GROWTH, COPPER-TOLERANT CELLS, AND EXTRACELLULAR PROTEIN-PRODUCTION IN COPPER-STRESSED CHEMOSTAT CULTURES OF VIBRIO-ALGINOLYTICUS

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 muM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis. The data demonstrate that V. alginolyticus can recover from challenge by high copper levels in a chemostat when copper is continuously added, that resistance to copper is maintained by the population when copper stress is removed for many generations, and that recovery is accompanied by the production of the extracellular, CuBPs which have been described previously in batch cultures. That all individual cells surviving copper challenge do not exhibit increased copper tolerance indicates that some cooperative phenomenon, such as medium conditioning, is responsible for copper detoxification by the bacterial population.