Dark anaerobic dinitrogen fixation by a photosynthetic microorganism.

Photosynthetic purple bacteria can grow with dinitrogen gas as the sole nitrogen source under anaerobic conditions with light as the energy source. The bacterium Rhodopseudomonas capsulata can fix nitrogen in darkness with alternative energy conversion systems, namely, anaerobic sugar fermentation and aerobic respiration at low oxygen tension. Although growth on dinitrogen is optimal under photosynthetic conditions, the results show that reduction of dinitrogen is not obligatorily coupled to activity of the photosynthetic apparatus.

Energy flux and membrane synthesis in photosynthetic bacteria.

Synthesis of the energy-converting membrane complex of the photosynthetic purple bacterium Rhodopseudomonas capsulata during growth under different conditions of energy flux was studied by examining the disorganizing effects of polymyxin B, with or without lysozyme, on integrity of the cell envelope. Cells growing with a limited supply of energy show an elevated bacteriochlorophyll content and increased resistance to breakdown of the "permeability barrier" by these agents. It seems that purple bacteria respond to energy restriction by preferentially synthesizing excess bacteriochlorophyll-membrane which, in effect, toughens the cell integument.

Interchangeability of phosphorylation coupling factors in photosynthetic and respiratory energy conversion.

The nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata can obtain energy for growth either by anaerobic photophosphorylation or dark oxidative (aerobic) phosphorylation. Successful resolution of phosphorylation coupling factors from energy-converting membranes of this bacterium permitted tests for reciprocal function of such protein factors in oxidative-and photophosphorylation processes. Evidence was obtained for the interchangeability of coupling factor preparations from dark-grown and photosynthetically grown cells in both kinds of energy conversion.

Evolutionary roots of the citric acid cycle in prokaryotes.

Advances in biochemistry, microbiology, and molecular biology suggest new approaches for exploring the early evolution of bioenergetic systems. These approaches, still in their infancy, are necessarily directed to detection of 'molecular fossils' in diverse extant prokaryotes. Since the Earth was devoid of atmospheric oxygen during early cellular evolution, it is likely that 'precursor fragments' of the classical citric acid cycle are to be found in contemporary anaerobic bacteria. Accumulating evidence indicates that such fragments originally served biosynthetic roles of one kind or another, before they were recruited for assembly of the energy-yielding aerobic cycle. The extraordinary versatility of citric acid cycle intermediates and reactions for multiple uses raises the possibility that origination of the aerobic cycle, viewed as an evolutionary event, occurred more than once.

Bicentenary homage to Dr Jan Ingen-Housz, MD (1730-1799), pioneer of photosynthesis research.

On September 7, 1999, the 200th anniversary of the death of Dr Jan Ingen-Housz was commemorated by ceremonies in Calne, England. Ingen-Housz discovered the action of light in photosynthesis in 1779, following Joseph Priestley's demonstration that green plants had the capacity to produce oxygen. Priestley's claim for priority in discovering the light requirement of photosynthesis is examined.

Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum.

The purple photosynthetic bacterium Chromatium vinosum, strain D, catalyzes several oxidations of reduced sulfur compounds under anaerobic conditions in the light: e.g., sulfide --> sulfur --> sulfate, sulfite --> sulfate, and thiosulfate --> sulfur + sulfate. Here it is shown that no sulfur isotope effect is associated with the last of these processes; isotopic compositions of the sulfur and sulfate produced can differ, however, if the sulfane and sulfonate positions within the thiosulfate have different isotopic compositions. In the second process, an observed change from an inverse to a normal isotope effect during oxidation of sulfite may indicate the operation of 2 enzymatic pathways. In contrast to heterotrophic anaerobic reduction of oxidized sulfur compounds, anaerobic oxidations of inorganic sulfur compounds by photosynthetic bacteria are characterized by relatively small isotope effects.

A novel directly coupled gradostat.

The original bidirectional compound chemostat (gradostat) described by Lovitt and Wimpenny has been simplified by making a more compact apparatus in which chemical gradients are established by diffusion between adjacent culture chambers. The experimental model (diffusion coupled (DC) gradostat) consisted of five chambers whose contents could be agitated by turbines rotating in the horizontal plane on a common shaft. Two biological experiments were designed to reveal the value of the DC gradostat. A methylotroph (Methylophilus methylotrophus) grown in a methanol gradient showed expected changes in cell viability as a function of position in the five vessel array. Cells of two species of photosynthetic bacteria (Rhodobacter capsulata and Rhodopseudomonas marina/agilis) with different salt sensitivities could be mixed and subsequently separated by the DC gradostat operating with a NaCl gradient of 0-3% w/v.

[Normal intrahepatic vessels. Remarks on their echographic image]. / Les vaisseaux intra-hépatiques normaux. Considération sur leur image échographique.

The sonographic appearances of intrahepatic vessels are demonstrated by the basic principles of ultrasonography. All intrahepatic vessels have a sonolucent lumen which may be blurred because of the slice-thickness effects. The vascular walls are detected only when they are perpendicularly struck by the ultrasound beam. The portal branches are visualized with satellite echoes which are reflections from the intraparenchymal hepatic arteries and/or biliary ducts, and from the collegenous sheath of the portal triad. These echoes are blended with the wall-echoes of the portal veins as to the major branches. For smaller branches they remain visible when the incidence of the ultrasound beam becomes less accurate, whereas the echoes from the portal veins tend to disappear. The echoes related to the hepatic veins only come from the walls of the veins, which are usually of medium size.

A serendipic legacy: Erwin Esmarch's isolation of the first photosynthetic bacterium in pure culture.

During the 1880's, Erwin von Esmarch was a junior associate ('Assistent') of Robert Koch studying bacteria of medical significance. In 1887, he isolated the first example of spiral-shaped bacteria in pure culture, from the dry residue of a dead mouse that he had suspended sometime earlier in Berlin tap-water. Under certain conditions, colonies of the organism were the color of red wine, and this led Esmarch to name the bacterium Spirillum rubrum. Twenty years later, Hans Molisch demonstrated that S. rubrum, an apparent heterotroph, was in fact a non-oxygenic purple photosynthetic bacterium, and it was renamed Rhodospirillum rubrum. Esmarch was a careful investigator and his classic paper of 1887 details the serendipitous isolation and general characteristics of the first pure culture of an anoxyphototroph, which later played a prominent role as an experimental system for study of basic aspects of bacterial photosynthesis. This report includes an English translation of his original paper (in German), a commentary on the historical significance of 'Esmarch's spirillum', and a summary of Esmarch's career.

The legacy of Hans Molisch (1856-1937), photosynthesis savant.

Hans Molisch (1856-1937) was an exceptionally gifted and productive researcher who had broad interests in plant biology, physiology and biochemistry. In addition, he pioneered in isolating a number of species of purple photosynthetic bacteria in pure culture (including Rhodobacter capsulatus), which facilitated his discovery of basic aspects of bacterial photosynthesis. Molisch demonstrated conclusively that molecular oxygen is not produced by photosynthetic bacteria, and discovered the photoheterotrophic growth mode. The range of Molisch's research accomplishments was impressive, and he emerges as a major figure in the history of photosynthesis research. This essay reviews the numerous research contributions made by Molisch, particularly in regard to advancing knowledge of the several forms of photosynthetic metabolism. An English translation of his 1914 paper on the photosynthetic creation of visual images on leaves is included as an Appendix.

Discovery of the heliobacteria.

The first photosynthetic bacterium obtained in pure culture wasRhodospirillum rubrum, isolated by Erwin Esmarch in 1887. The organism appeared to be an aerobic heterotroph, and Esmarch was unaware of its photosynthetic capability. The overall general characteristics of a number of major species of photosynthetic bacteria were described by Molisch and van Niel before 1945. Subsequently, our knowledge of the anoxygenic phototrophs increased greatly through the systematic study of numerous new species isolated from enrichment cultures in which capacity for anaerobic (and anoxygenic) growth with light as the energy source was a primary selective factor. A further refinement of the enrichment technique required ability to use N2 as the sole source of nitrogen for growth under anaerobic photosynthetic conditions, and this led to the isolation of additional new species, including the heliobacteria. The first recognition of the heliobacteria was facilitated by serendipity, which was a significant factor in a number of other researches on photosynthetic bacteria (Gest 1992).

A microbiologist's odyssey: Bacterial viruses to photosynthetic bacteria.

Perspective can be defined as the relationships or relative importance of facts or matters from any special point of view. Thus, my Personal perspective reflects the threads I followed in a 50-year journey of research in the complex tapestry of bioenergetics and various aspects of microbial metabolism. An early interest in biochemical and microbial evolution led to the fertile hunting grounds of anoxygenic photosynthetic bacteria. Viewed as a physiological class, these organisms show remarkable metabolic versatility in that certain individual species are capable of using all the known major types of energy conversion (photosynthetic, respiratory, and fermentative) to support growth. Since such anoxyphototrophs are readily amenable to molecular genetic/biological manipulation, it can be expected that they will eventually provide important clues for unraveling the evolutionary relationships of the several kinds of energy conversion. I gradually came to believe that understanding the evolution of phototrophs would require detailed knowledge not only of how light is converted to chemical energy, but also of a) pathways of monomer production from extracellular sources of carbon and nitrogen and b) mechanisms cells use for integrating ATP regeneration with the energy-requiring biosyntheses of biological macromolecules. Serendipic observation of photoproduction of H2 from organic compounds by Rhodospirillum rubrum in 1949 led to discovery of N2 fixation by anoxyphototrophs, and this capacity was later exploited for the isolation of hitherto unknown species of photosynthetic prokaryotes, including the heliobacteria. Recent studies on the reaction centers of the heliobacteria suggest the possibility that these bacteria are descendents of early phototrophs that gave rise to oxygenic photosynthetic organisms.

History of concepts of the comparative biochemistry of oxygenic and anoxygenic photosyntheses.

Experiments of Hans Molisch in 1907 demonstrated that purple bacteria do not evolve molecular oxygen during photosynthetic metabolism, and can use organic compounds as sources of cell carbon for anaerobic 'photoheterotrophic' growth. Molisch's conclusion that he discovered a new photosynthetic growth mode was not accepted for some 30 years because of the prevailing definition of photosynthesis as light-dependent conversion of carbon dioxide and inorganic reductants to cell materials. Meanwhile, during the decade of the 1930s, Cornelis van Niel formulated the 'comparative biochemical watercleavage hypothesis' of photosynthesis, which enjoyed great popularity for about 20 years. According to this concept, photolysis of water yielded 'H' and 'OH', the former acting as the hydrogen donor for CO2 reduction in all modes of photosynthesis. Oxygenic organisms were presumed to contain a unique biochemical system capable of converting 'OH' to water and O2. To explain the absence of O2 formation by purple and green photosynthetic bacteria, it was supposed that such organisms lacked the oxygen-forming system and, instead, 'OH' was disposed of by reduction with an inorganic H(e) donor (other than water) according to the general equation:[Formula: see text] where H2A is H2 or an inorganic sulfur compound.Critical tests of van Niel's hypothesis could not be devised, and his proposal was abandoned soon after the discovery of in vitro photophosphorylation by green plant chloroplasts and membranes of purple bacteria in 1954. Photophosphorylation was then viewed as one key common denominator of oxygenic and anoxygenic photosyntheses. From later research it became clear that light-dependent phosphorylation of adenosine diphosphate was a consequence of photochemical charge separation and electron flow in reaction centers embedded in membranes of all photosynthetic organisms. The similarities, as well as the differences, in fine structure and function of reaction centers in anoxygenic and oxygenic organisms are now believed to reflect the course of evolution of oxygenic organisms from anoxygenic photosynthetic precursors. Thus, with the acquisition of new knowledge, concepts of the comparative biochemistry of photosynthetic processes have been radically altered during the past several decades. This paper describes highpoints of the history of these changes.

Sun-beams, cucumbers, and purple bacteria : Historical milestones in early studies of photosynthesis revisited.

Discovery of the general outlines of plant and bacterial photosyntheses required the efforts of a large number of gifted scientists over the course of two centuries. The first to suggest that sunlight might affect plants in some way other than through conversion of light to heat was Stephen Hales, in 1725, and this notion was promptly satirized by Jonathan Swift in his description of the "cucumber project" inGulliver's Travels (1726). Considerably later, in 1772, Joseph Priestley reported the first experiments showing the production of "dephlogisticated air" (oxygen gas) by plants, and the interdependence of animal and plant life mediated by gases. Priestley and others, however, had difficulty repeating these experiments, mainly because they were unaware of the requirement for light in photosynthesis. The latter was clearly demonstrated in 1779 by Jan Ingen-Housz, who also determined that leaves were the primary sites of the photosynthetic production of oxygen by plants. When purple bacteria were first studied in the late 19th century by Theodor Engelmann, light-dependent O2 formation could not be detected. Contradictory observations in this connection were reported for a number of decades, but eventually the absence of O2 production in photosynthesis by purple bacteria was conclusively established. Attempts to explain why the bacteria do not evolve O2 led Cornelis van Niel to propose a "unified, comparative biochemical" explanation of photosynthetic processes that was widely accepted. This hypothesis, however, was abandoned soon after photophosphorylation by membranes from purple bacteria and plant chloroplasts was discovered in 1954. Unexpectedly, rapid progress in molecular biological and genetic studies of the membrane-bound reaction centers of purple bacteria indicate that current investigations are on the verge of revealing the detailed mechanisms by which energy conversion occurs in the reaction centers of all photosynthetic organisms.

Longevity of microorganisms in natural environments.

Bacterial endospores are exceptional among living systems in respect to their resistance to adverse environmental conditions, notably to heat and dessication, and consequently might be expected to show crytobiosis of very long duration. This report considers recent observations indicating that the maximal longevity of bacterial spores is probably much greater than previously believed.

Regulation of bacteriochlorophyll synthesis by oxygen in respiratory mutants of Rhodopseudomonas capsulata.

Respiratory mutants of the facultative photosynthetic bacterium Rhodopseudomonas capsulata were used to investigate the mechanism of (reversible) inhibition of bacteriochlorophyll (BChl) synthesis by molecular oxygen. Although mutant strain M5 lacks cytochrome oxidase activity, it closely resembles the parental wild-type strain in respect to the effect of O(2) on BChl formation. This observation does not support an earlier hypothesis that O(2) regulates BChl synthesis through an effect on the redox state of a component of the respiratory electron transport system. Mutant strain M2 shows normal cytochrome oxidase activity, but lacks both reduced nicotinamide adenine dinucleotide and succinate dehydrogenase activities; relative to the parental strain, BChl synthesis in M2 is more sensitive to O(2) inhibition. The foregoing and results of related experiments can be accounted for by a revised interpretation of the O(2) effect, which proposes that O(2) directly inactivates a "factor" necessary for BChl formation and that, at relatively low O(2) tension, the inactivation can be reversed by a flow of electrons (derived from reduced nicotinamide adenine dinucleotide and succinate) diverted from a portion of the electron transport system delimited by the mutational blocks in M2 and M5.

Genetic mutations affecting the respiratory electron-transport system of the photosynthetic bacterium Rhodopseudomonas capsulata.

Alternative energy-converting systems permit the nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata to grow either with light or (dark) respiration as the source of energy. Respiratory mutants, unable to grow aerobically in darkness, can be readily isolated and the defective step(s) in their respiratory mechanisms can be identified by study of biochemical activities in membrane fragments derived from photosynthetically grown cells. Such analysis of appropriate mutants and revertants permits construction of a model for the respiratory electron-transport system of the wild type. The results obtained indicate differential channeling of electrons derived from succinate and reduced nicotinamide adenine dinucleotide, and are interpreted in terms of a branched electron-transport scheme. The scheme provides a guide for further, more refined analysis of the respiratory mechanisms through biochemical genetic approaches, and several of the mutants isolated can be exploited for investigation of unsolved problems relating to interactions between respiratory and photosynthetic electron transport and the mechanism of inhibition of bacteriochlorophyll synthesis by molecular oxygen.

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures.

Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.