Cloning and characterization of the goadsporin biosynthetic gene cluster from Streptomyces sp. TP-A0584

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Abstract
Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of O2 consumption. The Vmax of ctb mutant cells for O2, calculated using either globin, was greater than that of wild-type cells at extracellular O2 concentrations up to ∼1 μM, suggesting a role for Ctb in moderating O2 supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed O2 in an O2 electrode (10–200 μM O2) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external O2 concentrations. However, a lack of sensitivity of the mutant to paraquat or H2O2 indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. O2 affinities of cells (Km values of approximately 40 nM and 1 μM) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating O2 flux within C. jejuni.
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