ABSTRACT
The syntrophic obligate proton-reducing bacterium Syntrophus buswellii DSM 102354T, isolated from anaerobic digester sludge, degrades benzoate and possibly hydrocinnamate (phenyl-3-propionate) when grown with a suitable hydrogenotrophic partner. The complete genome sequence data provide insight regarding aromatic compound degradation under thermodynamically limiting conditions when exogenous electron acceptors are absent.
KEYWORDS: Syntrophus, genome analysis, syntrophs, aromatic compounds
ANNOUNCEMENT
The genus Syntrophus belongs to the family Syntrophaceae and members live in many natural and man-made anaerobic habitats where they participate in anaerobic biomass recycling (1–3). Syntrophus buswellii DSM 102354T (=DSM 2612MT) was isolated from a tri-culture enrichment obtained from the DSMZ German Collection (DSM 2612BT) by serial dilution using the Hungate technique and a bicarbonate-buffered mineral-salts medium containing crotonate as the sole carbon supply (4). Taxonomic identification was carried out by PCR-based 16S rRNA gene sequencing (EMBL data library accession number X85131) and subsequent comparative analysis with 1,600 bacterial 16S rRNA reference sequences (4). The above-described S. buswellii DSM 2612BT tri-culture enrichment was previously derived from an anaerobically collected sewage sample from the primary anaerobic digester at the Urbana, Illinois, waste digestion facility (40.109 N, 88.204 W) prior to 1982 (5, 6). When S. buswellii DSM 102354T is cultured syntrophically with a suitable hydrogenotrophic partner such as Methanospirillum hungatei JF-1T, it metabolizes benzoate and presumably other aromatic substrates (2, 7).
S. buswellii DSM 102354T was obtained from the McInerney laboratory culture collection and cultivated anaerobically in a mineral-salts medium using the Hungate method with 20 mM crotonate as the sole carbon supply (3, 7). Cells were grown statically at 37°C to late log phase, harvested anaerobically, and stored at −70°C until processing. DNA (15.3 ng/µl) was extracted from cell pellets using the Zymo D4081 Quick-DNA Magbead Plus Kit as per the manufacturer’s instructions. DNA was then fragmented using Covaris g-Tubes in an Eppendorf 5424 centrifuge for 60 s at 3,000 g for a total of 3 spins according to the manufacturer’s protocol. The average size of the sheared DNA was then calculated using the genomic DNA Assay (TapeStation, Agilent Technologies) with an average of 12.9 Kb (median = 12.4 Kb, min = 10.4 Kb, and max = 19.8 Kb) for the construction of genomic libraries. Sheared DNA was then concentrated using AMPure PB beads according to the manufacturer’s recommendations (PacBio—multiplexed microbial libraries using SMRTbellExpress Template Prep Kit 2.0, #101-696-100 version 07, July 2020). The purified sheared DNA had an average size of 11.8 Kb (median = 11.7 Kb, min = 9.5 Kb, and max = 14.7 Kb) and a concentration of 74.4 ng/µl. Library preparation was performed according to the PacBio protocol. DNA sequencing was performed using the PacBio Sequel IIe platform, which generated 62,884 raw reads. Demultiplexing and adapter trimming were done using Lima v2.9.0 (https://github.com/pacificbiosciences/barcoding). All reads were then targeted for assembly by Canu v2.2 (8). Assembled genomes were further refined by Circlator v1.5.5 (9) to identify circular contigs, remove redundant non-circular contigs, and rotate circular contigs to start with dnaA. A completeness check was performed by CheckM v1.0.18 (10), and the N50 quality was determined by Assembly stats v1.01 (https://github.com/sanger-pathogens/assembly-stats). Genome ORF calling and annotation were performed by NCBI PGAP v6.9 (11).
The properties of the circular S. buswellii DSM 102354T genome are shown in Table 1.
TABLE 1.
S. buswellii DSM 102354T genome information
| Feature | Genome value |
|---|---|
| Topology | Circular |
| Size (bp) | 3,252,431 |
| GC% | 55.23% |
| Protein coding genes | 2,834 |
| 16S number | 1 |
| tRNA number | 48 |
| Coverage | 35.58× |
| Raw reads | 62,884 |
| Average read length | 8,186 bp |
| High-quality reads | 9,290 |
| N50 quality value (bp) | 3,252,431 |
| Completeness value | 97.1% |
| 16S similaritya | 99.62% |
| Sample collection site | 40.109 N, 88.204 W |
| NCBI taxonomy ID | 43774 |
GENOME HIGHLIGHTS
The genome contains two paralogs each of benzoyl-CoA ligase and benzoyl-CoA reductase for aromatic ring activation and reduction, suggesting the ability to degrade additional aromatic compounds besides benzoate (3, 13). It lacks pta and ack genes for substrate-level ATP formation from acetyl-CoA and instead contains AMP-forming acetyl-CoA synthetase genes for ATP synthesis as demonstrated in Syntrophus aciditrophicus (14).
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation grant no. NSF 1911781 and the Department of Energy BER award DE-FC02-02ER63421 to the UCLA DOE Institute of Genomics and Proteomics. We thank the UCLA Institute for Quantitative and Computational Biosciences (QCB) for resources.
Contributor Information
Robert P. Gunsalus, Email: robg@microbio.ucla.edu.
Elinne Becket, California State University San Marcos, San Marcos, California, USA.
DATA AVAILABILITY
The complete genome sequence of Syntrophus buswellii DSM 102354T has been deposited in the GenBank assembly accession GCA_046097065.1, and the raw sequencing reads have been deposited under the accession no. SRR31591383.
The IMG genome OID number is 8025829216 and the IMG submission ID is 303293.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The complete genome sequence of Syntrophus buswellii DSM 102354T has been deposited in the GenBank assembly accession GCA_046097065.1, and the raw sequencing reads have been deposited under the accession no. SRR31591383.
The IMG genome OID number is 8025829216 and the IMG submission ID is 303293.