Facultative Autotroph Research Articles

Regulation of the Thiobacillus intermedius glucose uptake system by thiosulfate.

Cells of the mixotrophic chemolithotroph (facultative autotroph) Thiobacillus intermedius which have been grown on a glucose-yeast extract medium, a condition in which glucose is used as a source of energy, accumulate the non-metabolizable analogue 2-deoxy-d-glucose against a concentration gradient in a predominantly unchanged state. On the other hand, cells grown mixotrophically on a thiosulfate-glucose medium, a condition in which glucose provides cell carbon but is not used extensively for energy, and in which enzymes of the Entner-Doudoroff pathway are repressed, do not accumulate 2-deoxy-d-glucose significantly. Similarly, cells grown chemolithotrophically on thiosulfate-carbonate do not take up this sugar. Transfer of thiosulfate-yeast extract-grown cells, which lack the capacity to accumulate 2-deoxy-d-glucose, to a glucose-yeast extract medium results in the induction of the concentrative sugar uptake system. The capacity of induced cells to take up 2-deoxy-d-glucose is inhibited by thiosulfate. Thus, the transport system for glucose appears to be regulated in this organism so that the sugar is accumulated only under conditions where it is utilized as a source of energy, and the presence of the preferred energy source leads to both repression and inhibition of the uptake system.

Replication of Bacteriophage SH-133 in the Facultative Autotroph Hydrogenomonas facilis II. Bacteriophage Synthesis and Hydrogenase Induction Under Step-Up and Step-Down Growth Conditions

Autotrophically grown infected cells are able to replicate phage SH-133 after being switched to a heterotrophic environment (step-up growth). The effect of step-down growth on phage replication varies with the choice of organic substrate. Phage replication and the induction of cellular hydrogenase occur under step-down growth from acetate but not peptone broth. The requirement of a continued source of energy for phage replication in either heterotrophically or autotrophically grown cells could be uniquely demonstrated in this phage-host system by the deletion of hydrogen from the growth medium.

Effect of growth substrate on enzymes of the citric and glyoxylic acid cycles in Thiobacillus novellus.

A quantitative study was conducted of enzymes involved in the citric acid cycle and associated systems of the facultative autotroph Thiobacillus novellus grown on five different substrates. Irrespective of the growth substrate the organism possessed complete citric and glyoxylic acid cycles and the specific activity of α-ketoglutarate dehydrogenase was always quite low. Also, the activities of the enzymes of both cycles were usually lowest in extracts from autotrophic cells, and highest in extracts from acetate-grown cells. The three remaining extracts had activities that were between the two extremes with those from glucose-grown cells generally lower than those from pyruvate and succinate. Several exceptions should be noted among these generalizations. For example, the activity of aconitate hydratase and malic dehydrogenase was lowest in extracts from glucose-grown cells while that of isocitric dehydrogenase was lowest in extracts from pyruvate-grown cells. Transhydrogenase activity was virtually absent from extracts of pyruvate- and succinate-grown cells while NADH oxidase, which was identical in these two extracts, was also relatively low. Of interest is the large amount of cytochrome c found in high-speed supernatants. In extracts from autotrophic cells this was about 2.3% of the soluble protein and is suggestive of a significant role being played by the electron-transport system during growth of the organism.

PH-conditional, ammonia assimilation-deficient mutants of Hydrogenomonas eutropha: isolation and growth characteristics.

Two mutants of the facultative autotroph Hydrogenomonas eutropha were isolated by using a modified penicillin selection method. The mutation involved was unusual in that its effect on cellular growth was conditional with regard to extracellular pH and the type of substrate employed. Growth of both mutants was abnormal under autotrophic conditions and during heterotrophic cultivation in the presence of organic substrates which lacked an amino group. Abnormal growth was characterized by linear growth rates which were low at pH 6.0 and moderate at pH 7.2. In contrast, growth of the mutants was normal on most amino acids. Those substrates yielding abnormal growth were oxidized at normal rates by the mutants, indicating the mutation did not impair their uptake or metabolism. The data suggest that the mutants are defective in their ability to assimilate inorganic nitrogen into organic forms, and this defect is strongly influenced by pH.

The tricarboxylic acid cycle in Thiobacillus denitrificans and Thiobacillus-A2.

SUMMARY: The enzymes involved in the tricarboxylic acid cycle and some other related reactions were measured quantitatively in cell-free extracts from the obligate autotroph Thiobacillus denitrificans grown anaerobically, and in the facultative autotroph Thiobacillus-A 2 grown under various conditions. The presence of an incomplete tricarboxylic acid cycle in T. denitrificans and in Thiobacillus-A 2 (when grown autotrophically on thiosulphate) is described. The cell-free extracts from these two organisms lacked α-ketoglutarate dehydrogenase and succinyl CoA synthetase. The data from Thiobacillus-A 2 grown on succinate, aerobically or anaerobically, and on glutamate aerobically suggest that both the enzymes are repressed during autotrophic growth. Results are further discussed in relation to possible reasons for autotrophy.

Chemoautotrophic sulfur bacteria from the marine environment. II. Characterization of an obligately marine facultative autotroph.

Marine strain D10-B, a Gram-negative, non-motile, facultative chemoautotrophic sulfur bacterium, oxidized thiosulfate to sulfate with the production of energy which was used for 14CO2 fixation. The assimilation of 14CO2 was almost completely inhibited by 5 × 10−4 M 2,4-dinitrophenol. Thiosulfate oxidation was inhibited by 47% in the presence of 5 × 10−4 M KCN. The thiosulfate-oxidizing system appeared to be inducible. Elemental sulfur, sulfite, and tetrathionate were also respired, but only thiosulfate served as a substrate for growth. Strain D10-B grew heterotrophically on a variety of single and complex organic substrates. The organism was compared to Thiobacillus novellus and Thiobacillus intermedins. It was classified as a marine strain of T. novellus.

Biochemical basis of obligate autotrophy in Nitrosomonas europaea.

The specific activities of isocitric dehydrogenase, alpha-ketoglutaric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) oxidase were determined in extracts of Nitrosomonas europaea and compared with the corresponding values for Anacystis nidulans and autotrophically grown Hydrogenomonas eutropha. In common with other obligate autotrophs and in contrast to facultative autotrophs, Nitrosomonas extracts lacked alpha-ketoglutaric dehydrogenase and KCN-sensitive NADH oxidase activity and had low succinic dehydrogenase activity. The Nitrosomonas NADH oxidase appeared to be of the peroxidase type.

Ultrastructure of Nitrobacter agilis grown under autotrophic and heterotrophic conditions.

Nitrobacter agilis, grown through seven transfers heterotrophically in the absence of nitrite, was examined in the electron microscope. The ultrastructure of such cells closely resembled that of autotrophically grown N. agilis. It was thus futher established that the organisms growing heterotrophically were indeed N. agilis and, therefore, that N. agilis is a facultative autotroph. Acetate incorporation into poly-beta-hydroxybutyrate was confirmed cytologically.

Activity of sulphur-oxidizing microorganisms in some Australian soils

The percentage oxidation of sulphur and the activity of Thiobacillus thiooxidans in a red-brown earth and a chernozem were followed at monthly intervals over a period of a year, but no obvious seasonal pattern emerged, and neighbouring sites rarely synchronized. Th. thiooxidans occurred more often in the red-brown earth than in the chernozem. In a survey of 273 composite samples of Australian soils oxidation of sulphur was good in approximately one-third only, moderate to poor in a half, and nil in the remaining one-sixth. There was no obvious correlation between sulphofication and several soil properties tested; but it was often higher in soils containing sulphides and lower in more alkaline soils. Th. thiooxidans was not detected in one-third of 288 soils examined, and it was seldom very active in the remaining two-thirds. There was no relationship between the incidence of these bacteria and the Great Soil Group or the locality from which the sample came, and only poor correlations between initial pH, final pH, and titratable acidity of incubated samples. The most numerous sulphur-oxidizing organisms were heterotrophs, including nine bacterial genera and two yeasts. Facultative autotrophic thiobacilli were encountered more often than obligately autotrophic species. Green or purple sulphur bacteria were seldom isolated from arable soils. Seventy-five isolates were tested for their ability to oxidize thiosulphate and sulphur, with and without peptone, and in general heterotrophs were inferior to autotrophs and facultative autotrophs. Autoclaved soils when reinoculated with autotrophic or heterotrophic bacteria or mixtures of both types oxidized most sulphur when Th. thiooxidans and Th, thioparus were present. Soil inoculum could be imitated by a mixture of autotrophic and heterotrophic bacteria. No photochemical oxidation of sulphur was detected in soil exposed to sunlight. After enrichment of soil with seven inorganic sulphur compounds only sodium thiosulphate and sodium sulphite stimulated subsequent oxidation of elementary sulphur, while two organic sulphur compounds, cysteine and particularly methionine, were inhibitory. Starch supplement had no effect on sulphur oxidation, but asparagine depressed it. Preliminary incubation of soil for 1 to 4 weeks prior to the addition of sulphur accelerated its oxidation. The incidence of Th. thiooxidans was unaffected by eight sulphur compounds, was reduced by methionine, also by asparagine and starch, and was increased by periods of incubation prior to the addition of elemental sulphur.

Acetate assimilation by Nitrobacter agilis in relation to its "obligate autotrophy".

Acetate (1 to 10 mm) had no effect on the rate of nitrite oxidation or exponential growth by Nitrobacter agilis. However, acetate-1-(14)C and -2-(14)C were both assimilated by growing cultures, and acetate carbon contributed 33 to 39% of newly synthesized cell carbon. Carbon from acetate was incorporated into all of the major cell constituents, including most of the amino acids of cell protein and poly-beta-hydroxybutyrate (PHB). Cultures grown in the presence of acetate showed a significant increase in turbidity, attributable in part to protein synthesis and the accumulation of PHB in the "post-exponential phase," when the supply of nitrite was completely exhausted. Cell suspensons of N. agilis assimilated acetate in the absence of bicarbonate and even in the absence of nitrite. However, the addition of nitrite increased the rate of acetate assimilation by cell suspensions. The distribution of (14)C-acetate incorporated by cell suspensions was qualitatively similar to that found with growing cultures. Cell suspensions of N. agilis slowly oxidized acetate to CO(2). Addition of nitrite suppressed CO(2) production from acetate but increased the assimilation of acetate carbon into cell material. N. agilis contained all the enzymes of the tricarboxylic acid cycle. Growth of N. agilis in the presence of acetate did not significantly affect the levels of the enzymes of the tricarboxylic acid cycle, but did result in a 100-fold increase in the specific activity of isocitratase. In contrast, carboxydismutase was partially repressed. N. agilis was grown heterotrophically through seven transfers on a medium containing acetate and casein hydrolysate. The addition of nitrite increased the rate of heterotrophic growth. Heterotrophically grown organisms still retained their ability to grow autotrophically with nitrite. However, these organisms oxidized nitrite at a slower rate. Organisms from autotrophic and heterotrophic cultures were analyzed to determine the mean guanine plus cytosine content of their deoxyribonucleic acid; in both cases this mean was 61.2 +/- 1%. We concluded that N. agilis is not an obligate autotroph; it appears to be a facultative autotroph which resembles the novel facultative autotroph, Thiobacillus intermedius, very closely.

Respiratory carriers involved in the oxidation of hydrogen and lactate in a facultative autotroph

A number of respiratory carriers have been identified in intact cells of H 2-oxidizing bacteria from observation of their characteristic absorption spectra. These are: flavines, cytochromes of the b and c type, and two CO-binding pigments showing bands at 416–417, 535, and 570 mμ, and 429 mμ, respectively, in CO minus reduced difference spectra. In anaerobiosis the carriers are reduced by H 2 or lactate to the same extent. Addition of H 2 or lactate to oxidized cell suspensions caused a change in the steady-state oxidation-reduction level of cytochrome c which was related to the rate of respiration. However, the rates of respiration with these substrates, and the concentration of pigments which combine with CO vary, depending on the previous cultural history of the cells, indicating quantitative differences in the pathway of oxidation of these substrates. Inhibitor studies showed that cyanide and azide accelerated the rate of cytochrome c reduction while antimycin A and Amytal inhibited reduction. These compounds affected respiration of H 2 and lactate similarly, suggesting no qualitative difference in pathway of electron transport. A proposed sequence of reactions between substrate and O 2 was presented.

Iron sulphide ratios in corrosion by sulphate-reducing bacteria.

THE stimulation of iron corrosion under anaerobic conditions by Vibrio desulfuricans is well known, and the organism has recently been shown1 to be a facultative autotroph. Several laboratory investigations of this type of corrosion have been described2–4. But, to our knowledge, there has, as yet, been no published comparison of the corrosion under heterotrophic and under autotrophic conditions. We have felt that such a comparison should throw further light on the mechanism of this type of corrosion and on the explanation first proposed by von Wolzogen Kuhr2. This worker considered that the stimulation of corrosion was due to the utilization by the bacteria of cathodically produced hydrogen, by which the corrosion reaction would otherwise be polarized.

The Isolation and Cultivation of Sulphate-Reducing Bacteria

Summary: There are many strains and perhaps several species of sulphate-reducing bacteria. They may be isolated by using a variety of media over a wide range of temperature. Crude cultures are readily obtained, but isolation of absolutely pure cultures is usually difficult. Considerable simplification in procedure may sometimes be effected by including 3% Na2SO3.7H2O in the media; the sulphite eliminates most of the contaminating organisms in crude cultures and facilitates subsequent purification. A method of maintaining stock cultures on sterile clay is preferred to the use of artificial media. No growth takes place in media rendered ‘biologically free’ of iron, but the traces normally present in media constituents as impurities are sufficient for good development. Rapid and abundant growth free from ferrous sulphide has been obtained in a mineral salts + lactate + yeast extract medium containing ferrous ions below the saturation concentration for ferrous sulphide. One thermophilic and four mesophilie strains have been shown to be facultative autotrophs, using the oxidation of hydrogen as energy source, with sulphate, sulphite, thiosulphate and elementary sulphur as hydrogen acceptors. No autotrophic growth takes place in the absence of hydrogen. Hydrogen can be provided in vitro by immersing metallic iron in the medium.

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.