Desulfatiglans anilini Initiates Degradation of Aniline With the Production of Phenylphosphoamidate and 4-Aminobenzoate as Intermediates Through Synthases and Carboxylases From Different Gene Clusters.

Xiaoman Xie,Thomas Huhn,Bernhard Schink,Dieter Spiteller,Nicolai Müller

doi:10.3389/fmicb.2020.02064

Abstract

The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn2+, K+, and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.

Highlights

To date only few microorganisms are known that can degrade aniline
The proteins that were highly abundant after growth with aniline or 4-aminobenzoate were analyzed: Among them, the proteins encoded by a putative gene cluster identified in this study, which we termed gene cluster ani, owing to the fact, that the corresponding genes were higher expressed during growth with aniline or 4-aminobenzoate compared to growth with phenol or benzoate as judged by their Z-scores (locus tag from H567DRAFT_03866 to H567DRAFT_03876 (Figures 1–3)
Differential proteomics indicated that at least three gene clusters are involved in aniline degradation (ani gene cluster and hcr gene cluster, as well as the gene coding for an aromatic carboxylase subunit of the phenol degradation phe gene cluster (Figures 1, 2)

Summary

Introduction

To date only few microorganisms are known that can degrade aniline. Several aerobic bacteria degrade aniline to the central intermediate catechol using oxygen-dependent reactions. The enzymes required in the aerobic degradation of aniline were characterized by heterologous expression of the enzymes in E. coli (Takeo et al, 2013). The key intermediate of aerobic aniline degradation, catechol, undergoes either meta-cleavage or ortho-cleavage, depending on the type of bacterium (Fujii et al, 1997; Fukumori and Saint, 1997; Murakami et al, 1998; Murakami et al, 2003; Liang et al, 2005; Xiao et al, 2009). In the absence of oxygen, bacteria have to use completely different strategies to degrade aniline

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