shown in Fig. 1;a light-dark transition after exposure to 14COzfor lOmin is followed by a very rapid disappearance from the pool of ribulose 1,s-diphosphate, correlated with a rise in the pool concentration of 3-phosphoglycerate, and by the appearance in the pool of a previously undetectable intermediate, 6-phosphogluconate. These observations suggest that a light-dark transition brings into play the oxidative pentose phosphate cycle, used to generate maintenance energy at expense of the cellular glycogen 'reserve. Biggins (1969) demonstrated that daik endogenous respiration proceeds via NADP+, an observation in accord with the nicotinamide nucleotide specificities of cyanobacterial glucose 1975
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0
.-*m
B&
Time (min) Fig. 1. Intracellular pool concentrations of 14C-labelledphosphorylatedintermediates in a unicellular cyanobacterium, Aphanocapsa 6308
''COz was introduced at zero time, and the cells were incubated for a period of lOmin in the light, followed by a period of 8min in the dark. 8,3-Phosphoglycerate; A , glucose 6-phosphate; 0,ribulose 1,s-diphosphate; A, 6-phosphogluconate; 0 , fructose 1,6diphosphate; EU, sedoheptulose 1,7-diphosphate. The data are taken from Pelroy & Bassham (1 972).
11
Glucose I-phosphate
J
4J-
4
2 Glyceraldehyde 3-phosph~te
Ti. NADPH
j-c
Ribulose 1,5diphosphate
Scheme 1. Primary pathways of carbon metabolism in cyanobacteria Primary substrates and metabolic end products are enclosed in square boxes. Reactions specific to photosynthetic CO, assimilation are designated by white arrows, and reactions specific to dark respiratory metabolism by heavy black arrows. The site of regulation of the respiratory pathway by ribulose 1,s-diphosphate is indicated by the symbol: (Ribulose 1,5-diphosphate) =. The scheme is adapted from Pelroy et al. (1972).
6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Doolittle & Singer (1 974) have shown that mutational elimination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in an obligately photoauto-
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15
-
20
-
I5
-
10 5
0 0 1
0.5
I .o
I0
Concn. of glucose (mM) Fig. 2. Comparison of the rates of 14C02formation from [U-'4Clp1ucose as a function of glucose concentration by three unicellular cyanobacteria
m, Aphanocapsa 6714, a facultative chemoheterotroph; orAphanocapsa 6308, an obligate photoautotroph; 0 , Synechococcus 6301, also an obligate photoautotrophs. The data are taken from Pelroy et al. (1972).
trophic unicellular cyanobacterium decreases endogenous respiration to an undetectable level. The primary pathways of carbon metabolism of cyanobacteria in the light and in the dark are schematized in Scheme 1. Photoinhibition of respiration The photoinhibition of the respiration of both endogenous and exogenous substrates, a distinctive attribute of cyanobacteria, reflects the alosteric inhibition of glucose 6-phosphate dehydrogenase by ribulose 1,5-diphosphate, the only phosphorylated intermediate unique to the reductive pentose phosphate cycle (Pelroy et a f . , 1972). The oxidative pentose phosphate pathway is thus held in check during photosynthetic metabolism, but comes almost instantly into play in the dark, as a result of the very rapid disappearance of ribulose 1,5-diphosphate from the metabolic pool (see Fig. 1). Doolittle & Singer (1974) have provided a striking demonstration of the operation of this control mechanism in an obligately photoautotrophic cyanobacterium. They isolated mutants with greatly impaired ability to survive in the dark (less than 1% survival after 30min without illumination!). Without exception, these mutants had lost 6-phosphogluconate dehydrogenase activity. Their rapid death in the dark is attributable to the intracellular accumulation of 6-phosphogluconate, after the release of the ribulose 1,5-diphosphate-mediatedinhibition of glucose 6-phosphate dehydrogenase activity. Selection of phenotypic revertants with restored capacity to survive in the dark resulted in the isolation of double mutants, which had also lost glucose 6-phosphate dehydrogenase activity. Phenomenon of obligate autotrophy If all cyanobacteria use the oxidative pentose phosphate pathway for dark endogenous metabolism, why are some, but not others, obligate photoautotrophs? The inability to utilize exogenous glucose could in principle reflect either absence of hexokinase or absence of a specific glucose permease. Pelroy et al. (1972) found hexokinase activities in two obligate photoautotrophs to be 25-50 % of those in a heterotrophic cyanobacterium; however, there was a very marked difference between the obligate autotrophs and the facultative heterotroph in the affinity of intact cell for glucose (Fig. 2). These authors accordingly suggested that the absence of specific permeases, required to mediate entry
1975
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of the relatively small number of substrates that can feed into the oxidative pentose phosphate cycle, is the most probable explanation for the widespread occurrence of obligate photoautotrophy among cyanobacteria. However, certain cyanobacteria that are facultative photoheterotrophs, able to grow'in the light in the presence of 3-(3,4dichloropheny1)-1,I-dimethylurea at the expense of glucose, seem wholly incapable of dark growth with glucose. This phenomenon obviously cannot be accounted for by postulating a permeability barrier; its explanation remains unknown. Biggins, J. (1969) J. Bucteriol. 99, 570-575 Bishop, N. I. (1958) Biochim. Biophys. Acra 27, 205-206 Brown, A. H. & Webster, G. C. (1953) Amer. J. Bot. 40,753-757 Cheung, W. Y. & Gibbs, M. (1966) Plant Physiol. 41, 731-737 Doolittle, W. F. & Singer, R. A. (1974) J. Bacteriol. 119, 677-683 Hoare, D. S. & Moore, R. B. (1965) Biochim. Biophys. Acta 109, 622-625 Hoare, D. S., Hoare, S. L. & Moore, R. B. (1967) J. Cen. Microbiol. 49, 351-370 Hoare, D. S., Hoare, S. L. & Smith, A. J. (1969) in Progress in Photosynthesis Research (Metzner, H., ed.), vol. 3, pp. 1570-1573, Laupp, Tubingen Hoare, D. S., Ingram, L. O., Thurston, E. L. & Walkup, R. (1971) Arch. Mikrobiol. 78,310-321 Ingram, L. O., Van Baalen, C. & Calder, J. A. (1973)f. BacterioL 114, 701-705 Jones, L. W. & Myers, J. (1963) Nature (London) 199, 670-672 Kindel, P. & Gibbs, M. (1963) Nature (London) 200,260-261 Pearce, J. & Carr, N. G. (1969) J. Gen. Microbiol. 54, 451-462 Pelroy, R. A. & Bassham, J. A. (1972) Arch. Mikrobiol. 86, 25-38 Pelroy, R. A. & Bassham, J. A. (1973) J. Bucteriol. 115,937-942 Pelroy, R. A., Rippka, R. & Stanier, R. Y. (1972) Arch. Mikrobiol. 87, 303-322 Rippka, R. (1972) Arch. Mikrobiol. 87, 93-98 Smith, A. J., London, J. & Stanier, R. Y. (1967) J. Bacteriol. 94, 972-983 Uffen, R. L. & Wolfe, R. S. (1970) J. Bacteriol. 104, 462-472 Van Baalen, C., Hoare, D. S. & Brandt, E. (1971) J. Bacteriol. 105, 685-689
Nitrogen Fixation and the Weterocyst in Blue-Green Algae W. D. P. STEWART, P. ROWELL and E. TEL-OR Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, U.K. The blue-green algae, or cyanobacteria, are Oz-evolving prokaryotes, many species of which fix Nz. Greatest morphological differentiation occurs in some Nostocales, and in the Stigonematales where, in addition to the typical vegetative cells of blue-green algae, there are two additional cell types: akinetes and heterocysts. The role of akinetes as perennating structures is well established (see Fogg et al., 1973), and, though various views have been proposed on the role of the heterocyst, it is accepted generally that its main function is in N, fixation. The close correlation between heterocysts and N, fixation is seen at the genetic and molecular levels. In genetic studies on the heterocystous Nostoc muscorum we have obtained various nitrogenase-less mutants (i.e. mutants unable to grow or reduce acetylene under aerobic or microaerobic conditions) and usually these mutants also lack heterocysts. By using one such mutant into which resistance to streptomycin (1000pg/ml) was introduced (nif-st-R),evidence of nif-gene transfer has been obtained on mixing it with wild-type (nif+st-S) Nosfocmuscorum. In such studies the prototrophic streptomycin-sensitive donor was counter-selected, and when this was done nif+st-R colonies developed at a frequency of 2.2 in lo5colonies, 4.6 in los colonies and 5.0 in lo6 colonies in three different experiments. These values are significantly higher than those attributable to spontaneous mutation (1 in lo7 or less), and the higher yield of new genotypes may be interpreted as evidence of nif-gene transfer, although in genetic studies of this type the possibility cannot be ruled out that what appears to be nif-gene transfer may be transfer of some factor that allows the expression of latent rzifgenes in the non-N2-
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Smith, A. J., London, J. & Stanier, R. Y . (1967) J. Bacteriol. 94, 972-983 Smith, K. M., Brown, R. M., Jr., Goldstein,D. A. & Walne, P. L. (1966) ViroIogy28,580-591 Stanier, R. Y . (1964) in The Bacteria (Gunsalus,I. C . & Stanier, R. Y . ,eds.), vol. 5, pp. 445-464, Academic Press, London Stanier, R. Y . & van Niel, C. B. (1941) J. Bacteriol. 42, 437-466 Stanier, R. Y . & van Niel, C. B. (1962) Arch. Mikrobiol. 42, 17-35 Stewart, W. D. P. & Lex, M. (1970) Arch. Mikrobiol. 73, 250-260 Stitzenberger, E. (1860) in Dr. L . Rabenhorsts AlgenSachsensresp. Mittel Europa's Systematische Geordnet mit Zugrundlegung Eines Neuen Systems, Decaden I-C, Dresden Stewart, W. D. P., Rowell, P. & Tel-Or, E. (1975) Biochem. SOC.Trans. 3, 357-361 Sugiyama, T. & Akazawa, T. (1967) J. Biochem. (Tokyo) 63, 4 7 4 8 2 Tabita, R. F., Stevens, S. E. & Quijano, R. (1974) Biochem. Biophys. Res. Commun. 61,45-52 Taylor, B. F. & Hoare, D. S. (1969) J. Bacteriol. 100, 487-497 Taylor, M. M. & Storck, R. (1964) Proc. Nut. Acad. Sci. US.52, 958-965 Tolbert, N. E. (1971) Annu. Rev. Plant Physiol. 22, 45-74 Tolbert, N. E. (1973) Curr. Top. Cell. Regul. 7, 21-50 Van Baalen, C., Hoare, D. S. & Brandt, E. (1971) J. Bacferiol. 105, 685-689 Zelitch, I. (1964) Annu. Rev. Plant Physiol. 15, 121-139
The Utilization of Organic Substrates by Cyanobacteria ROGER Y . STANIER Service de Physiologie Microbienne, Institut Pasteur, Paris 7501 5 , France The dominant mode of energy-yielding metabolism in cyanobacteria is oxygenic photosynthesis, and the dominant nutritional mode of these organisms is photoautotrophy. Many cyanobacteria are obligate photoautotrophs, dependent on light as energy source and on CO, as principal carbon source. Other cyanobacteria are facultative photo- or chemo-heterotrophs. Facultative photoheterotrophs can use one or more organic compounds in place of CO, as a principal carbon source in the light; they do not, however, necessarily derive energy from the metabolism of the organic substrate, since light can in principle supply all energy requirements for growth. Facultative chemoheterotrophs can derive both carbon and energy from the dark metabolism of organic substrates. All facultatively chemoheterotrophic cyanobacteria have so far also proved to be facultative photoheterotrophs; however, the converse is not always true (Van Baalen et al., 1971 ; Rippka, 1972). The unambiguous demonstration of photoheterotrophy in cyanobacteria and in eukaryotes that perform oxygenic photosynthesis poses a methodological problem. I n this mode of photosynthesis, the reductive assimilation of CO, is intimately related to photochemical events, since reductant (NADPH) is generated through the complex sequence of reactions triggered by the photo-oxidation of water, known as non-cyclic photophosphorylation. To show that an organic compound can serve as the principal carbon source in the light, it is necessary to prevent or inhibit photosynthetic CO, assimilation. This has been achieved in several ways. The most direct means is to eliminate CO, from the culture medium; there is a risk, however, that minor but perhaps essential anaplerotic C0,-fixing reactions (e.g. synthesis of oxaloacetate from pyruvate or phosphoenolpyruvate) will also be prevented. Nevertheless, by the use of this technique Ingram et al. (1973) demonstrated photoheterotrophic growth of a cyanobacterium at the expense of glycerol. Photoheterotrophy in cyanobacteria has also been demonstrated in cultures exposed to very low light-intensities, insufficient to support measurable photoautotrophic growth. Van Baalen et al. (1971) observed that, under these conditions, the addition of glucose to the medium permitted growth. Presumably the lightflux permitted a rate of A W synthesis just sufficient to supply the maintenance energy 1975
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required under photoautotrophic conditions; growth on the addition of glucose is possible because the overall ATP requirement for biosynthesis from this carbon source is considerably less than that from COz. A third method of demonstrating photoheterotrophy, which has been developed and extensively applied in our laboratory (Rippka, 1972), involves the use of the herbicide 3-(3,4-dichlorophenyl)-l,l-dimethylurea. As shown by Bishop (1958), low concentrations of this compound inhibit Photosystem 2 activity, thereby blocking the photochemical generation of reducing power and the assimilation of COz, but still permit ATP synthesis by cyclic photophosphorylation. At a concentration of 1 0 , 3-(3,4-dichloro~ ~ pheny1)-1,l -dimethylurea completely arrests photoautotrophic growth of cyanobacteria. Reversal of the growth inhibition by glucose or other organic substrates constitutes prima facie evidence that these compounds can replace COz as the principal carbon source for photosynthetic growth. The growth rate at the expense of glucose in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea and in the light is much higher than the growth rate at the expense of glucose in the dark; indeed, some cyanobacteria that grow well under the former conditions appear to be incapable of chemoheterotrophic growth (Rippka, 1972). The phenomenon of obligate photoautotrophy, so prevalent in cyanobacteria, has long presented a challenge to the biochemist. Further, some aspects of the organic nutrition of cyanobacteria, summarized below, raise additional biochemical questions. (1) The range of substrates able to support heterotrophic growth is very limited. Glucose is the most widely used substrate, closely followed by fructose and sucrose. Other compounds that permitheterotrophicgrowth of a few strains are glycerol (Ingram et al., 1973), ribose and gluconate (R. Rippka, unpublished work). (2) Growth rates in the dark are very low, always far lower than the phototrophic growth rate of the same strain (Hoare et al., 1971; Rippka, 1972). (3) Even obligately photoautotrophic cyanobacteria readily assimilate amino acids and certain organic acids (notably pyruvate and acetate) in the light (Hoare & Moore, 1965; Hoare et al., 1967; Smith et al., 1967). Assimilation is strictly light-dependent, and none of these compounds can serve as a principal carbon source. Smith et al. (1967) found that acetate, the most rapidly assimilated of the compounds examined, contributed only about 10% to the carbon of newly synthesized cell compounds, the rest being derived from COz. (4) During photoautotrophic growth, assimilated carbon in excess of immediate biosynthetic requirements is stored as glycogen (Kindel & Gibbs, 1963). Glycogen also accumulates as the major assimilatory product during the light or dark metabolism of sugars (Cheung & Gibbs, 1966; Pelroy et al., 1972). Placed in the dark in the absence of a n exogenous substrate, all cyanobacteria perform an endogenous respiration, presumably at the expense of their glycogen reserve. This respiratory activity is coupled with ATP synthesis (Biggins, 1969). Endogenous respiration is sharply diminished by exposure to light, even of low intensity (Brown & Webster, 1953; Jones & Myers, 1963). Light has also been shown to inhibit severely the respiration of exogenous glucose, an effect almost completely abolished by 10p~-3-(3,4-dichlorophenyl)-1,l-dimethylurea (Pelroy et al., 1972). ( 5 ) The dark energy-yielding metabolism of many cyanobacteria appears to be strictly respiratory: several attempts to grow chemoheterotrophic strains anaerobically in the dark at the expense of sugars, or to demonstrate biosynthesis under these conditions, have given negative results (Pelroy et al., 1972; Pelroy & Bassham, 1973; R. Rippka, unpublished work). Hoare et al. (1971) reported very low rates of dark anaerobic growth at the expense of sugars by a Nostoc sp.; however, it is not certain that the cultures were rigorously 02-free. The possibility that certain cyanobacteria may possess weak fermentative capacity capable of supporting a very low rate of dark anaerobic growth merits further systematic exploration, particularly in view of the long-overlooked ability of some non-sulphur purple bacteria to grow anaerobically in the dark by fermentation (Uffen & Wolfe, 1970). VOL
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Recent biochemical studies make it possible to account for many of the physiological data outlined above in metabolic terms. The relevant biochemical findings are described below. Absence of a functional tricarboxylic acid cycle
Smith et al. (1967) observed that the photoassimilation of amino acids or organic acids by obligately photoautotrophic cyanobacteria results in entry of substrate carbon into a rather limited number of biosynthetic pathways. Further, the specific patterns of incorporation suggested that the tricarboxylic acid cycle was non-functional. This inference was confimed by enzymic analysis: in cell-free extracts of the three cyanobacteria examined, 2-oxoglutarate dehydrogenase activity was undetectable, and the activities of both malate dehydrogenase and succinate dehydrogenase were very low. There is also evidence for the absence of a functional tricarboxylic acid cycle in other cyanobacteria (Hoare et al., 1969; Van Baalen et al., 1971; Pelroy et al., 1972). This deficiency is probably characteristic of the group as a whole, and accounts for the inability of such compounds as acetate, pyruvate and dicarboxylic acids to serve as sole sources of carbon and energy for growth in the dark, even though they can be readily assimilated in the light. Respiratory metabolism of sugars
The pathway of glucose respiration has been studied in three facultatively heterotrophic cyanobacteria (Cheung & Gibbs, 1966; Pearce & Carr, 1969; Pelroy et al., 1972). The data of Pelroy et al. (1972) on a unicellular cyanobacterium, Aphanocapsu 6714, are the most complete. In all three cases the oxidative pentose phosphate pathway appears to be the major (and very probably the sole) route of glucose oxidation. Thus radiorespirometric experiments show that oxidation of glucose carbon atoms is highly asymmetric, COz arising preferentially from position 1.The cells always contain high activities of the two dehydrogenases operative in this pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). On the other hand the activities of enzymes specific to the Embden-Meyerhof pathway (phosphofructokinase) and the Entner-Doudoroff pathway (6-phospho-2-keto-3-deoxygluconatealdolase and 6-phosphogluconate dehydratase) are either very low or undetectable. Finally, in cells respiring glucose, glucose 6-phosphate and 6-phosphogluconate together account for over half the intracellular pool of phosphorylated intermediates. The two dehydrogenases of the oxidative pentose phosphate pathway are NADP+specific, in the cyanobacteria so far examined. This implies that respiratory electron transfer proceeds almost wholly through NADP+ rather than through NAD+. Interplay between the reductive and oxidative pentose phosphate cycles
Detailed kinetic studies on 14COzincorporation into components of the metabolic pool, conducted on four unicellular cyanobacteria, reveal that COz fixation proceeds viathe reductive pentose phosphate(Ca1vin-Benson) pathway(Pe1roy &Bassham, 1972). Typical data are shown in Fig. 1. The first stable product, and the only polymer formed in significant amounts during short-term fixation experiments, is glycogen. As shown in Fig. 1;a light-dark transition after exposure to 14COzfor lOmin is followed by a very rapid disappearance from the pool of ribulose 1,s-diphosphate, correlated with a rise in the pool concentration of 3-phosphoglycerate, and by the appearance in the pool of a previously undetectable intermediate, 6-phosphogluconate. These observations suggest that a light-dark transition brings into play the oxidative pentose phosphate cycle, used to generate maintenance energy at expense of the cellular glycogen 'reserve. Biggins (1969) demonstrated that daik endogenous respiration proceeds via NADP+, an observation in accord with the nicotinamide nucleotide specificities of cyanobacterial glucose 1975
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0
.-*m
B&
Time (min) Fig. 1. Intracellular pool concentrations of 14C-labelledphosphorylatedintermediates in a unicellular cyanobacterium, Aphanocapsa 6308
''COz was introduced at zero time, and the cells were incubated for a period of lOmin in the light, followed by a period of 8min in the dark. 8,3-Phosphoglycerate; A , glucose 6-phosphate; 0,ribulose 1,s-diphosphate; A, 6-phosphogluconate; 0 , fructose 1,6diphosphate; EU, sedoheptulose 1,7-diphosphate. The data are taken from Pelroy & Bassham (1 972).
11
Glucose I-phosphate
J
4J-
4
2 Glyceraldehyde 3-phosph~te
Ti. NADPH
j-c
Ribulose 1,5diphosphate
Scheme 1. Primary pathways of carbon metabolism in cyanobacteria Primary substrates and metabolic end products are enclosed in square boxes. Reactions specific to photosynthetic CO, assimilation are designated by white arrows, and reactions specific to dark respiratory metabolism by heavy black arrows. The site of regulation of the respiratory pathway by ribulose 1,s-diphosphate is indicated by the symbol: (Ribulose 1,5-diphosphate) =. The scheme is adapted from Pelroy et al. (1972).
6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Doolittle & Singer (1 974) have shown that mutational elimination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in an obligately photoauto-
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15
-
20
-
I5
-
10 5
0 0 1
0.5
I .o
I0
Concn. of glucose (mM) Fig. 2. Comparison of the rates of 14C02formation from [U-'4Clp1ucose as a function of glucose concentration by three unicellular cyanobacteria
m, Aphanocapsa 6714, a facultative chemoheterotroph; orAphanocapsa 6308, an obligate photoautotroph; 0 , Synechococcus 6301, also an obligate photoautotrophs. The data are taken from Pelroy et al. (1972).
trophic unicellular cyanobacterium decreases endogenous respiration to an undetectable level. The primary pathways of carbon metabolism of cyanobacteria in the light and in the dark are schematized in Scheme 1. Photoinhibition of respiration The photoinhibition of the respiration of both endogenous and exogenous substrates, a distinctive attribute of cyanobacteria, reflects the alosteric inhibition of glucose 6-phosphate dehydrogenase by ribulose 1,5-diphosphate, the only phosphorylated intermediate unique to the reductive pentose phosphate cycle (Pelroy et a f . , 1972). The oxidative pentose phosphate pathway is thus held in check during photosynthetic metabolism, but comes almost instantly into play in the dark, as a result of the very rapid disappearance of ribulose 1,5-diphosphate from the metabolic pool (see Fig. 1). Doolittle & Singer (1974) have provided a striking demonstration of the operation of this control mechanism in an obligately photoautotrophic cyanobacterium. They isolated mutants with greatly impaired ability to survive in the dark (less than 1% survival after 30min without illumination!). Without exception, these mutants had lost 6-phosphogluconate dehydrogenase activity. Their rapid death in the dark is attributable to the intracellular accumulation of 6-phosphogluconate, after the release of the ribulose 1,5-diphosphate-mediatedinhibition of glucose 6-phosphate dehydrogenase activity. Selection of phenotypic revertants with restored capacity to survive in the dark resulted in the isolation of double mutants, which had also lost glucose 6-phosphate dehydrogenase activity. Phenomenon of obligate autotrophy If all cyanobacteria use the oxidative pentose phosphate pathway for dark endogenous metabolism, why are some, but not others, obligate photoautotrophs? The inability to utilize exogenous glucose could in principle reflect either absence of hexokinase or absence of a specific glucose permease. Pelroy et al. (1972) found hexokinase activities in two obligate photoautotrophs to be 25-50 % of those in a heterotrophic cyanobacterium; however, there was a very marked difference between the obligate autotrophs and the facultative heterotroph in the affinity of intact cell for glucose (Fig. 2). These authors accordingly suggested that the absence of specific permeases, required to mediate entry
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of the relatively small number of substrates that can feed into the oxidative pentose phosphate cycle, is the most probable explanation for the widespread occurrence of obligate photoautotrophy among cyanobacteria. However, certain cyanobacteria that are facultative photoheterotrophs, able to grow'in the light in the presence of 3-(3,4dichloropheny1)-1,I-dimethylurea at the expense of glucose, seem wholly incapable of dark growth with glucose. This phenomenon obviously cannot be accounted for by postulating a permeability barrier; its explanation remains unknown. Biggins, J. (1969) J. Bucteriol. 99, 570-575 Bishop, N. I. (1958) Biochim. Biophys. Acra 27, 205-206 Brown, A. H. & Webster, G. C. (1953) Amer. J. Bot. 40,753-757 Cheung, W. Y. & Gibbs, M. (1966) Plant Physiol. 41, 731-737 Doolittle, W. F. & Singer, R. A. (1974) J. Bacteriol. 119, 677-683 Hoare, D. S. & Moore, R. B. (1965) Biochim. Biophys. Acta 109, 622-625 Hoare, D. S., Hoare, S. L. & Moore, R. B. (1967) J. Cen. Microbiol. 49, 351-370 Hoare, D. S., Hoare, S. L. & Smith, A. J. (1969) in Progress in Photosynthesis Research (Metzner, H., ed.), vol. 3, pp. 1570-1573, Laupp, Tubingen Hoare, D. S., Ingram, L. O., Thurston, E. L. & Walkup, R. (1971) Arch. Mikrobiol. 78,310-321 Ingram, L. O., Van Baalen, C. & Calder, J. A. (1973)f. BacterioL 114, 701-705 Jones, L. W. & Myers, J. (1963) Nature (London) 199, 670-672 Kindel, P. & Gibbs, M. (1963) Nature (London) 200,260-261 Pearce, J. & Carr, N. G. (1969) J. Gen. Microbiol. 54, 451-462 Pelroy, R. A. & Bassham, J. A. (1972) Arch. Mikrobiol. 86, 25-38 Pelroy, R. A. & Bassham, J. A. (1973) J. Bucteriol. 115,937-942 Pelroy, R. A., Rippka, R. & Stanier, R. Y. (1972) Arch. Mikrobiol. 87, 303-322 Rippka, R. (1972) Arch. Mikrobiol. 87, 93-98 Smith, A. J., London, J. & Stanier, R. Y. (1967) J. Bacteriol. 94, 972-983 Uffen, R. L. & Wolfe, R. S. (1970) J. Bacteriol. 104, 462-472 Van Baalen, C., Hoare, D. S. & Brandt, E. (1971) J. Bacteriol. 105, 685-689
Nitrogen Fixation and the Weterocyst in Blue-Green Algae W. D. P. STEWART, P. ROWELL and E. TEL-OR Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, U.K. The blue-green algae, or cyanobacteria, are Oz-evolving prokaryotes, many species of which fix Nz. Greatest morphological differentiation occurs in some Nostocales, and in the Stigonematales where, in addition to the typical vegetative cells of blue-green algae, there are two additional cell types: akinetes and heterocysts. The role of akinetes as perennating structures is well established (see Fogg et al., 1973), and, though various views have been proposed on the role of the heterocyst, it is accepted generally that its main function is in N, fixation. The close correlation between heterocysts and N, fixation is seen at the genetic and molecular levels. In genetic studies on the heterocystous Nostoc muscorum we have obtained various nitrogenase-less mutants (i.e. mutants unable to grow or reduce acetylene under aerobic or microaerobic conditions) and usually these mutants also lack heterocysts. By using one such mutant into which resistance to streptomycin (1000pg/ml) was introduced (nif-st-R),evidence of nif-gene transfer has been obtained on mixing it with wild-type (nif+st-S) Nosfocmuscorum. In such studies the prototrophic streptomycin-sensitive donor was counter-selected, and when this was done nif+st-R colonies developed at a frequency of 2.2 in lo5colonies, 4.6 in los colonies and 5.0 in lo6 colonies in three different experiments. These values are significantly higher than those attributable to spontaneous mutation (1 in lo7 or less), and the higher yield of new genotypes may be interpreted as evidence of nif-gene transfer, although in genetic studies of this type the possibility cannot be ruled out that what appears to be nif-gene transfer may be transfer of some factor that allows the expression of latent rzifgenes in the non-N2-
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