Complete Genome Sequences of Two Acetylene-Fermenting Pelobacter acetylenicus Strains

GENOME ANNOUNCEMENT

The fermentation of acetylene (C₂H₂) to ethanol and acetate is a rare metabolic pathway (1). Only three well-characterized microbial strains, all within the Pelobacter genus, can grow on C₂H₂ via acetylene hydratase (AH), a W-containing, exothermic, and low-redox-potential enzyme (1, 2). However, little is known about the regulatory controls and evolution of this metabolism.

Here, we present complete genome sequences of two acetylene-fermenting isolates, P. acetylenicus DSM 3246 and DSM 3247. Both strains were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Freshwater strain DSM 3246 was grown in DSMZ medium 298 using 0.01 mM acetoin (CH₃COCHOHCH₃). Estuarine strain DSM 3247 was grown in DSMZ medium 293 using either acetoin or C₂H₂ (1.5%).

The genomes were sequenced at the University of California at Davis Genome Center (Davis, CA USA) using a Pacific Biosciences (PacBio) single-molecule real-time (SMRT) RSII instrument (Pacific Biosciences of California, Inc., Menlo Park, CA). Briefly, cultures were grown to high density and pelleted by centrifugation. Genomic DNA was extracted from multiple pellets with the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions but modifying the protocol by inverting in place of vortexing to reduce DNA shearing. DNA from multiple pellets was concentrated using either the Power Clean Pro kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) or the Zymo Spin genomic DNA (gDNA) concentrator (Zymo Research Corporation, Irvine, CA, USA).

A sequencing-ready library with 10-kb inserts was prepared for each strain and sequenced with C4 chemistry on one SMRT cell. Sequencing of DSM 3246 produced 71,385 sequence reads, with an N₅₀ size of 19,001 bp, and sequencing of DSM 3247 produced 62,650 sequence reads, with an N₅₀ size of 18,239 bp. The genome of P. acetylenicus DSM 3246 was assembled into a 3,192,352-bp circular chromosome with 57.4% G+C content (222× coverage) and a 13,658-bp circular plasmid with 57.4% G+C content (36× average coverage) using the Hierarchical Genome Assembly Process (HGAP), which includes polishing with Quiver (3). The genome of P. acetylenicus DSM 3247 was assembled into a 3,176,363-bp circular chromosome (110× average coverage) with 57.4% G+C content, using the same procedure.

Annotation was performed using the Prokaryotic Genome Annotation Pipeline (PGAP) from the National Center for Biotechnology Information (NCBI) (4). DSM 3246 and DSM 3247 contained 2,976 and 2,914 genes, respectively. Functional annotation identified 2,774 coding genes, 16 rRNAs, 53 tRNAs, five noncoding RNAs, and 132 pseudogenes in DSM 3246. DSM 3247 contained 2,805 coding genes, nine rRNAs, 51 tRNAs, five noncoding RNAs, and 44 pseudogenes. Both DSM 3246 and DSM 3247 contained one type I clustered regularly interspaced short palindromic repeat (CRISPR) array.

Gene sequences for the five enzymes necessary for the fermentation of acetylene to ethanol and acetate (AH, aldehyde dehydrogenase, alcohol dehydrogenase, phosphate acetyltransferase, and acetate kinase) were annotated in both strains. DSM 3246 and DSM 3247 each contained one copy of the ahy gene encoding AH. The DSM 3246 ahy is 100% identical on the amino acid level to that of DSM 3247. Comparative analyses of these and other Pelobacter species may provide insight into the regulation and evolution of acetylene fermentation.