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Title:Metabolism of methylated sulfur compounds in methanosarcina
Author(s):Fu, He
Director of Research:Metcalf, William W
Doctoral Committee Chair(s):Metcalf, William W; Cann, Isaac
Doctoral Committee Member(s):Olsen, Gary J; Whitaker, Rachel J
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Methylated sulfur compounds
Metabolism
Methanogens
Methanosarcina
Abstract:Methylated sulfur compounds play pivotal roles in the global sulfur and carbon cycles and contribute to global temperature homeostasis. Although the degradation of these molecules by aerobic bacteria has been well studied, relatively little is known regarding their fate in anaerobic ecosystems. The methanogenic archaeon Methanosarcina acetivorans uses a variety of methylated sulfur compounds as carbon and energy sources. Previous studies implicated the mtsD, mtsF and mtsH genes in catabolism of dimethylsulfide, but the genes required for use of other methylsulfides have yet to be established. In particular, the molecular mechanisms for metabolism of dimethylsulfide, methanethiol and methylmercaptopropionate (MMPA) by strictly anaerobic methanogens of the genus Methanosarcina have yet to be identified. Here, we show that a four-gene locus, designated mtpCAP-msrH, is specifically required for the growth on MMPA. The mtpC, mtpA and mtpP genes encode a putative corrinoid protein, coenzyme M (CoM) methyltransferase and Major Facilitator Superfamily (MFS) transporter, respectively, while msrH encodes a putative transcriptional regulator. Mutants lacking mtpC or mtpA display a severe growth defect in MMPA medium, but are unimpaired during growth on other substrates. The mtpCAP genes comprise a transcriptional unit that is highly and specifically up-regulated during growth on MMPA, whereas msrH is monocistronic and constitutively expressed. Mutants lacking msrH fail to transcribe mtpCAP and grow poorly in MMPA medium, consistent with its assignment as a transcriptional activator. The mtpCAP-msrH locus is conserved in numerous marine methanogens, including eight Methanosarcina species that we showed are capable of growth on MMPA. Mutants lacking the mtsD, mtsF and mtsH genes display a 30% reduction in growth yield when grown on MMPA, suggesting that these genes play an auxiliary role in MMPA catabolism. A quadruple mtpCAP, mtsD, mtsF, mtsH deletion strain was incapable of growth on MMPA. Reanalysis of mtsD, mtsF and mtsH mutants suggests that the preferred substrate for MtsD is dimethylsulfide, while the preferred substrate for MtsF is methanethiol. Moreover, we demonstrated that MtpA catalyzes a robust methylcob(III)alamin: mercaptopropionate(MPA), generating equal molar of MMPA and cob(I)alamin, but not methylcob(III)alamin: CoM methyl transfer reaction, suggesting that MtpA is an MMPA-specific MMPA:cobalamin methyltransferase. MtpA catalyzed the methylcobalamin: MPA methyl transfer reaction with an apparent Km for MPA at 12 uM, kcat at 0.315 s-1, with equal molar consumption of MPA and production of MMPA. Co-streptavidin precipitation of MtpA revealed that it co-purifies with MtpC and MtsF, raising the possibility that MtsF is a methyltransferase for methylcobalamin: CoM methyl transfer reaction. Our genetic analysis of deletion mutants of Methanosarcina acetivorans also revealed that ramS2 is necessary for DMS metabolism, and contribute to MMPA metabolism. Taken together, these discoveries established the molecular mechanisms of metabolism of methylated sulfur compounds in Methanosarcina. These new pieces of knowledge will aid the development of predictive sulfur cycle models, and enable molecular ecological approaches for the study of methylated sulfur compounds in anaerobic ecosystems.
Issue Date:2017-04-20
Type:Thesis
URI:http://hdl.handle.net/2142/97717
Rights Information:Copyright 2017 He Fu
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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