Benzylsuccinate Synthase is Post-Transcriptionally Regulated in the Toluene-Degrading Denitrifier Magnetospirillum sp. Strain 15-1.

Ingrid Meyer-Cifuentes,Nico Jehmlich,Hermann J Heipieper,Sven-Bastiaan Haange,Martin Von Bergen,Sylvie Gruhl,Jochen A Müller,Vanessa Lünsmann

doi:10.3390/microorganisms8050681

Abstract

The facultative denitrifying alphaproteobacterium Magnetospirillum sp. strain 15-1 had been isolated from the hypoxic rhizosphere of a constructed wetland model fed with toluene. This bacterium can catabolize toluene anaerobically but not aerobically. Here, we used strain 15-1 to investigate regulation of expression of the highly oxygen-sensitive glycyl radical enzyme benzylsuccinate synthase, which catalyzes the first step in anaerobic toluene degradation. In cells growing aerobically with benzoate, the addition of toluene resulted in a ~20-fold increased transcription of bssA, encoding for the catalytically active subunit of the enzyme. Under anoxic conditions, bssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate. Proteomics showed that abundance of benzylsuccinate synthase increased in cells growing anaerobically with toluene. In contrast, peptides of this enzyme were never detected in oxic conditions. These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes.

Highlights

Microbial toluene degradation has been extensively studied, resulting in the detailed description of one anaerobic and various aerobic catabolic pathways for this aromatic compound [1,2,3,4]
BssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate
These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes

Summary

Introduction

Microbial toluene degradation has been extensively studied, resulting in the detailed description of one anaerobic and various aerobic catabolic pathways for this aromatic compound [1,2,3,4]. Anaerobic degradation is initiated by benzylsuccinate synthase (BSS, encoded by bss genes), which adds a molecule of fumarate to the methyl group of toluene [5,6]. The generated benzylsuccinate is transformed to benzoyl-CoA in a β-oxidation-like scheme involving enzymes encoded by the bbs genes [7]. The large α subunit (BssA) when activated contains the glycyl radical [6], whereas the small β (BssB) and γ (BssC) subunits are potentially needed for formation, solubility and stability of a catalytically competent enzyme [5,6]. The glycyl radical is generated post-translationally by an activating enzyme, BssD [5,6]. The active form of BSS is oxygenolytically cleaved at the glycyl radical site [13]. Its inactive form is already oxygen sensitive, likely due to the presence of a [4Fe4S]-cluster of apparently very low midpoint potential in each of the small subunits [14,15]

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