Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T

PLOS ONE, Dec 2019

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 μmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

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Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T

March Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T Christina Meinert 0 1 2 Ulrike Brandt 0 1 2 Viktoria Heine 0 1 2 Jessica Beyert 0 1 2 Sina Schmidl 0 1 2 Jan Hendrik WuÈ bbeler 0 1 2 Birgit Voigt 0 2 Katharina Riedel 0 2 Alexander SteinbuÈ chel 0 1 2 0 Funding: We acknowledge support by Open Access Publication Fund of the University of Muenster 1 Institut fuÈr Molekulare Mikrobiologie und Biotechnologie, WestfaÈlische Wilhelms-UniversitaÈt , MuÈnster, Germany , 2 Institut fuÈr Mikrobiologie, Ernst-Moritz-Arndt-UniversitaÈt , Greifswald, Germany , 3 Environmental Science Department, King Abdulaziz University , Jeddah , Saudi Arabia 2 Editor: Olaf Kniemeyer, Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie eV Hans-Knoll-Institut , GERMANY 2-Mercaptosuccinate (MS) and 3,3Â-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 μmol mg-1 min-1) and is controlled by LysR (MIM_c37410). - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing interests: The authors have declared that no competing interests exist. Introduction Advenella mimigardefordensis strain DPN7T was first described by WuÈbbeler et al. in 2006 [1]. Initially, it was designated as Tetrathiobacter mimigardefordensis and in 2009 reclassified to the genus Advenella, which currently consists of five species [2±7]. Strains of this genus belong to the family Alcaligenaceae and have been detected in a variety of habitats [8]. They can perform diverse metabolic reactions, and some strains of this genus degrade xenobiotics [1,9]. A. mimigardefordensis strain DPN7T was isolated because of its capability to utilize organic sulfur compounds such as the xenobiotic 3,3Â-dithiodipropionic acid (DTDP), dibenzothiophene, taurine or 2-mercaptosuccinic acid (MS) as sole carbon source. Interestingly, A. mimigardefordensis strain DPN7T is the only known strain known to metabolize DTDP as well as MS. This is interesting as both compounds were discussed as precursor substrates for polythioester (PTE) production [10]. PTEs represent an interesting class of biopolymers, whose constituents are linked by thioester bonds. Therefore, they comprise a sulfur-containing polymer backbone, which alters the thermophysical properties of the polymer, resulting in increased thermal stability and a higher degree of crystallinity in comparison to the structurally related polyhydroxyalkanoates [ 11,2 ]. PTEs could replace synthetic plastics derived from petrochemicals, especially if biologically persistent polymers are required. However, the production costs must be lowered significantly and the yield of the polymer must be increased [12]. The first chemical production of PTEs was described in 1951 by Marvel and Kotch [13]. In 2001, biotechnical production of PTEs has been reported by LuÈtke-Eversloh and colleagues [14] in strains of Ralstonia eutropha and Escherichia coli. In 2012, PTE homopolymer production starting from DTDP was successfully implemented [15] in A. mimigardefordensis strain DPN7T. The cellular conte (...truncated)


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Christina Meinert, Ulrike Brandt, Viktoria Heine, Jessica Beyert, Sina Schmidl, Jan Hendrik Wübbeler, Birgit Voigt, Katharina Riedel, Alexander Steinbüchel. Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T, PLOS ONE, 2017, Volume 12, Issue 3, DOI: 10.1371/journal.pone.0174256