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Cleavage of poly(cis-1,4-isoprene) by the latex clearing protein (Lcp) and involvement of a molybdenum-dependent oxidoreductase (OxiBA) in early steps of rubber degradation

Fachliche Zuordnung Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Förderung Förderung von 2007 bis 2011
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 42348726
 
Erstellungsjahr 2017

Zusammenfassung der Projektergebnisse

This study focused on three aspects of polyisoprene degradation in bacteria and the involvement of the latex clearing protein Lcp. (1) At the beginning Lcp from the clear zone forming rubber degrading bacterium Streptomyces sp. strain K30 was investigated. Initial hints for the excretion of Lcp by the tat System were confirmed by generation of a tatC disruption mutant of Streptomyces lividans 10-164 harboring lcp from Streptomyces sp. strain K30. In contrast to the parent strain, this mutant was impaired in forming clear zones on rubber overlay agar plates. Unfortunately, it was not possible to generate a tatC mutant of Streptomyces sp. strain K30. Therefore, and due to several other constraints regarding the application of molecular methods to strain K30, which became obvious during the study, we continued our studies on rubber degradation and Lcp with Gordonia polyisoprenivorans strain VH2. (2) The analysis of the genome sequence of G. polyisoprenivorans strain VH2 unraveled the presence of two lcp genes coding for Lcp1 and Lcp2, respectively. Either gene gave a rubber degrading phenotype, and a rubber-negative phenotype was only obtained in a Δlcp1Δlcp2 double deletion mutant. A method was developed to produce purified Lcp2 protein in sufficient quantities for further biochemical analysis including its metal ion content and of prosthetic groups. Purified Lcp of Gordonia polyisoprenivorans VH2 (Lcp1VH2) exhibited an intense red color indicating the presence of a cofactor. Inhibitor studies showed that Lcp1VH2 activity decreased when incubated with agents specifically chelating ferrous iron and copper, with agents chelating copper inhibited the enzyme most significantly. However, first analyses of the metal content with inductively coupled plasma optical emission spectrometry as well as an analysis with a bathocuproine sulfonate assay showed only substoichiometric amounts of copper. In addition, non-covalently bound heme-b was detected in Lcp1VH2 as cofactor in pyridine hemochrome spectra and by 3+ LC/ESI-ToF-MS. This heme-cofactor contained iron, most likely in the Fe state. We characterized the heme-cofactor. Changes were detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 “as isolated” occurs in an open state, directly being accessible for external ligands. Three highly conserved histidines (H195, H200 and H228) presumably acting as ligands coordinating the heme within the heme pocket, were mutagenized by replacing each of them with alanine by site-directed mutagenesis. By complementing the rubber-negative lcp-deletion mutant of G. polyisoprenivorans VH2 with lcp1VH2H195A, lcp1VH2H200A, and lcp1VH2H228A, the impact of the substitutions was monitored during mineralization of rubber. Only mutant lcp1VH2H195A had lost the ability to mineralize poly(cis-1,4-isoprene). (3) In parallel and by other studies, we obtained several independent axenic cultures of bacteria capable of utilizing poly(trans-1,4-isoprene), which is Gutta Percha, as sole carbon and energy source for growth. It was the first time that such microorganisms were obtained. Nocardia nova strain SH22a, which also degraded poly(cis-1,4-isoprene) (Natural ruber), was chosen as model organism for further analysis of Gutta Percha degradation, and its genome was sequenced. The sequence revealed the presence of one lcp gene. When lcp was disrupted, the mutant exhibited a Natural Rubber-negative as well as a Gutta Percha-negative phenotype thus indicating that both isomers of polyisoprene were cleaved by Lcp. We obtained first evidence that two different Mce transporters are responsible for the uptake of the resulting trans-oligoisoprenes and the cis-oligoisoprenes in strain SH22a, respectively.

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