Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants

Carlos M. Téllez, Kristen P. Gaus, David W. Graham, Robert G Arnold, Roberto Z Guzman

Research output: Contribution to journalArticle

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Abstract

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 x 1016 M-1. 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.

Original languageEnglish (US)
Pages (from-to)1115-1122
Number of pages8
JournalApplied and Environmental Microbiology
Volume64
Issue number3
StatePublished - Mar 1998

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methane monooxygenase
Methylosinus trichosporium
Copper
copper
methane
mutants
ligand
Ligands
methane monooxygenase (soluble)
mutant

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants. / Téllez, Carlos M.; Gaus, Kristen P.; Graham, David W.; Arnold, Robert G; Guzman, Roberto Z.

In: Applied and Environmental Microbiology, Vol. 64, No. 3, 03.1998, p. 1115-1122.

Research output: Contribution to journalArticle

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abstract = "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 x 1016 M-1. 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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