Mycobactericum smegmatis ATCC 607 became iron starved and did not reach maximum population density when grown at an iron concentration of 0.1 microM, or less. Iron deficient cells were more susceptible than iron replete cells to H2O2 killing; 9 mM H2O2 killed about 80% of the population of cultures grown at 0.05 microM iron, while about 25 mM H2O2 was required for similar killing of cultures grown at 1 or 20 microM iron. In response to H2O2, iron sufficient cells produced major oxidative stress proteins of molecular masses of 90, 75, 65, 62, and 43 kDa (the 75 and 65 kDa proteins were identified as DnaK and GroEL homologs, respectively). Iron deficient M. smegmatis did not upregulate the DnaK and GroEL proteins when stressed with H2O2. Both iron deficient and iron sufficient M. smegmatis produced (at 48 degrees C) major heat shock proteins of molecular masses of 90, 75 (DnaK), 65 (GroEL), 62, 43, and 16 kDa. The stress protein response induced by 2 M ethanol challenge was similar to the heat shock response except that ethanol induced a unique 55 kDa protein and the 16 kDa heat shock protein was not apparent. Induction of ethanol stress proteins was identical in high iron and low iron cells. All of the stress agents induced expression of a 62 kDa protein which may also be induced by iron insufficiency. The heat and ethanol shock responses of M. smegmatis were unchanged by iron deficiency; therefore, the absence of DnaK and GroEL from the response of iron starved M. smegmatis to H2O2 may be due to a specific defect (or alteration) of the oxidative stress response during iron starvation.