UBC Theses and Dissertations
Consequences of bacillus subtilis in iron deficiency Peters, Walter Joseph
Cultures of Bacillus subtilis growing in an iron-deficient medium produced coproporphyrin III (coproporphyrin) and phenolic acids. (2,3-dihydroxybenzoylglycine (DHBG), 2,3-dihydroxybenzoic acid (DHB), or both). (DHB(G) refers to DHB or DHBG, or both compounds). Phenolic acid production was proportional to the amount of iron present, and occurred logarithmically, parallelling growth. In the presence of DHB, lower levels of iron inhibited phenolic acid production, so that the actual inhibition of synthesis may involve the Fe(+3): (DHB(G))₃ complex. Accumulatlon of DHB(G) was influenced by the levels of aromatic amino acids, anthranilic acid, and histidine in the medium. In vitro experiments demonstrated that DHB was formed from chorismic acid. In vivo and in vitro experiments with strain B-1471 showed that DHB was coupled to added glycine to form DHBG. Disappearance of DHB(G) was observed in all strains studied, but oxidation did not occur. Phenolic acid production always preceded coproporphyrin production. Phenolic acids have very strong affinities for ferric iron. Their production may therefore allow the scavenging of the last traces of iron from the medium for hemin synthesis. The relationship between phenolic acid and coproporphyrin production was borne out by the following observations: (i) a higher level of iron was required to prevent coproporphyrin production than phenolic acid production (ii) the Fe(+3) (DHB(G))₃ complex was a more potent inhibitor of coproporphyrin production than iron alone (iii) a mutant blocked at σ-aminolevulinic acid synthetase did not produce phenolic acids during iron-deficient growth (iv) serine auxotrophs produced much lower levels of coproporphyrin and phenolic acids than the wild-type strain (v) some mutants defective in phenolic acid production produced low levels of coproporphyrin, whereas one strain of this type produced elevated levels of coproporphyrin. Compounds known to inhibit normal functioning of the tricarboxylic acid cycle decreased coproporphyrin production in all strains studied. These inhibitors reduced DHBG excretion, but had no effect on DHB production. A number of analogs of DHB inhibited DHB(G) accumulation to varying degrees, depending upon their structure. The most potent inhibitors were m-substituted derivatives of benzoic acid. Two sideramines, ferrichrome and ferrioxamine, inhibited DHBG production in strain B-1471. The inhibitory action of ferrichrome was shown to be due to its ability to mediate cellular uptake of low levels of iron. The capacity of B. subtilis for iron uptake was increased about 20-fold by growing the cells in an iron-deficient medium. Under these conditions, the addition of low levels of phenolic acids increased both the rate and extent of iron uptake. Mutants unable to synthesize normal levels of phenolic acid were shown to have a reduced capacity for iron uptake after growth in an iron-deficient medium. Mutants resistant to 8-hydroxyquinoline had an increased capacity for iron-uptake under these conditions.
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