Date of Award
Doctor of Philosophy (PhD)
Biomedical Sciences - Microbiology & Immunology
Andrew S. Gordon
Supernatant proteins in Vibrio alginolyticus batch cultures were analyzed by SDS-PAGE before copper was added, 24 and 48 hours after the addition of copper, and in 24 hour control (no Cu) cultures. Two proteins, one 21 kilodalton (kDa) and one 19 kDa, were found to be copper-induced, and were designated copper-binding protein 1 (CuBP1) and CuBP2. CuBP1 and CuBP2 became detectable in supernatants during the Cu-induced lag phase, and increased in concentration over the following 48 hours. Chloramphenicol inhibited production of these proteins. Gel-to-gel variability was implicated as the dominant factor determining whether one or two Cu-induced proteins were detected in Vibrio alginolyticus supernatants, and ca. 20 kDa Cu-induced proteins were quantitated together in subsequent analyses.
Experiments in continuous (chemostat) cultures of Vibrio alginolyticus demonstrated that the bacteria could survive copper stress in an open system. Copper stress reversibly inhibited swarming in most colonies from long-term copper-stressed cultures, and permanent inhibition of swarming was observed in some isolates. Mutation to an oxidase negative phenotype, which was not reversible, occurred at high frequency in copper-stressed continuous cultures.
The stability of two Cur mutants isolated from continuous culture was demonstrated by subculturing each isolate ten times on nonselective marine agar (10° MA), and comparing plate counts on unamended and 40μM Cu-amended agar to corresponding plate counts of isolates freshly passed on Cu-amended agar.
One Cur isolate, Cu40B3, constitutively produced a ca. 21 kDa protein which displayed the same chromatographic behavior (immobilized metal ion affinity chromatography followed by reverse phase high performance liquid chromatography) as CuBP. After fifteen nonselective subcultures, a revertant Cus derivative of Cu40B3 (Cu40B3(SW)) was isolated. Cu40B3(SW) lost the mutation to constitutive CuBP production and copper resistance simultaneously, indicating that constitutive CuBP production in Cu40B3 is necessary for maintenance of its copper-resistant phenotype.
Copper-sensitive Vibrio alginolyticus mutants displayed a range of alterations in supernatant protein profiles, and two of the seven mutants were indistinguishable from the wild-type in terms of supernatant proteins with and without copper stress. One Cus mutant was isolated which contained no CuaP in supernatants from 50 μM copper-stressed cultures.
Harwood, Valerie J..
"A Genetic and Physiological Study of the Role of Extracellular Copper-Binding Proteins in Copper Detoxification by the Marine Bacterium Vibrio alginolyticus"
(1992). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/bt7w-3x13