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Appl Environ Microbiol, March 1998, p. 1115-1122, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Carlos M. Téllez,¹ Kristen P. Gaus,^2, David W. Graham,³ Robert G. Arnold,^1,* and Roberto Z. Guzman¹

Department of Chemical and Environmental Engineering¹ and Department of Soil and Water Science,² University of Arizona, Tucson, Arizona 85721, and Department of Civil Engineering, University of Kansas, Lawrence, Kansas 66049³

Received 6 June 1997/Accepted 17 December 1997

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 µM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant's inability to take up CBL-complexed copper, not to a defective CBL structure.

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^* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of Arizona, Tucson, AZ 85721. Phone: (520) 621-6044. Fax: (520) 621-6048. E-mail: arnold{at}bigdog.engr.arizona.edu.

Present address: Texas Natural Resource Conservation Commission, Ft. Worth, TX 76116.

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