Harwood V J, Gordon A S
Department of Biology, Old Dominion University, Norfolk, Virginia 23529.
Appl Environ Microbiol. 1994 Jun;60(6):1749-53. doi: 10.1128/aem.60.6.1749-1753.1994.
Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed 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 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-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 was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.
将溶藻弧菌野生型的细胞外蛋白与耐铜和对铜敏感的突变体的细胞外蛋白进行了比较。一个耐铜突变体（Cu40B3）组成型产生一种细胞外蛋白，其表观分子量（21 kDa）和色谱行为与铜结合蛋白（CuBP）相同，CuBP是一种铜诱导的上清蛋白，与野生型溶藻弧菌的铜解毒有关。对铜敏感的溶藻弧菌突变体在上清蛋白谱上表现出一系列变化。在用50 microM铜胁迫培养后，在一个对铜敏感的突变体的上清液中未检测到CuBP。在野生型或耐铜突变体Cu40B3中，用亚抑制水平的铜进行预培养不会诱导对铜的抗性增加。耐铜突变体在非选择性琼脂上进行10次或更多次传代培养后，仍保持在添加铜的琼脂上生长的能力，表明该表型具有稳定性。分离出了一个对铜具有野生型敏感性且不再组成型表达CuBP的Cu40B3衍生物。Cu40B3中组成型CuBP产生和耐铜性的同时丧失表明，组成型CuBP产生是该突变体耐铜所必需的。这些数据支持了这样一种假设，即溶藻弧菌的细胞外约20 kDa蛋白是该生物体在铜浓度升高时存活和生长的重要因素。在耐铜和对铜敏感的溶藻弧菌中观察到的一系列表型表明，对铜敏感性的改变是由多种生理变化介导的。
