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MmoD regulates soluble methane monooxygenase and methanobactin production in Methylosinus trichosporium OB3b

ABSTRACT: Methane oxidation by aerobic methanotrophs is well-known to be strongly regulated by the availability of copper, i.e., the “copper-switch”. That is, there are two forms of the methane monooxygenase: a cytoplasmic or soluble methane monooxygenase (sMMO) and a membrane-bound or particulate methane monooxygenase (pMMO). sMMO is only expressed and active in the absence of copper, while pMMO requires copper. Previous work has also shown that one gene in the operon of the soluble methane monooxygenase – mmoD – also plays a critical role, but its function is still vague. Herein we show that MmoD is not needed for expression of genes in the sMMO gene cluster but is critical for formation of sMMO polypeptides and sMMO activity in Methylosinus trichosporium OB3b, indicating that MmoD plays a key post-transcriptional role in maturation of sMMO. Further, data also show that MmoD controls expression of methanobactin, a unique copper-binding compound used by some methanotrophs for copper collection. Collectively these results provide greater insights into the components of the “copper-switch” and thus provide new strategies to manipulate methanotrophic activity.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Methylosinus Trichosporium Ob3b

SUBMITTER: Henriette Remmer

LAB HEAD: Henriette Remmer

PROVIDER: PXD045741 | Pride | 2023-09-29

REPOSITORIES: Pride

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Project description:Copper-limiting growth conditions were thought to cause an induction of genes possibly involved in copper uptake and sorting. This rationale in mind, we performed microarray analyses on B. japonicum cells grown in three variations of the BVM minimal medium. Variant 1 contained 2 M-NM-<M CuSO4 (copper excess). Variant 2 was prepared in HCl-treated glassware without any copper added (copper starvation). The residual copper concentration in this copper-starvation medium was analyzed by GF-AAS and determined to be 5 nM. Variant 3 (extreme copper limitation) was prepared like variant 2 but with the addition of 10 M-NM-<M BCS and 1 mM ascorbic acid where BCS chelates Cu(I) selectively, and ascorbic acid reduces any Cu(II) to Cu(I). Changes in the transcription profiles were recorded by the pairwise comparison of cells grown in variant 2 vs. 1, and variant 3 vs. 2. Only a small set of genes were differentially up- or down-regulated when copper-starved cells were compared with cells grown in copper excess. Most notably, five genes located adjacent to each other on the B. japonicum genome displayed an increased expression: bll4882 to bll4878. The five genes were named pcuA, pcuB, pcuC, pcuD, and pcuE (mnemonic of proteins for Cu trafficking). The genes with decreased expression are either of unknown function or M-bM-^@M-^S not surprisingly M-bM-^@M-^S play a role in copper resistance. Extreme copper limitation (variant 3 vs. 2) did not further enhance the expression of the five pcu genes. Instead, another cluster of adjacent genes was strongly up-regulated: bll0889 to bll0883, which code for unidentified transport functions. Incidentally, the list also includes the copper chaperone ScoI. Taken together, copper-limiting growth conditions have led to the de-repression of genes potentially involved in copper acquisition. Microarray-based transcriptome analysis of B. japonicum 110spc4 wild-type cells grown under normal, copper-limiting and copper excess conditions

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MmoD regulates soluble methane monooxygenase and methanobactin production in Methylosinus trichosporium OB3b

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