Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC

Abstract

The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

“…All the AD gene clusters found so far encode GS-like and GAT-like proteins, which have not been found in other dioxygenases, with one exception (34). In this study, we proposed an aniline oxidation mechanism for Acinetobacter sp.…”

“…Hydrogenophaga sp. PBC has the sadABD gene cluster for 4-aminobenzenesulfonate oxidation (34), which encodes a GSlike protein but not a GAT-like protein. However, intriguingly, this strain contains a DNA region encoding a GAT-like protein and a part of an AD large-subunit homolog (whole-genome shotgun sequence contig 46; accession no.…”

“…In particular, the GS-like and GAT-like proteins are quite unique; these proteins have so far not been identified in other aromatic compound dioxygenases, with the only exception being a GS-like protein gene (sadB) in the 4-aminobenzenesulfonate 3,4-dioxygenase gene cluster (sadABD) of Hydrogenophaga sp. strain PBC (34). However, the function of the gene product is still unknown.…”

Nanopore Long-Read Guided Complete Genome Assembly of Hydrogenophaga intermedia, and Genomic Insights into 4-Aminobenzenesulfonate, p-Aminobenzoic Acid and Hydrogen Metabolism in the Genus Hydrogenophaga

“…Transposon mutagenesis of strain PBC led to the identification of the sad operon that is responsible for the initial deamination step of 4-aminobenzenesulfonate to 4-sulfocatechol. The deamination step was presumed to operate similarly H. intermedia strain S1 given its near identical 16S rRNA sequence to strain PBC (Gan et al, 2011a,b, 2012c). In addition, both Hydrogenophaga strains could only be grown in axenic culture with 4-aminobenzenesulfonate as the sole carbon and nitrogen source if they were supplemented with p -aminobenzoate and biotin (Kampfer et al, 2005; Gan et al, 2011a,b), providing insight into the syntrophic relationship between the Hydrogenophaga strains and their helper strains in addition to suggesting that the biosynthesis pathways for these compounds were either missing or non-functional in H. sp.…”

Genome Sequence of Hydrogenophaga sp. Strain PBC, a 4-Aminobenzenesulfonate-Degrading Bacterium

“…The recent identification of the genetic components for the oxidation of 4-ABS to 4-sulfocatechol from Hydrogenophaga sp. PBC added another crucial piece of the puzzle to the metabolism of 4-ABS (6).…”