Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd' mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd' mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(ohydroxypheny1acetic acid) (EDDHA), whose complex with Fe(lll) has a large stability constant (log K = 33*9), also inhibited the growth of Cyd' mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(lll) or boiled catalase prevented the inhibition of Cyd' mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(lll), ascorbate plus Ga(lll), or Ga(lll) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(ll) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd' mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd' mutants overproduce siderophores, presumably reflecting intracellular iron deprivation. 2TN, UK

Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity.