Evidence for a putative isoprene reductase in Acetobacterium wieringae

Abstract

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome assembled genome (MAG) of the most abundant (89% rel. abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and RT-PCR identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 x HypA-like, HypB-like) and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, FAD and two pairs of [4Fe-4S]-clusters. Other known Acetobacterium strains do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase ([~]47-49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons. ImportanceIsoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction. By comparing a genome from our isoprene reducing enrichment culture, dominated by Acetobacterium wieringae, with genomes of other Acetobacterium lineages that do not reduce isoprene, we shortlisted candidate genes for isoprene reduction. Using comparative proteogenomics and reverse transcription PCR we have identified a putative five-gene operon encoding an oxidoreductase referred to as putative isoprene reductase.