Some of the problems posed by the biochemistry of the chemo- and photoautotrophic bacteria and algae have been examined in order to attempt an explanation for the obligate auto trophy of some microbes. The dependence of these organisms on carbon dioxide and light or on inorganic respiratory substrates may be due to a requirement for special energy sources if the organisms are unable to obtain sufficient energy for growth from the respiration of organic compounds. The influence of organic compounds on autotrophs, and the enzymes found in themy are considered, and alternative possibilities of special features of the regulation ofautotrophic metabolism or of particular enzyme deficiencies are examined as possible causes of obligate autotrophy.
Introduction — The autotrophic microbes
Literally ‘autotroph* means ‘self-nourishing’ and is a term used to describe organisms which are capable of growing in the absence of any of the organic compounds that are generally required for the growth of bacteria or Protozoa and all other animals. Autotrophs can use carbon dioxide as their only source of carbon and from it are able to build up the multitude of substances which con- stitute a living organism. Their only other requirements are nitrogen, which is gained from inorganic sources like ammonium or nitrate ions or in some cases from elementary nitrogen gas; mineral salts, water and, of course, chemically available energy. While all autotrophs use the same carbon source, carbon dioxide, different types use a variety of different sources of energy to support growth. This allows a division of the autotrophs into two broad classes. Firstly, those using light energy, the photoautotrophs.
These include the algae and the photosynthetic bacteria. Of course, all other green plants are also photoauto- trophs but we are concerned here only with microbial autotrophy. The second group of autotrophs, on an energy-classification basis, is the chemoautotrophs. These are a number of specialized bacterial groups which do not require light at all, but obtain energy from oxidizing inorganic compounds like ammonia or nitrite or sulphur compounds to products such as nitrite, nitrate and sulphate.
Obviously these groups pose a number of problems to the biochemist. The most obvious questions to be asked of them are: (i) What biochemical mechanisms allow them to grow with such simple nutritional requirements ? (ii) How do they obtain energy from their unique inorganic oxidations or from light? (iii) What is the relation of autotrophic organisms to the organic nutrients which support the growth or rather are essential to the growth of heterotrophic organisms (i.e. all those that are not autotrophic)? That is perhaps to ask whether there is any particular property which makes the autotroph any more different from heterotrophs than can be explained simply by assuming that autotrophs have a greater capacity for biosynthesis than do heterotrophs. These first two questions are clearly ones of great scope and answers to them have been partly made during the past 20 or 40 years with the chemoautotrophs and photoautotrophs respectively, although the greatest advances with both have come in the past 10 or 20 years.
Advances during this period have accompanied the development of techniques such as the use of radioactive tracers and advanced spectrophotometric methods to follow reaction mechanisms in intact and disrupted organisms. The outcome of such work seems to show that autotrophs seem to have a common mechanism, absent in heterotrophs, for the fixation of carbon dioxide1 into compounds from which the rest of the cell substances can be synthesized. The energy-trapping processes in both photo- and chemoautotrophs may show some similarities because in all cases much of the production and capture of chemical energy depends on the transfer of elec- trons from reduced compounds to oxidized ones. These electron transfers are accompanied by decreases in energy levels, and energy becomes available for reactions which couple it to the synthesis of ‘energy storage* compounds such as adenosine-5′-triphosphate (ATP). The other form of energy required for biochemical reactions is the power to reduce oxidized compounds: for example, carbon dioxide assimilation is a reductive process.
The source of this reducing power is again from the electrons made available from oxidizing reduced com- pounds such as ammonia in a chemoautotroph, hydrogen gas or sulphur by a photosynthetic bacterium or water by algae. The results of recent work on these two aspects of autotrophic metabolism are discussed in brief in the next section. However, fascinating as these problems are, the autotrophs present a further problem which may be far more unusual than those of the problems already outlined. This is the problem posed by the obligate autotrophs. These are organisms which are not only able to grow under the simple cultural conditions outlined, but which are quite unable to grow under any other conditions.
Thus they are incapable of heterotrophic growth and are entirely dependent on carbon dioxide and their particular special energy source. The problem of why there are such obligate autotrophs is far from solved at present. One immediate conclusion one might draw is that the whole basis of the metabolism of such autotrophs is different from all other nonautotrophic organisms and hence one could not expect anything but auto-trophy from them. This view will be seen to be untenable in subsequent discussion, showing the basic similarity between autotrophic and heterotrophic metabolism.
The problem to which a solution is most required is: ‘Why are certain auto- trophs unable to grow heterotrophically?*
Carbon dioxide fixation and energy metabolism in autotrophs
First, the principal groups of autotrophic micro-organisms to be mentioned are listed so that their relations to each other may be clearer.
Algae. Certain green algae (Chlorophyceae) have been used in studying carbon dioxide fixation, and some are obligate autotrophs, e.g. some Chlorella and Chlamydomonas species. The blue-green algae (Cyanophyceae) are probably generally obligate autotrophs, although an insufficient number has been studied to be positive on this. Obligate photoautotrophy is also shown by some members at least of other algal groups (e.g. Monodus sp. and diatoms). Bacteria. The purple (Thiorhodaccae) and green (Chlorobacteriaccae) sulphur bacteria use inorganic sulphur compounds (thiosulphate, sulphide) as sources of electrons in photosynthesis and can use carbon dioxide as principal carbon sources. The green bacteria (e.g. Chlorobium), however, are obligate autotrophs while the purple ones are not necessarily so.
Some other autotrophs are known but are at present less thoroughly studied. The filamentous Beggiatoa oxidizes sulphide to accumulate sulphur in its cells, which then oxidize the sulphur to sulphate. Gallionella seems to obtain energy and to grow in a medium containing iron filings.5 Hydrogenomonas oxidizes hydrogen to water and fixes carbon dioxide, but can also grow heterotrophically. An organism oxidizing carbon monoxide and fixing carbon dioxide is known (Carboxydomonas) but has been little studied.
The mechanism of autotrophic C02-fixation was first established in green algae and follows the pattern shown in Fig. 1. These reactions have been established also by I4C02 incorporation and by enzyme studies in photosynthetic bacteria and in the principal groups of autotrophs. The key reaction (lacking in heterotrophs) is the condensation of C02 with ribulose diphosphate (RUDP) to give two molecules of phosphoglyceric acid (C3) by the so-called ‘carboxydismutase’ reaction. These undergo cyclic transformations to regenerate RUDP, but of course the carbon gained is one C3 molecule for every three C02 fixed. This C3, phosphoglyceric acid or glyceraldehyde-3-phosphate, can enter biosynthesis via pyruvate or be converted to hexoses by the condensation of two C3 units by a reaction which is the reverse of the aldol cleavage of fructose diphosphate in glycolysis. Some carbon dioxide is also fixed, as in heterotrophs, by combination with pyruvate or phosphoenolpyruvate.
The photosynthetic pigment (e.g. chlorophyll) acts as a primary source of electrons for the reduction of ferredoxin by a process driven by light energy. The ferredoxin may then reduce a coenzyme such as NADP or its electrons may pass by a series of intermediate carriers back to the pigment molecule.
This electron flow back to the pigment yields chemically available energy. Similarly the supply of electrons from the donors (to replace those lost in reducing NAD, etc.) are transferred to the pigment through carriers whose oxidation and reduction is probably also linked to ATP synthesis. The mechanism of ATP formation during these electron transfer probably resembles the oxidative phosphorylation seen in bacterial and mitochondrial respiration.
In the chemoautotrophs the principal source of ATP is again probably from processes resembling oxidative phosphorylation. Cytochromes are involved in all the chemoautotrophic electron transfers, but in Thiobacillus, at least, some ATP synthesis probably occurs by a ‘substrate-level’ phosphorylation.4 A further problem with the oxidation of sulphur and nitrogen compounds is that none of the oxidation couples is sufficiently electronegative to be linked to direct coenzyme reduction. However, reduced coenzymes or ferredoxin are essential for carbon dioxide fixation. Recent work indicates that cytochromes reduced during, say, nitrite or thiosulphate oxidation are used in the cell to reduce NAD, but this process requires energy.7 So, some of the ATP formed during the oxidation of the inorganic compounds is used to ‘drive’ electrons as shown in this hypothetical scheme where Nitrobacter is used as an example.
Clearly not all the ATP formed during the oxidation can be used in the coen-zyme-reducing system, nor is the site of ATP formation rigorously established in Nitrobacter, but this outline is almost certainly the type of process involved. Evidence for this kind of process has been obtained using cell-free extracts of nitrifiers and of Thiobacillus. This kind of mechanism for NAD reduction has been well studied in mitrochondria, where succinate is the reductant, instead of inorganic compounds.
To conclude, the energy-trapping processes in autotrophs involve electron transfer, and in the chemoautotrophs the oxidation reactions are probably analogous to the respiratory processes of heterotrophs. Basically, therefore, the generalized processes of autotrophic and heterotrophic metabolism could be similar, with chemotrophic energy production being used as a variation on a common type of respiratory mechanism to drive a metabolism otherwise similar to the heterotrophic metabolism. Regarding carbon metabolism, all work on carbon dioxide fixation indicates that autotrophs have a special system for incorporating carbon dioxide into a few particular compounds, which then enter into metabolic processes such as are common in heterotrophs; e.g. the tricarboxylic acid cycle.
The autotrophs thus seem to emerge not as ‘primitive’ or ‘simple’ organisms as was once thought, but as creatures likely to have a bio- synthetic capacity like that of heterotrophs, to which is coupled the additional carbon-dioxide fixing system and the specialized light- or chemotrophic-energy utilizing reactions. In general terms, therefore, one might expect the autotrophs to be biosynthetically superior to the heterotrophs and to contain a greater complex of enzymes. This certainly seems true for those which are ‘facultative heterotrophs* such as Hydrogenomonast Thiobacillus novelus or many algae (e.g. Chlorella pyrenoidosa), which can grow on nutrient media in the dark or without their inorganic substrates.