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

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 (sMMOc) 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 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 sMMOcphenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.

Copper-Binding Compounds from Methylosinus trichosporium OB3b

“…This paper is also the first report of a second CBC, CBC-L 2 , which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO C mutant strains of M. trichosporium OB3b.…”

“…The Nterminal amino acid sequence of CBC-L 2 , SPMP?S, differed from that of CBC-L 1 . The amino acid compositions of both CBCs show some similarity, especially in the multiple S, to the iron-chelating pseudobactins, ferribactins, and pyoverdins produced by some species of Pseudomonas and Azotobacter (20,53,55) but showed no sequence similarities. Unlike many iron-binding polypeptides, the identified amino acids of both CBCs are all L-amino acids (20,53,58).…”

“…Fitch et al (17) found that in M. trichosporium OB3b, these sMMO C mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 ϫ 10 16 M Ϫ1 (55). Other evidence for a specific copper uptake system was provided by the copperbinding cofactor (CBC) from M. capsulatus Bath (63).…”

Methanobactin: a potential novel biopreservative for use against the foodborne pathogen Listeria monocytogenes

“…In addition, because pMMO activity was lost when CBCs were removed from the pMMO polypeptides, it was suggested that CBCs may function as a cofactor for its activity or stability, scavenge oxygen radicals, as well as fulfilling possible secondary roles previously mentioned. Earlier that same year, Téllez et al (1998) isolated what they referred to as a copper-binding ligand (CBL) from M. trichosporium OB3b, thought to be a same compound described by DiSpirito et al (1998). This study described a ~500-MW compound that had a ligand preferring copper over nickel despite 20-fold concentrations of the latter.…”

Bioremediation of selenium species in solution by methanotrophic bacteria

“…Methanobactin is known to play a significant role in the copper-switch that controls expression of the two forms of MMO. Indeed, the first form of methanobactin characterized was from Methylosinus trichosporium OB3b (Figure 1-8), and it was found to be a small modified polypeptide of 1,154 Da (DiSpirito et al 1998;Tellez et al 1998;Kim et al 2004). Reprinted with permission from (Semrau et al 2013).…”