Pyrimidines Staph, aureus

Fig. 20. Effect of the addition of a mixture of purines and pyrimidines on the rate of synthesis of protein in Staph, aureus incubated with a complete mixture of amino acids at different concentrations. (Gale and Folkes, 1953a.)...

Figure 22 also shows the effect of modifying the components of the purine-pyrimidine mixture in the presence of a complete amino acid mixture. Addition of any single purine or pyrimidine has little effect compared with the action of the complete mixture. Omission of single purines or pyrimidines, with the exceptions of uracil and xanthine, does not significantly decrease the nucleic acid synthesis omission of uracil decreases the synthesis to the level obtained with amino acids alone. This can be correlated with the finding that Staph, aureus is unable to syn-... 

Fig. 26. Progress curves for (a) nucleic acid synthesis and (b) protein synthesis in washed suspensions of Staph, aureus incubated with 1, glucose and a complete mixture of amino acids 2, as (1) - - complete mixture of purines and pyrimidines 3, as (2) + 30 Mg. chloramphenicol/ml. 4, glucose and mixture of purines and pyrimidines 5, as (4) -t- 30 Mg. chloramphenicol/ml. (Gale and Folkes, 1953b.)...

The addition of purines and pyrimidines accelerates the rate of protein synthesis in washed Staph, aureus as shown above. If penicillin in high concentrations is added to the incubation mixture, the additional protein synthesis due to the presence of purines and pyrimidines is abolished, although the basal protein synthesis is not significantly affected, as shown in Fig. 29. This effect is accompanied by a decrease in the purine-stimulated nucleic acid formation, but the concentration of penicillin which abolishes the additional protein synthesis does no more than halve (and frequently less than halve) the additional nucleic acid synthesis. Estimation of the proportions of the purine and pyrimidine bases in the nucleic acid fraction by the method of Smith and Markham (1950) modified by Wyatt (1951) shows that the changes lie in the ribonucleic acid fraction and that there is no significant alteration in the proportions of the various bases whether penicillin is present or not (Gale and Folkes, 1953b). 

Hotchkiss (1949) found that suspensions of Staph, aureus incubated with mixtures of amino acids, purines, and pyrimidines in the presence of penicillin took up more uracil than in the absence of penicillin. It is possible that there is a connection between these various investigations. Since there is no obvious alteration in the uracil content of the staphylococcal nucleic acid formed in the presence of penicillin (Gale and Folkes, 1953a Park, private communication) it seems that the formation of the uridine-5 -pyrophosphate derivatives may represent a stimulation of a side reaction rather than a side-tracking of the incorporation of uracil into nucleic acid. On the other hand, should the incorporation of an essential base into nucleic acid be inhibited, it is probable that the whole nucleic acid synthesis would cease rather than that the relative proportions of the bases within the nucleic acid should change. The true answer must probably await the development of a method for the study of nucleic acid synthesis in detail. George and Pandalai (1948) have claimed that the action of penicillin can be reversed by the addition of nucleic acid to cultures. 

Penicillin has no effect on protein synthesis by washed Staph, aureus under the author s experimental conditions until the concentration is raised to a value several orders above the hmiting bactericidal level. It does, however, prevent the increase in rate of protein synthesis that normally follows the addition of purines and pyrimidines to the external medium. Its action is therefore somewhat similar to that of bacitracin except that it does not inactivate the protein-synthesizing mechanism of the cell which is not synthesizing nucleic acid. For the present its action is indicated in Fig. 33 as a blocking of the pre-protein template concerned with nucleic acid synthesis but the high concentrations and partial effects obtained would suggest that, if it does act in this way, it affects a specific part of the template system rather than the whole mechanism. This will be elaborated below. 

For Staphylococcus aureus, thiamin (or its constituent pyrimidine and thiazole components together) was found by Knight to be an essential nutrient (155,156). Unlike the propionic acid bacteria, all the typical strains of Staph, aureus which have been examined (156, 289) drppear to need thiamin as an essential nutrient. No observations have been recorded of strains of Staph, aureus which can synthesize thiamin, as with the propionic and lactic acid bacteria such strains may yet be found. O Kane (273) records strains of Staph, flavus which grew in a simple ammonia medium without added growth factors. 

When it was found that the pyrimidine and thiazole components of thiamin could often replace thiamin, the opportunity was taken to examine the question of the biological specificity of the molecule, since a large number of related pyrimidines and thiazoleshad been prepared by the workers engaged in the synthesis of thiamin. Detailed studies on the specificity of the pyrimidine and thiazole portions of thiamin and of thiamin analogs have been made for Staph, aureus (169) Phycomyces blakesleeanus (38, 305, 306, 333, 334, 348) for various protozoa (236, 237) and for pea-roots (29, 30, 37). [Comparative summaries are given in 159, 211, 309]. 

When an equimolar mixture of pyrimidine and thiazole was given to Staph, aureus instead of thiamin the nature of the substitution at 5 in the pyrimidine was important. Thus, —CH2 NH2, —CH2 OH and —CH2 NH CSH permitted growth but —CH2 CO -NHa or — CH3 did not. The relative effects of these various substituents at position 5 was interpreted (159) to mean that for use the pyrimidine and thiazole portions were united by Staph, aureus to give the thiazolium compound thiamin itself, and that the two components were not used separately. With pea-roots similar differences are found in the availability of the substitution at 5 these also appear to be related to the ability of the root-cells to form the link with the thiazole. Thus —CH,Br (100%), —CH2-NH-CSH (100%) —CH2 NH2 (95%) and —CH2-OEt (25%) decrease in availability in the order shown, while —H, —CH2 COOH and —CH2 CO NH2 could not be used (30). 

Direct evidence for the synthesis of thiamin by joining the pyrimidine and thiazole components was shown for Phycomyces blakesleeanus by Bonner and Buchman (38). They further showed that after growth the resting mycelium broke down the thiamin, with destruction of the thiazole and liberation of the free pyrimidine. With Staph, aureus Hills (127) found similarly that the destruction of the thiazole was more rapid than that of the pyrimidine. 

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