REGULATION OF EXTRACELLULAR COPPER-BINDING PROTEINS IN COPPER-RESISTANT AND COPPER-SENSITIVE MUTANTS OF VIBRIO-ALGINOLYTICUS

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copp er-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatogra phic behavior as copper-binding protein (CuBP), a copper-induced super natant protein which has been implicated in copper detoxification in w ild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants d isplayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 mu M copper. Increased resistance t o copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Cop per-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP w as isolated. The simultaneous loss of both constitutive CuBP productio n and copper resistance in Cu40B3 indicates that constitutive CuBP pro duction is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) o f V. alginolyticus is an important factor in survival and growth of th e organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus ind icate that altered sensitivity to copper was mediated by a variety of physiological changes.