| Summary: | <p><strong>Section 1</strong>. <em>Acetobacter suboxydans</em> (American Type Culture Collection No. 621) is an organism of the Pseudomonas family found in spoiled beer. Among the substances it requires for growth is <em>p</em>-aminobenzoic acid (<em>p</em>-AB), and since this substance is known to prevent the inhibition of bacterial growth by sulphonamides, the inter-relations of <em>p</em>-AB and a sulphonamide drug in the growth of <em>A. suboxydans</em> were investigated. The main approach was through a study of the variations which enabled the organism (1) to grow without <em>p</em>-AB and (2) to resist inhibition by sulphonamides. Such changes were likely to be associated with interesting variations in the metabolism of <em>p</em>-AB, since there is evidence, for instance, that in certain organisms sulphonamide resistance is due to an increased ability to synthesize <em>p</em>-AB.</p> <p>For the majority of organisms <em>p</em>-AB overcomes sulphonamide inhibition in a competitive manner: for a given degree of inhibition the ratio of the concentrations of <em>p</em>-AB an sulphonamide is constant, though the absolute amounts of these may be varied considerably. Competitive relationships of this kind suggest that both metabolite and inhibitor react with the enzyme normally using the metabolite, and that the proportion of each reacting is governed by the law of mass action. As a contrast, in non-competitive relationships, a small amount of metabolite will overcome Inhibition over a wide range of concentrations of inhibitor. It is likely that a non-competitive antagonist of an inhibitor is, or readily becomes, a product of the cell's utilisation of a competitive antagonist.</p> <p>As a preliminary to this study it was necessary to obtain certain information about the properties of the unchanged strain of <em>A. suboxydans</em>, and the following points were established:</p> <ol> <li>The growth of the organism was very sensitive to the air supply: optimal aeration gave optical growth.</li> <li>The organism was normally cultured in a medium of glycerol, casein hydrolysate, vitamins and salts (Medium 2, Appendix 2), but it grew to a limited extent in a medium in which glucose replaced glycerol. The limited growth in glucose medium was probably due to rapid acid formation from this substrate. Growth in glycerol medium was also affected by addition of glucose: at low concentrations glucose stimulated growth, but at higher levels it was inhibitory.</li> <li>The strain used in this laboratory would not grow satisfactorily on a synthetic medium similar to that used by Stokes & Larsen (1945).</li> <li>The organism required between 4 × 10<sup>-9</sup> and 1.6 × 10<sup>-9</sup> M <em>p</em>-AB to show visible growth in Medium 2, <em>p</em>-AB could be replaced by synthetic pteroylglutamic acid when added at about 100-fold greater molar concentration.</li> <li>Adenine replaced <em>p</em>-AB entirely, but not only was it required at a greater concentration than 10<sup>-5</sup> M, but only a small stationary population of cells was reached in adenine cultures.</li> <li>The response to <em>p</em>-AB was affected by the amount of casein hydrolysate added to the medium, and certain amendments had to be made to the original medium as published by Landy & Dicken (1942). The effect was not due to <em>p</em>-AB contained as impurity in the casein hydrolysate.</li> <li>Both <em>p</em>-AB and pteroylglutamic acid overcame sulphonamide inhibition of growth in a competitive manner. With 10<sup>-7</sup> M <em>p</em>-AB the organism was able to resist between 10<sup>-4</sup> and 2 × 10<sup>-4</sup> M sulphathiazole. Adenine, however, acted non-competitively, permitting growth in all concentrations of drug up to 2 × 10<sup>-3</sup> M.</li> <li>A synergism between pteroylglutamic acid and a mixture due to Lampen, Roepke & Jones (1946) of purines, methionine and thymine was traced to the adenine in the mixture.</li> <li>Extracts of the organism were prepared which replaced folic acid for <em>Lactobacillus casei</em> and adenine for an "adenine-less" mutant of <em>Neurospora crassa</em>. The folic acid content of cells of <em>A. suboxydans</em> was independent of their age and of the amount of <em>p</em>-AB in which they were grown.</li> </ol> <p>Extracts of the kind mentioned above were used for comparison of the quantitative properties of the variant strains described below.</p> <p>"Sub-strains" of <em>A. suboxydans</em> 621 were obtained trained to dispense with <em>p</em>-AB (strain A) and to resist some 200 times the normal amount of sulphathiazole for a given amount of <em>p</em>-AB (strain C). Throughout this work sulphathiazole was used as the sulphonamide type drug in preference to sulphanilamide amide as the latter is known to effect the CO<sub>2</sub> enzymes of cells in a manner not shown by its analogues.</p> <p>The <strong>sulphathiazole-resistant strain</strong>, strain C, was shown to have undergone no change in its requirement for <em>p</em>-AB in its normal medium. That is to say that it required the same amount of <em>p</em>-AB to give visible growth after a fixed time as did the parent strain, though its medium contained 2 × 10<sup>-3</sup> M sulphathiazole. Its growth curve in a medium containing this amount of sulphathiazole and 10<sup>-7</sup> M <em>p</em>-AB was similar to that of the parent strain growing in the same conditions but without sulphathiazole. On the other hand, strain C could grow in the absence of added <em>p</em>-AB if sulphathiazole were also omitted; the extent of growth was then limited.</p> <p>The relationship between <em>p</em>-AB and sulphathiazole in strain C was still competitive; there had merely been a change in the molar ratio of these two substances required to cause inhibition. The strain did not destroy sulphathiazole.</p> <p>The <strong>non-exacting strain</strong>, strain A, grew without added <em>p</em>-AB, but still resembled the parent organism in that it required nicotinic and pantothenic acids for growth. It had an increased resistance to sulphathiazole compared with the parent organism when both were tested with 10<sup>-7</sup> M <em>p</em>-AB, though this resistance was not as great as that of strain C. The relationship between <em>p</em>-AB and sulphathiazole was also competitive in this strain.</p> <p>The growth of strain A was affected by the amount of "vitamin-free" casein hydrolysate provided in its medium: growth in Medium 2 was limited compared with the other strains. In this medium its extent of growth was increased by: <ol> <li><em>p</em>-AB</li> <li>Pteroylglutamic acid at some 100 times the molar concentration at which <em>p</em>-AB was active.</li> <li>High concentrations of aspartic acid.</li> <li>Glucose at some concentrations.</li> <li>Increased amounts of vitamin-free casein hydrolysate. This last effect was not shown, however, by a second strain of this type called strain A<sub>2</sub>. The stimulation of growth by casein hydrolysate was not due to the presence of <em>p</em>-AB in this material.</li> </ol> <p>Many pure vitamins, amino-acids and other compounds of importance in general cell metabolism were tested for ability to stimulate the growth of strain A; only those mentioned above were active.</p> <p><strong>Other strains</strong>. An unsuccessful attempt was made to train strain A to resist sulphathiazole further by serial subculture in partially inhibitory concentrations of drug. Similarly, an attempt was made to reduce to zero the <em>p</em>-AB requirement of strain C in the presence of its normal concentration of sulphathiazole (2 × 10<sup>-3</sup> M). This was also unsuccessful, though a peculiar strain (strain CD) was obtained after prolonged subculture with suboptimal amounts of <em>p</em>-AB.</p> <p>The possible synthesis of <em>p</em>-AB, folic acid and adenine by the trained and normal strains of <em>A. suboxydans</em> was investigated.</p> <p>Attempts to detect <strong>synthesis of <em>p</em>-AB</strong> by strains A and C were made by ether extraction of cultures grown without this growth factor. The extracts were concentrated and assayed with a strain of normal <em>A. suboxydans</em> trained to grow in the presence of the extracts. Ho synthesis of <em>p</em>-AB was detected.</p> <p><strong>Synthesis of folic acid</strong> did occur with strain C, and the folic acid content of cells of this strain (assayed with <em>Lb. casei</em>) was the same as the content of cells of the parent strain. This was true of cells grown both with and without sulphathiazole. The folic acid content of cells of strain A was very much diminished compared with the other two strains. It is unlikely that any of the strains of <em>A. suboxydans</em> synthesised the pteroylglutamic acid molecule as such, because the shapes of the assay curves with <em>Lb. casei</em> using bacterial extracts were different from the control curves obtained with a sample of pteroylglutamic acid.</p> <p>Assays of whole cultures of <em>A. suboxydans</em> with <em>Lb. casei</em> were complicated by the presence of an inhibitor of <em>Lb. casei</em> in the assay material, and no conclusive results were obtained.</p> <p><strong>Synthesis of adenine</strong> occurred in strains A and C as well as in the parent strain; it was detected by assay with an "adenine-less" mutant of <em>N. crassa</em>. The adenine content of cells of all three strains was similar, and its presence probably accounted for the non-competitive anti-sulphathiazole activity of cell extracts for the normal strain.</p> <p><strong>Comparisons with <em>Escherichia coli</em> 273-384</strong>. This was a mutant of <em>Esch. coli</em> requiring <em>p</em>-AB for growth, and some variant strains analogous to A and C in <em>A. suboxydans</em> were isolated and investigated. The organism was readily trained to grow in the absence of vitamin and the resulting variant grew to the same extent as its parent growing optimally and its growth was not stimulated by <em>p</em>-AB. Its resistance to sulphathiazole was the same as that of its parent when both were tested with 10<sup>-7</sup> M <em>p</em>-AB.</p> <p>A strain was obtained having a sulphathiazole resistance increased by about 50-fold for a given amount of <em>p</em>-AB, but its resistance was not successfully increased further. In the absence of sulphathiazole it still required <em>p</em>-AB for growth (unlike strain C of <em>A. suboxydans</em>), and in the presence of drug its requirement for <em>p</em>-AB was considerable. The training was in a sense incomplete, since the growth rate of the resistant organism was less than that of the parent strain even after many subcultures in the training medium.</p> <p><strong>Discussion</strong>. The results outlined above were discussed in an attempt to understand further the mechanisms of the variations. that had taken place in <em>A. suboxydans</em>. It was considered to be unlikely that either strain had developed an ability to synthesise <em>p</em>-AB, though the possibility that extremely small amounts were synthesised could not be eliminated entirely. Strain A was regarded as having developed a mode of growth independent of this vitamin, and probably also of folic acid; its growth was probably dependent on the supply of amino-acids.</p> <p>The view was taken that, while strain C may possibly have undergone a change of the same type as that undergone by strain A, this was not the change responsible for its high sulphathiazole resistance. The primary change was considered to be a quantitative expansion of <em>p</em>-AB-utilising enzyme in the cell, at the surface of which <em>p</em>-AB and sulphonamides can be regarded as competing. An increase in affinity of the enzyme for <em>p</em>-AB as compared with sulphathiazole would also account for the experimental data. The point was made that further York on the rates of utilisation of <em>p</em>-AB, or rates of formation of products from <em>p</em>-AB, would be of interest.</p> <p>A brief mathematical consideration of this view was given to show that a roughly 80-fold expansion of <em>p</em>-AB-utilising enzyme would account for the 200-fold increase in sulphathiazole resistance observed.</p> <p><strong>Section 2</strong>. Concurrently with the work on the variations of <em>A. suboxydans</em> some experiments were undertaken on the interactions of artificially induced mutants requiring <em>p</em>-AB for growth. In view of the results of other workers on induced mutants it is likely that mutations resulting in a growth factor requirement are due to damage to one stage in a sequence of biochemical reactions leading normally to the synthesis of the factor required. This may be represented:</p> <p>A → B → C → D → E → F..... → metabolite.</p> <p>In various organisms with a similar requirement these interruptions may well be at different stages in the sequence, and, as Beadle (1945) and his co-workers have shown with <em>N. crassa</em>, a mutant damaged, for example, between A and B will sometimes respond to extracts if one damaged between C and D, since C may accumulate in cultures of the latter.</p> <p>Ten mutant organisms requiring <em>p</em>-AB for growth were obtained from other laboratories and tested with this possibility in view, and interactions between the mould <em>Ophiostoma multiannulatum</em> 617 and two mutants of <em>Esch. coli</em> were recorded. Interactions between two mutants of <em>N. crassa</em> and <em>A. suboxydans</em> were shown to be due to formation of glucose by <em>N. crassa</em> from sucrose provided in its medium. This was formed at concentrations which stimulated the growth of <em>A. suboxydans</em> when fluid from a grown mould culture was added to its medium.</p></p>
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