Genome sequence of Methylocystis hirsuta CSC1, a polyhydroxyalkanoate producing methanotroph

Abstract Polyhydroxyalkanoates (PHAs) are biodegradable plastics that can be produced by some methanotrophic organisms such as those of the genus Methylocystis. This allows the conversion of a detrimental greenhouse gas into an environmentally friendly high added‐value bioproduct. This study presents the genome sequence of Methylocystis hirsuta CSC1 (a high yield PHB producer). The genome comprises 4,213,043 bp in 4 contigs, with the largest contig being 3,776,027 bp long. Two of the other contigs are likely to correspond to large size plasmids. A total of 4,664 coding sequences were annotated, revealing a PHA production cluster, two distinct particulate methane monooxygenases with active catalytic sites, as well as a nitrogen fixation operon and a partial denitrification pathway.


| INTRODUC TI ON
Anthropogenic emissions of methane currently account for up to 30% of the global emissions of greenhouse gases (considering that methane has a 25 times greater global warming potential than CO 2 ; Desai & Harvey, 2017). Despite the fact that methane can be used as an industrial energy source via combustion at concentrations higher than 20%, more than 56% of its emissions have concentrations lower than 5% (Lebrero et al., 2016). Biological methane abatement is a very attractive alternative to treat diluted methane emissions based on its high effectiveness and environmentally friendliness. In addition, biological methane abatement can be coupled to the production of added-value compounds. Polyhydroxyalkanoates (PHAs) are intracellular biopolyesters produced under nutrient-limiting conditions by a wide range of methane-consuming organisms (Pieja, Morse, & Cal, 2017).
Methanotrophs are organisms able to use methane as the sole energy and carbon source, some of them use methane exclusively and others are facultative methanotrophs, able to grow also in other carbon sources. We focus here on bacterial methanotrophic species using oxygen as electron acceptor. Even if anaerobic methane oxidation can also occur coupled to sulfate, nitrate and nitrite reduction, this phenomenon plays a minor ecological role compared to aerobic oxidation. Aerobic methanotrophic bacteria are usually classified into type I and type II methanotrophs, which are different in their membrane arrangement, fatty acid composition, and methane assimilation pathways (Hanson & Hanson, 1996).
Type II methanotrophs such as Methylocystis, Methylosinus, and Methylocella are considered the main methanotrophic PHA-synthesizing genera. For instance, Methylocystis hirsuta has been shown to accumulate PHB up to 45% of its total biomass (García-Pérez et al., 2018), which makes it a very interesting cell factory. This value is higher than those previously reported for other methanotrophs (Pieja, Rostowski, & Criddle, 2011), including its close relative Methylocystis sp. SC2.

| RE SULTS
The assembly's results are summarized in Table 1. A phylogenetic analysis was carried out using JSpeciesWS (Richter, Rosselló-Móra, Glöckner, & Peplies, 2015), which calculates the average nucleotide identity (ANI) comparing all shared orthologous protein-coding genes of two genomes (Richter & Rosselló-Móra, 2009). The phylogenetic tree ( Figure 1) containing the 10 closest species identified by JSpeciesWS was built using the function dendrogram from SciPy after defining the distance between species as 100 minus their ANI value.
The genome was annotated using the NCBI Prokaryotic Genome annotation pipeline (Tatusova et al., 2016). A general summary of the biological functions coded in the genome was obtained using RAST (Overbeek et al., 2014). Approximately, 25% of the annotated genes corresponded to RAST subsystems (Table 2).
Despite the presence of plasmids has not been assessed experimentally, a cluster of three plasmid replication genes (RepA, RepB and RepC) was detected in the fourth contig (which is 158,363 bases long). This suggests that this contig could correspond to a large plasmid. The locus tags of the genes forming this cluster of plasmid replication genes are as follows: D1030_20715, D1030_20710, and D1030_20705. The fourth contig also contains several repeat regions that could be binding sites for the plasmid-encoded repeat proteins (Sekine et al., 2006). The third contig (260,028 bp) contains also two plasmid replication genes (RepA and RepC) separated by a protein that could not be annotated and has repeated regions in their proximity, which suggests the possibility of this contig being also a large size plasmid. The chromosome also contains two genes annotated as RepA proteins and two other putative RepC proteins.
Overall, it appears very likely that M. hirsuta is able to sustain plasmid replication. The closest strain Methylocystis sp. SC2 does contain two large plasmids.
In order to identify gene clusters involved in the synthesis of secondary metabolites, the platform antiSMASH 4.0 was used (Blin et al., 2017). The two clusters showing higher similarity (to known clusters) were a PHA biosynthetic gene cluster and an enterobactin biosynthetic cluster (Table 3)  cluster, pmoCAB2, which codes an enzyme with higher affinity for methane at low concentrations. This cluster was absent in M. hirsuta CSC1, which contained instead a second cluster (Figure 2b) with the pMMO subunits arranged in the order ABC and that is designated as pmoABC3 cluster. In order to identify the evolutionary relations among these three gene clusters, a multiple alignment of each of their proteins was performed using the software MUSCLE (Edgard, 2004). Figure 2c shows that the proteins in the pmoABC3 cluster are more evolutionary distant than those in the two other clusters (pmoCAB1 and 2  (Hanson & Hanson, 1996).
A complete nif operon (involved in nitrogen fixation) was found (with genomic coordinates 818892-827543

CO N FLI C T O F I NTE R E S T
The authors declare not to have any conflict of interest.

AUTH O R S CO NTR I B UTI O N
SB performed the bioinformatics analysis and wrote the manuscript. ER carried out the microbial cultures and sample preparation.
RM conceived and supervised the work. All the authors edited the manuscript.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
This whole genome shotgun project has been deposited at DDBJ/ ENA/GenBank under the accession number QWDD00000000 F I G U R E 2 PHB synthesis and methane oxidation cluster. (a) PHB synthesis cluster. (b) Two clusters coding pMMO enzymes. The pmoCAB1 cluster is duplicated and is identical to the cluster found in Methylocystis sp. SC2. The pmoABC3 cluster differs in the order in which the subunits are arranged. (c) Evolutionary distance between proteins in the pmoCAB1, pmoABC3, and pmoCAB2 clusters. pmoCAB2 is present in Methylocystis sp. SC2 but is absent in Methylocystis hirsuta CSC1