Draft Whole-Genome Sequence of the Anthracene-Degrading Strain Mycolicibacterium frederiksbergense LB501T, Isolated from a Polycyclic Aromatic Hydrocarbon-Contaminated Soil

Here, we report the draft whole-genome sequence of an anthracene-degrading bacterium, Mycolicibacterium frederiksbergense strain LB501T, using the PacBio and Illumina sequencing platforms. The complete genome sequence of strain LB501T consists of 6,713,618 bp and provides new insights into its metabolic capabilities, including aromatic conversion pathways with promiscuous activities.

ABSTRACT

Here, we report the draft whole-genome sequence of an anthracene-degrading bacterium, Mycolicibacterium frederiksbergense strain LB501T, using the PacBio and Illumina sequencing platforms. The complete genome sequence of strain LB501T consists of 6,713,618 bp and provides new insights into its metabolic capabilities, including aromatic conversion pathways with promiscuous activities.

ANNOUNCEMENT

Mycolicibacterium frederiksbergense strain LB501T was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated soil (Belgium) using PAH-sorbing Teflon membranes (1, 2). Strain LB501T, previously belonging to the genus Mycobacterium, has been reclassified into the genus Mycolicibacterium (3, 4). The strain was grown in a mineral medium (5) containing 0.5 g liter⁻¹ of anthracene as the sole carbon source. DNA was extracted using the DNeasy PowerMax soil kit (Qiagen, Carlsbad, CA) with a modified protocol that included a pretreatment with lysozyme (20 mg ml⁻¹, at 37°C for 30 min) and proteinase K (1.8 mg ml⁻¹, at 56°C for 30 min) to improve cell lysis. The genome was sequenced using the PacBio RS II (Menlo Park, CA) and Illumina (San Diego, CA) sequencing platforms. Default parameters were used except where otherwise noted. For PacBio sequencing, genomic DNA was directly used for library preparation without fragmentation. A 20-kb SMRTbell library was generated and sequenced on one single-molecule real-time (SMRT) cell at the Génome Québec Innovation Centre (McGill University, Montréal, Canada), which generated 1,238,955,776 bp from 124,389 subreads, with an average subread length of 9,960 bp. Raw subreads shorter than 500 bp or having a quality score lower than 0.75 were filtered out, and the remaining reads were de novo assembled using the Hierarchical Genome Assembly Process (HGAP.3)/Quiver protocol from the SMRT Analysis software suite version 2.3.0.140936.p4 (6). The resulting assembly was subsequently scaffolded using SSPACE-Longread version 1.1 (7). To further correct for potential assembly artifacts, paired-end sequencing (2 × 75 bp) was performed on the Illumina NextSeq 500 sequencing platform at the Utrecht Sequencing Facility (Utrecht, The Netherlands). A new DNA extraction was carried out as described above, and a DNA library was prepared using a TruSeq Nano DNA low-throughput library prep kit with a target insert size of 350 bp (Illumina). Illumina raw reads were trimmed using Trimmomatic version 0.32 (palindromic mode, headcrop:16, minlen:32, slidingwindow:4:15, trailing:30), yielding a total number of 5,959,272 (5,959,272 R1 + 5,959,272 R2) Illumina quality-controlled paired-end reads. The Illumina reads were aligned against scaffolds, and a consensus sequence of each scaffold was generated using bcftools version 1.9 (8). The final corrected scaffolds were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.8 (9). The final assembly consists of four scaffolds, with a total size of 6,713,618 bp. One scaffold corresponds to a circular chromosome with a length of 6,086,872 bp and a G+C content of 67.3%. The chromosome was circularized using SSPACE-Longread version 1.1. The other three scaffolds correspond to plasmids, one 341,437-bp plasmid (G+C content, 65.8%), one 268,343-bp plasmid (G+C content, 65.2%), and one 16,966-bp plasmid (G+C content, 66.1%). The chromosome and the 341,437-bp plasmid each contain one within-scaffold gap of 2,142 bp (3,206,107 to 3,208,248) and 491 bp (862 to 1,352), respectively. The whole-genome sequence encompasses 6,440 coding sequences (CDS), 2 rRNA operons (16S, 23S, and 5S rRNAs), 2 noncoding RNAs, and 46 tRNA genes. Two chromosomally encoded catabolic regions of approximately 99 and 33 kb were identified at positions 737,825 to 836,329 and 3,866,266 to 3,899,381, respectively, which contain several gene clusters required for complete PAH biodegradation, such as phenanthrene, anthracene, and pyrene. In particular, the 99-kb catabolic region presents a gene structure and organization similar to those of the 150-kb catabolic region A from Mycobacterium vanbaalenii PYR-1 (10, 11). These regions contain several ring-hydroxylating dioxygenases (RHDs), including nidAB, nidA3B3, and pdoA2B2, responsible for initial dioxygenation in the ring cleavage process of low- and high-molecular-weight PAHs (12, 13). The pht cluster (phtRAaAbBAcAd) (14) and the β-ketoadipate pathway cluster (pcaJICDBGHR) (15, 16) were identified in the major catabolic region, which demonstrates that anthracene degradation by strain LB501T proceeds through the o-phthalate degradation pathway (phthalate 3,4-dioxygenase) and ortho-cleavage of protocatechuate (protocatechuic acid [PCA] 3,4-dioxygenase), as previously suggested (17). Moreover, the genome sequence harbors multiple genes involved in heavy metal resistance, such as copper (copA, copC, and copD), arsenic (arsRBCDA), and mercury (merA and merB). The presence of these metal resistance genes suggests that Mycolicibacterium frederiksbergense strain LB501T could be a promising microorganism for bioaugmentation of soils contaminated with both PAHs and heavy metals.

The genome sequence of strain LB501T will enable transcriptomic and proteomic analyses under different environmental conditions and the development of specific biomarkers for bioaugmentation monitoring. The metabolic capabilities of strain LB501T are also relevant to biorefining processes of lignin fractions of lignocellulosic biomass (i.e., the bioaromatic platform) (18, 19).

Data availability.

This whole-genome shotgun project has been deposited at GenBank under the BioProject number PRJNA521839, the BioSample number SAMN10915336, and the accession numbers CP038799, CP038797, CP038798, and CP038796. The versions described in this paper are the first versions, CP038799.1, CP038797.1, CP038798.1, and CP038796.1. The PacBio and Illumina raw reads have been deposited in the Sequence Read Archive (SRA) under accession numbers SRR10121029 and SRR11515926, respectively.