Porphyrin formation and its regulation in Arthrobacter

Porphyrins (tetrapyrroles) are the basic compounds of a number of substances functioning in living organisms as carriers of oxygen (hemoglobin), carriers of electrons (cytochromes) or as a trap for radiant energy (chlorophyll). In these active forms the tetrapyrroles contain a metal and are bound to a specific protein.In addition to the protein-bound tetrapyrroles, free porphyrins are found in nature. These compounds may be considered as the oxidized form of porphyrinogens, the true intermediates in the biosynthesis of heme (one of the two basic compounds of hemoglobin and cytochromes) and chlorophyll. The free porphyrins have no known biological function; their accumulation in general indicates the presence of some metabolic block in the biosynthetic pathway leading to heme or chlorophyll. Therefore, the accumulation of free porphyrins may be used as a tool for studying the effect of various factors on tetrapyrrole synthesis.So far the effect of B vitamins, oxygen and iron on the synthesis of heme and chlorophyll has been studied in different types of organisms including men, animals, plants, algae (particularly Chlorella species), photosynthetic bacteria and some heterotrophic bacteria (particularly Corynebacterium diphtheriae and Micrococcus lysodeikticus).During the study of the physiology of a number of Arthrobacter strains by collaborators of the Laboratory of Microbiology, Wageningen, a red pigment was found to occur in certain strains and under certain conditions (Antheunisse, private communication). Continuing this investigation, the present author found the red pigment to be coproporphyrin III (CP III).Since iron in relatively large amounts in a number of Arthrobacter strains did not eliminate CP III accumulation, as it had been found by LASCELLES in Rhodopseudomonas spheroides, the present investigation started with experiments to study this phenomenon. In subsequent experiments the regulation of the porphyrin synthesis in Arthrobacter, strain 223, was studied.In Chapter III the results of experiments with this organism growing in a mineral salts medium with glucose as the carbon source (medium A) have been given. To obtain CP III accumulation, biotin, zinc, sulfate, phosphate and ammonium ions had to be present in the nutrient medium in considerably higher concentrations than those required for giving maximum cell yield. Iron was also required in small amounts for growth and CP III accumulation. Increasing amounts of Fe did not eliminate CP III accumulation as it has been described for a number of bacteria in the literature. Report of more detailed experiments concerning the effect of iron on porphyrin synthesis is given in chapters VI and IX. CaCO 3 supplied in amounts of 1 %, although favourably affecting cell yield, entirely prevented CP III accumulation. With decreasing amounts of CaCO 3 , CP III accumulation increased.The aeration rate of the culture was found to have a pronounced effect on the synthesis of the porphyrin by Arthrobacter, strain 223. Maximum values for CP III were obtained at an aeration rate of I11 mMol O 2 /liter hr. Doubling this aeration rate slightly decreased the amount of CP III accumulated, but a four times higher rate (44 mMol O 2 /liter hr) although not affecting cell yield, almost eliminated CP III accumulation. Decreasing the aeration rate to 4 mMol O 2 / liter hr, which considerably reduced cell yield, entirely eliminated CP III accumulation. The latter was restored by adding 3-aminolevulinic acid (ALA, a precursor of CPG III), succinic acid or glutamic acid, showing that lack of substrate, and not reduced activity or reduced formation of the enzymes involved in CPG III formation, was responsible for the reduced CP III accumulation at a low aeration rate.The failing of the CP III accumulation at a high aeration rate (44 mMol O 2 /liter hr) was partly restored by adding ALA but not by adding succinic acid or glutamic acid. That the eliminated activity of ALA synthetase at a high aeration was the cause of the eliminated CP III accumulation was concluded from the fact that CPG III - producing cells stopped their production upon transfer to a high aeration rate. The enzymes catalysing the conversion of ALA to CPG III were also unfavourably affected by a high aeration rate but to a smaller extent than ALA synthetase.Chapter IV deals with the results of experiments concerning the influence of sulphur-containing amino acids on the formation of porphyrins in cultures of Arthrobacter, strain 223. They were done because in chapter III relatively large amounts of sulfate were shown to be required for the accumulation of CP III. In this process sulfate was assumed to exert its effect as a result of its conversion to homocysteine. This assumption was based on the following observations. (1) In CP III-accumulating Arthrobacter cultures sulfate could be replaced by cysteine or homocysteine (traces of sulfate as contamination being present in the nutrient medium). Since Arthrobacter, strain 223, easily converted cysteine to homocysteine but did not catalyse the reverse process, cysteine could not be formed from homocysteine and consequently could not be the cause of the CP III accumulation. (2) Small amounts of threonine, an inhibitor of the formation of homoserine, prevented the accumulation of CP III in cultures with sulfate or cysteine. The threonine effect could be eliminated by adding homocysteine or homoserine, a precursor of homocysteine. (3) Addition of 1 % CaCO 3 to medium A prevented the CP III accumulation unless homocysteine was present.In addition to threonine, methionine when present in sulfate-containing medium A in amounts of 50 μg/ml or more, prevented the accumulation of CP III. Of a number of other compounds including amino acids, purines, pyrimidines B vitamins, none was able to eliminate CP III accumulation. The eliminating effect of methionine on CP III accumulation, might be explained by assuming the functioning of a feedback mechanism controlling the synthesis of homocysteine. That this hypothesis, however, was not valid was concluded from the observations that (a) 50 μg/ml methionine which eliminated CP III accumulation in a sulfate medium, did not prevent porphyrin accumulation when present in a medium with sulfate + cysteine, (b) the presence of homocysteine in an A medium containing approximately 50 μg/ml methionine did not give CP III accumulation. The elimination of CP III accumulation in medium A by methionine presumably depended on the synthesis of a compound which favoured the conversion of CPG III to PP IX and heme. Larger concentrations of methionine under certain conditions (A medium with cysteine or with homocysteine) brought about the decomposition of this compound and consequently enhanced CP III accumulation.In Chapter V the effect of methionine and homocysteine on the formation of porphyrins by Arthrobacter, strain 223, was studied in more detail. Since the methyl group of methionine may be converted to formaldehyde (via choline) a comparison was made between the effect of increasing amounts of methionine and that of increasing amounts of formaldehyde on CP III accumulation in A media containing sulfate, sulfate + cysteine and sulfate + homocysteine, respectively. A far-going similarity between the effect of methionine and that of formaldehyde on CP III accumulation and heme formation was observed (the smallest amount of formaldehyde required to prevent CP III accumulation equalled the amount of formaldehyde which could have been derived from the smallest amount of methionine preventing this accumulation). From this similarity it was concluded that the effect of methionine on CP III accumulation had to be ascribed to the improved supply of one carbon units (via choline, N.N-dimethylglycine, sarcosine). Evidence as to this hypothesis was provided by the fact that the effect of different amounts of choline on CP III accumulation resembled that of formaldehyde and methionine. A second way in which methionine may increase the one carbon units supply of the cells is by its sparing action on the utilization of these units (no methionine has to be formed from homocysteine + one carbon units).One of the most important reactions in Arthrobacter, strain 223, in which one carbon units are involved is the synthesis of serine from glycine. Since serine via phosphatidylserine (PS) is one of the precursors of phosphatidylethanolamine (PE), the latter compound was assumed to be required for the conversion of CPG III to PP IX Decrease of the amount of PE in the cell would automatically increase the amount of CP III.Large amounts of methionine (added to the A medium supplied with cysteine or homocysteine), formaldehyde or choline gave a resumed accumulation of CP III. This effect was thought to be due to the removal of PE from the cells, partly by methylation of PE to PC, partly by enhanced phospholipase C activity as a result of the formation of PC.The favourable effect of large amounts of methionine, formaldehyde or choline on CP III accumulation required the presence of homocysteine. This amino acid was found to inhibit the formation of one carbon units from choline by inhibiting the conversion of betaine to N.N-dimethylglycine. This resulted in a reduced one carbon units supply and consequently in a reduced formation of serine. More evidence as to this conclusion was obtained by the fact that N.N-dimethylglycine or sarcosine when added to media with large amounts of methionine or choline prevented CP III accumulation.In Chapter VI a report is given of a number of experiments dealing with the effect of the iron supply of the bacteria on the formation of porphyrins. The preliminary experiments on this subject, reported in Chapter III, have shown that excessive amounts of iron which according to literature recordings concerning several types of microbes, should eliminate CP III accumulation, did not show this effect when applied to Arthrobacter, strain 223, growing in medium A.The experiments described in the previous chapters have shown that CP III accumulation in Arthrobacter, strain 223, cultures can be prevented and concomitantly heme formation enhanced by improving the supply of one carbon units. Therefore, the resumed studies on iron supply were carried out with a modified nutrient medium (B 3 ) containing in addition to medium A, methionine, cysteine, ethanolamine and some additional manganese.Reducing the amount of iron added to medium B 3 brought about a decreased formation of the iron-containing heme in Arthrobacter, strain 223, attended with the accumulation of CP III in amounts several times larger than the maximum amount of heme formed with large amounts of iron. This overproduction of CP III implied the presence of a regulatory mechanism depending on heme. The correctness of this hypothesis was shown by adding increasing amounts of heme to the culture medium in which CP III accumulation occurred and by estimating enzyme activity in cell-free extracts of Arthrobacter, strain 223. The main site of the regulating effect of heme on CP III accumulation was found to be localized in the enzyme converting glycine and succinyl-CoA to ALA (ALA synthetase). The synthesis of this enzyme was strongly repressed by heme while its activity was moderately inhibited (feedback control).The activity of ALA dehydratase, one of the enzymes catalysing the conversion of ALA to CPG III, was not inhibited by heme; however, the synthesis of this enzyme was considerably repressed.Iron added to iron-deficient medium B 3 (in which CP III accumulation occurred) affected the synthesis of ALA synthetase and that of ALA dehydratase similarly to added heme, showing that iron affected porphyrin accumulation as a result of its incorporation into heme.In addition to its regulating effect on porphyrin accumulation via heme formation, iron may affect porphyrin synthesis by stimulating the conversion of CPG III to PP IX. This was found to be due to the favouring effect of iron on PE formation as a result of the stimulation of the formation of fatty acids, which are required for the synthesis of PE.The presence of small amounts of myristic and oleic acid in iron-deficient medium B 3 gave rise to the accumulation of PP IX by Arthrobacter, strain 223. Large amounts of iron added to this medium prevented the accumulation of PP IX and gave heme formation.The accumulation of CP III in iron-deficient cultures (B, medium) of Arthrobacter, strain 223, was affected by the aeration rate of the culture medium similarly to that in iron-sufficient cultures (A medium) as reported in Chapter III. A high aeration rate depressed the synthesis of ALA synthetase and that of ALA dehydratase.In Chapter VII the effect of the biotin supply of Arthrobacter, strain 223, on the formation of porphyrins was studied in more detail. Earlier experiments, reported in chapter III, had shown that CP III accumulation in medium A required considerably larger amounts of biotin than those required for optimal growth. For the investigations discussed in chapter VII, A medium containing 5 x 10 -5 μg/ml biotin was used. At this concentration of biotin no CP III accumulated and heme was formed.When 1 day-old biotin-deficient cultures of Arthrobacter, strain 223, were supplied with a large amount of threonine, the heme content of the cells was strongly reduced and CP III accumulated. This accumulation was not reduced by increasing the supply of iron, but it was eliminated by a number of compounds serving as donor for one carbon units, showing that threonine is promoting CPG III formation as a result of inhibiting serine formation and thus formation of PE. This conclusion was confirmed by the fact that the addition of serine along with threonine prevented the accumulation of CP III.A further possibility to restore the accumulation of CP III in biotin-deficient Arthrobacter cultures was to add a large amount of methionine to the nutrient solution. The CP III accumulation, similarly to that with threonine, was prevented by adding compounds serving as donor for one carbon units or by adding serine. This shows that methionine interfered with PE. (More details concerning this phenomenon in chapters V and IX).The fact that in biotin-deficient Arthrobacter cultures a ready accumulation of CP III can be achieved (addition of threonine or methionine) shows that the enzymes catalysing the formation of CPG III, like ALA synthetase, are functioning normally. This conclusion is in disagreement with literature recordings claiming that a reduced synthesis of ALA synthetase and a reduced activity of this enzyme are responsible for the absence of CP III accumulation in biotindeficient bacteria. More evidence against the latter explanation was obtained by the results of experiments with cell- free extracts of biotin-deficient CP III-accumulating arthrobacters grown in the presence of threonine. The ALA synthetase and ALA dehydratase activities in these cells were similar to those of biotin-sufficient cells, accumulating CP III.In Chapter VIII the integration of protein synthesis and heme formation has been discussed. In cells heme is associated with specific proteins (hemoglobin, cytochromes). Therefore, the regulation of the synthesis of these compounds not only involves the tetrapyrrole part but also the protein moiety. To study the relationship of heme formation and protein synthesis in Arthrobacter, strain 223, experiments with chloramphenicol have been carried out.Addition of chloramphenicol immediately stopped the synthesis of bacterial protein as well as the porphyrin synthesis (CP III accumulation and herne formation). No precursors of CPG III were detected in the cultures incubated with the antibiotic, eliminating the hypothesis that protein-bound precursors of CPG III would exist and would be involved in the inhibiting effect of chloramphenicol on porphyrin formation. Addition of ALA did not restore the porphyrin formation in Arthrobacter cultures incubated with the antibiotic.Decrease of the amount of nitrogen in the medium reduced the amount of synthesized porphyrins to a larger degree than that of bacterial protein. Addition of ALA restored the synthesis of porphyrins, indicating the absence of protein-bound precursors of herne in Arthrobacter.In Chapter IX a comparison was made of the phospholipid composition of Arthrobacter cells and the ability of these cells to form different types of porphyrin. Evidence of the functioning of phospholipids in heme formation in Arthrobacter, strain 223, has been obtained in chapters V, VI and VII. In these chapters the functioning of phosphatidylethanolamine (PE) in the conversion of CPG III to PP IX was emphasized.To study the relationship between phospholipids and porphyrin synthesis, cells of Arthrobacter, strain 223, either producing large amounts of iron-free porphyrins or synthesizing the iron-containing heme as the predominant tetrapyrrole have been analysed for phospholipid composition.To obtain cells accumulating CP III, PP IX, or forming heme, use was made of the experimental results recorded in chapters IV, V, VI and VIII.1. CP III-accumulating cells, grown in medium A with excessive amounts of biotin and iron, contained phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), and phosphatidic acid (PA).Suppression of the CP III accumulation (which was attended with heme formation) by supplying this medium with compounds serving as donor for one carbon units gave cells containing in addition to the three above-mentioned phospholipids, phosphatidylethanolamine (PE).2. A different medium in which Arthrobacter, strain 223, accumulated large amounts of CP III, was the biotin-rich medium B 3 without added iron. The phospholipid content of those cells was very low, and no PE was detectable.When this medium had been supplied with mixtures of long chain fatty acids (palmitoleic or oleic acid + a C 14 , C 15 , or C 18 saturated fatty acid), CP III accumulation was prevented whereas heme formation was strongly increased. The phospholipid content of these cells was much higher, with PE being the dominant phosphatide.When the biotin-rich iron-deficient medium B 3 had been supplied with small amounts of myristic acid + oleic acid, PP IX was the porphyrin which accumulated in the culture medium, and PE practically the only phospholipid of the cells.Supplied with excessive amounts of iron, no PP IX accumulated in the latter medium. Heme was the predominant porphyrin and PE and PA the most important phospholipids. The correlation between accumulation of PP IX and absence of PA suggested that the latter phospholipid is involved in the conversion of PP IX to heme.3. In biotin-deficient Arthrobacter cultures, grown in medium A with large amounts of iron, no CP III was found; heme was the dominant porphyrin formed and PE the prevailing phospholipid.When large amounts of threonine were added to Arthrobacter cultures grown for 1 day in this medium, or methionine was added to this medium before inoculation, CP III accumulated while the heme content of the cells remained low; no PE was detected in these cells, but increased amounts of PG and DPG.When the latter medium along with threonine was supplied with N,N-dimethyIglycine or serine, CP III accumulation was prevented, heme was formed in a considerable amount while PE was the predominant phospholipid. The same results were obtained when the latter medium was supplied with methionine + N, N- dimethyIglycine or serine.4. Biotin-deficient, iron-deficient Arthrobacter cultures grown in medium B 3 , accumulated CP III; the heme content as well as the phospholipid content of the cells was low.Addition of long chain fatty acids to this medium had the same effect as when these acids had been added to the biotin-sufficient, iron-deficient medium (see this summary, 2).When Arthrobacter, strain 223, in medium A with large amounts of biotin and iron (1), started to accumulate CP III, the phospholipid content dropped to a low level, apparently due to the activity of phospholipase C. With continued incubation, the phospholipid content returned to a normal level with PG and DPG, but not PE, as predominant phospholipids found.To confirm the results obtained in the above-mentioned experiments with whole cells that PE favours the conversion of CPG III to PP IX, experiments were performed with cell-free extracts of Arthrobacter, strain 223, grown for 72 hours in iron-deficient B 3 medium. These bacteria had alow content of phospholipids, while no PE was detectable.The conversion of ALA to PP IX by these extracts required the presence of PE isolated either from Arthrobacter or from mammalia. A mixture of PG + DPG + PA, or iron, had no effect. Since iron when supplied to Arthrobacter growing in the iron-deficient B 3 medium catalysed this reaction, it seemed to be obvious that in Arthrobacter cells this metal exerts its favourable effect as a result of its stimulating effect on PE formation.To confirm the conclusion that in Arthrobacter PA would be involved in the conversion of PP IX to heme, experiments were carried out with the membrane fraction of Arthrobacter, strain 223, grown in biotin-sufficient, iron-deficient medium B 3 containing small amounts of myristic acid and oleic acid. The conversion of PP IX + ferrous iron to heme by this membrane fraction was favoured by PA. The highest amount of heme was found with PA containing unsaturated + saturated fatty acids. PA with saturated fatty acids only and a mixture of PA + PG + DPG, isolated from Arthrobacter, strain 223, were also active be it to a less extent. PC + PE had no activity.