Anaerobic degradation of xenobiotic isophthalate by the fermenting bacterium Syntrophorhabdus aromaticivorans

Abstract

Syntrophorhabdus aromaticivorans is a syntrophically fermenting bacterium that can degrade isophthalate (3-carboxybenzoate). It is a xenobiotic compound which has accumulated in the environment for more than 50 years due to its global industrial usage and can cause negative effects on the environment. Isophthalate degradation by the strictly anaerobic S. aromaticivorans was investigated to advance our understanding of the degradation of xenobiotics introduced into nature, and to identify enzymes that might have ecological significance for bioremediation. Differential proteome analysis of isophthalate- vs benzoate-grown cells revealed over 400 differentially expressed proteins of which only four were unique to isophthalate-grown cells. The isophthalate-induced proteins include a phenylacetate:CoA ligase, a UbiD-like decarboxylase, a UbiX-like flavin prenyltransferase, and a hypothetical protein. These proteins are encoded by genes forming a single gene cluster that putatively codes for anaerobic conversion of isophthalate to benzoyl-CoA. Subsequently, benzoyl-CoA is metabolized by the enzymes of the anaerobic benzoate degradation pathway that were identified in the proteomic analysis. In vitro enzyme assays with cell-free extracts of isophthalate-grown cells indicated that isophthalate is activated to isophthalyl-CoA by an ATP-dependent isophthalate:CoA ligase (IPCL), and subsequently decarboxylated to benzoyl-CoA by a UbiD family isophthalyl-CoA decarboxylase (IPCD) that requires a prenylated flavin mononucleotide (prFMN) cofactor supplied by UbiX to effect decarboxylation. Phylogenetic analysis revealed that IPCD is a novel member of the functionally diverse UbiD family (de)carboxylases. Homologs of the IPCD encoding genes are found in several other bacteria, such as aromatic compound-degrading denitrifiers, marine sulfate-reducers, and methanogenic communities in a terephthalate-degrading reactor. These results suggest that metabolic strategies adapted for degradation of isophthalate and other phthalate are conserved between microorganisms that are involved in the anaerobic degradation of environmentally relevant aromatic compounds.

Fig. 1

In vitro enzyme assays performed with cell-free extracts of isophthalate-grown cells of S. aromaticivorans. a Representative LC-MS/MS chromatograms showing a time course of isophthalyl-CoA formation with coenzyme A, ATP, and isophthalate, monitoring the specific ion trace of m/z 409 of the quasimolecular ion m/z 916. b Kinetics of coenzyme A (0.25 mM) consumption and isophthalyl-CoA formation (no benzoyl-CoA was detected). c Representative LC-MS/MS chromatograms exhibiting the formation of benzoyl-CoA by decarboxylation of enzymatically formed isophthalyl-CoA, monitoring the specific ion trace of m/z 365 of the quasimolecular ion m/z 872. d Kinetics with excess amount of coenzyme A (0.5 mM) consumption, isophthalyl-CoA and benzoyl-CoA formation

Fig. 2

Comparative analysis of induced-proteins of S. aromaticivorans cells grown with isophthalate (black bars) or benzoate (gray bars)

Fig. 3

The identified gene cluster induced specifically during anaerobic degradation of isophthalate to benzoyl-CoA by S. aromaticivorans. a The predicted functions of induced genes involved in anaerobic isophthalate degradation to benzoyl-CoA included a putative isophthalate:CoA ligase (IPCL), isophthalyl-CoA decarboxylase (IPCD). b Representation of o-phthalate-induced gene cluster of nitrate-reducing bacteria: IcIR family transcriptional regulator (1), UbiX-like flavin prenyltransferase (2), UbiD-like o-phthalyl-CoA decarboxylase (PCD; 3), succinyl-CoA:o-phthalate CoA transferase (SPT; 4 and 5), and TRAP transporters (6). The % identities (BLASTp) with the corresponding products of genes are indicated at the bottom of the respective genes (80–90% coverage). The induced genes are shown with thick frames and black genes have no proposed function (b is adapted from [30, 32]). SU small subunit, LS large subunit

Fig. 4

Phylogenetic tree calculated by the Maximum Likelihood method showing the affiliation of the UbiD family isophthalyl-CoA decarboxylase (IPCD) of S. aromaticivorans with the related UbiD-related (de)carboxylase sequences of diverse microorganisms. Numbers at the nodes are shown only for percentage bootstrap (1000 replicates) values above 50. Accession numbers of sequences are given in parentheses. The scale bar represents 5% sequence divergence

Fig. 5

Multiple sequence alignment of isophthalate-induced UbiX-like putative FMN-binding protein of S. aromaticivorans (0377) and related experimentally characterized UbiX-like enzymes of bacterial and fungal strains: Escherichia coli strain K-12 (gi2507150), Aspergilus niger (ABN13117), S. cerevisiae (P33751), Bacillus subtilis (P94404), E. coli O157:H7 (P69772), P. aeruginosa (Q9HX08), and Azoarcus sp. strain PA01 (not characterized). Highly conserved amino acid residues are highlighted and those putatively involved in binding with flavin mononucleotide (FMN) and dimethylallyl phosphate (DMAP) are indicated with arrows (figure adapted from [62, 68])

Fig. 6

Pathway of anaerobic isophthalate conversion to benzoyl-CoA reconstructed using in vitro enzyme assays by S. aromaticivorans that involve activation of isophthalate to isophthalyl-CoA by an ATP-dependent isophthalate-CoA ligase (IPCL) followed by decarboxylation to benzoyl-CoA which is catalyzed by UbiD family isophthalyl-CoA decarboxylase (IPCD) and associated prFMN forming UbiX-like flavin prenyltransferase. The identified genes encoding the respective proteins are shown above the arrows. The functions of the gene products have not been directly shown, but inferred by the abundance of the proteins and their annotated functions. The downstream pathway involves the enzymes of the anaerobic benzoyl-CoA pathway (not shown)