Genome analysis provides insights into microaerobic toluene-degradation pathway of Zoogloea oleivorans BucT
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Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.
KeywordsZoogloea Toluene degradation Metatranscriptomics Biodegradation
At present, the genus Zoogloea (family Zoogloeaceae) contains five validly described species, which can be characterized as floc-forming, nitrogen-fixing bacteria. Members of the genus have been isolated from various habitats including activated sludge, soil or hydrocarbon contaminated groundwater (Xie and Yokota 2006; Shao et al. 2009; Farkas et al. 2015). Despite the fact that Zoogloea spp. play a crucial role in wastewater treatment by causing the flocculation of the activated sludge, limited genome sequence information is available regarding these bacteria. The first publicly available genome sequence was reported by Muller et al. (2017). Recent studies characterizing benzene- and toluene-degrading microbial communities have shown that Zoogloea genus-related bacteria could have an important role in the degradation of these contaminants in subsurface environments. Protein- and RNA-stable isotope probing (SIP) based analysis of an aerobic benzene-degrading microbial community revealed Zoogloea-related bacteria as predominant benzene-degraders (Jechalke et al. 2013). Our previous DNA- and transcriptome-SIP studies have shown that Zoogloea oleivorans is a highly efficient toluene degrader under microaerobic conditions (Bradford et al. 2018; Táncsics et al. 2018). We hypothesized that Z. oleivorans was capable of degrading toluene under microaerobic conditions due to the fact that it harbours a catechol 2,3-dioxygenase (C23O) gene which encodes a subfamily I.2.C-type extradiol dioxygenase enzyme (Farkas et al. 2015). Kukor and Olsen (1996) suggested that this group of extradiol dioxygenases was adapted to environments with low-oxygen concentrations, hinting at their role in ring-cleavage reactions under hypoxic conditions. On the other hand, it is known that ring-cleaving dioxygenases belonging to the same subfamily may show different oxygen affinities, as was observed in the case of chlorocatechol 1,2-dioxygenases (Balcke et al. 2008) and comparative analysis of aerobic and microaerobic BTEX-degrading enrichment cultures (Benedek et al. 2018). In the present study, to uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. In addition, metatranscriptomic (non-rRNA) sequence reads originated from our previous SIP study on microaerobic toluene degradation in aquifer samples with abundant Zoogloea spp. (Bradford et al. 2018) were mapped on the genome.
Materials and methods
Genomic DNA from Zoogloea oleivorans BucT was isolated using the DNeasy UltraClean Microbial Kit (Qiagen, Germany) according to the instructions of the manufacturer. The whole-genome sequencing was performed as described previously (Borsodi et al. 2019), briefly: Nextera Mate Pair Sample Preparation Kit (Illumina, USA) was used to generate mate-paired libraries according to the manufacturer’s protocol for gel-plus version with slight modifications. 13 µl of Mate-Paired Tagment Enzyme was used to produce a robust smear within the 7-11 kbp region. The 7-11 kbp DNA fraction was excised from the gel using the Zymoclean Large Fragment DNA Recovery kit (Zymo Research, USA) and the circularized DNA was sheared using Covaris S2. All quality measurements were performed on a TapeStation 2200 instrument (Agilent, USA). Final libraries were quantified using Qubit (ThermoFisher, USA) and sequenced on an Illumina MiSeq instrument using MiSeq Reagent Kit v2 (500 cycles) sequencing chemistry. De novo assembly and scaffolding were performed with CLC Genomics Workbench Tool v11 (Qiagen, Germany). The mate-paired reads were assembled into 107 contigs. Automatic annotation of the genome was performed by the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAP) v4.5 (Tatusova et al. 2016). The genome sequence of strain BucT has been deposited at the GenBank database under the WGS accession number SDKK00000000 (Bioproject: PRJNA516779; Biosample: SAMN10797634). Mapping of metatranscriptomic sequence reads (NCBI Gene Expression Omnibus accession number GSM3380032; sample name: 13CHunamp) on the de novo assembled genome of strain BucT was performed by CLC Genomics Workbench Tool v11 (Qiagen, Germany) using the following parameters: length fraction = 0.8; similarity fraction = 0.8. Phylogenetic tree was reconstructed using the maximum-likelihood algorithm using MEGA version 6.0. Tree topology and distances were evaluated by bootstrap analysis based on 1000 replicates. Graphical visualization of gene clusters was performed by using SnapGene v4.3.4.
Results and discussion
We have previously investigated a microaerobic, 13C-labelled toluene-degrading enrichment culture, in which Zoogloea oleivorans was an abundant toluene degrader, by RNA-stable isotope probing (Bradford et al. 2018). Metatranscriptomic data (non-rRNA sequence reads of the heavy RNA fraction) derived from this enrichment study were used to reveal which of the above-mentioned gene clusters of Z. oleivorans were involved in degradation. The partial meta-cleavage pathway encoding gene cluster was found to be expressed in the enrichment, especially the subfamily I.2.C-type C23O and the 2-hydroxymuconic semialdehyde dehydrogenase genes (86 and 88 gene reads in the metatranscriptome, respectively, and high-RPKM values). On the other hand, genes encoding the multicomponent phenol-hydroxylase system were mostly inactive or showed low detectability (0-5 gene reads in the metatranscriptome and low RPKM values). Similary, subfamily I.2.A-type C23O genes, which are part of the phenol-degradation gene cluster, also showed low activity (5 and 9 gene reads, respectively, and low-RPKM values). Thus, we excluded the formation of 3-methylcatechol through 2-hydroxytoluene (o-cresol) and the involvement of subfamily I.2.A-type extradiol dioxygenases as possible mechanisms in the ring-cleavage reaction. However, genes of the biphenyl-degradation gene cluster, especially genes encoding the toluene-dioxygenase enzyme appeared highly expressed (115 reads altogether in the metatranscriptome). Accordingly, the formation of 3-methylcatechol through toluene-cis-dihydrodiol can be postulated. The phenomenon that a toluene-dioxygenase enzyme played a role in the hydroxylation of the aromatic ring under microaerobic conditions can be explained via previous observations. It has been shown for Pseudomonas putida F1, that the concentration of dissolved oxygen did not significantly affect the expression and longevity of toluene dioxygenase, and the strain could also grow on toluene under microaerobic conditions (Costura and Alvarez 2000). The above-mentioned Thauera sp. strain DNT-1 was also able to degrade toluene aerobically when only trace amount of oxygen was present in the environment (Shinoda et al. 2004). On the other hand, ring monooxygenation is usually the predominant activation mechanism of toluene degradation under microaerobic conditions, instead of dioxygenation. Thus it has been observed for toluene-degrading chemostat cultures, that Burkholderia (formerly Pseudomonas) cepacia strain G4, which uses a monooxygenation mechanism for toluene activation, outcompeted Pseudomonas putida strain F1 (using dioxygenation) under oxygen limitation (Duetz et al. 1994). A predominance of ring monooxygenation was also observed in hypoxic, toluene-degrading constructed wetlands, linked to members of the Burkholderiaceae and Comamonadaceae (Martínez-Lavanchy et al. 2015).
In summary, results of the present study provide evidence that under microaerobic conditions a toluene dioxygenase-like enzyme of Zoogloea oleivorans was involved in the initial activation (aromatic ring-hydroxylation) of toluene, while the subfamily I.2.C-type extradiol dioxygenase catalysed the ring-cleavage reaction. The gene clusters encoding the tod-like and the subfamily I.2.C-type extradiol dioxygenase enzymes were flanked by mobile-genetic elements, suggesting that these gene clusters were acquired by strain BucT through HGT events. Thus, the capacity of microaerobic toluene degradation seems like a mosaic encoded in the genome of Zoogloea oleivorans BucT.
Open access funding provided by Szent István University (SZIE). This research was supported by the Higher Education Institutional Excellence Program (NKFIH-1159-6/2019) awarded by the Ministry for Innovation and Technology of Hungary within the framework of water related researches of Szent István University. Lauren M. Bradford was funded by the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP7/2007-2013), grant agreement 616644 (POLLOX) to Tillmann Lueders.
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