A Complete Genome of the Alphaproteobacterial Methanotroph Methylocystis parvus OBBP.

A complete genome is presented for Methylocystis parvus OBBP, a Gram-negative aerobic methanotroph of the phylum Alphaproteobacteria. M. parvus OBBP is the genus type strain and of interest in the production of polyhydroxybutyrate and environmental microbiology. The genome consists of two plasmids (248 kbp and 205 kbp) and a chromosome (4.076 Mbp).

In Vivo Genome Editing in Type I and II Methanotrophs Using a CRISPR/Cas9 System.

Methanotrophic bacteria are Gram-negative, aerobic organisms that use methane as their sole source of carbon and energy. In this study, we constructed and exemplified a CRISPR/Cas9 genome editing system and used it to successfully make gene deletions and insertions in the type I methanotroph Methylococcus capsulatus Bath and the type II methanotroph Methylocystis parvus OBBP. High frequencies of gene deletions and insertions were achieved in combination with homology-directed repair. In M. parvus OBBP, we also investigated the impact of several parameters on the CRISPR/Cas9 genome editing, where the ligD gene was targeted with various PAM sequences and guide RNA spacer sequences, homology arms of variable length, differences in the duration of mating during conjugation, and exploiting promoters of different strengths to control the expression of cas9 and sgRNA. Although not the first attempt to develop a CRISPR/Cas system in methanotrophs, this work demonstrated for the first time an efficient CRISPR/Cas9 system generating scarless clean gene deletions and insertions in methanotroph genomes.

Isolation and characterisation of Methylocystis spp. for poly-3-hydroxybutyrate production using waste methane feedstocks.

Waste plastic and methane emissions are two anthropogenic by-products exacerbating environmental pollution. Methane-oxidizing bacteria (methanotrophs) hold the key to solving these problems simultaneously by utilising otherwise wasted methane gas as carbon source and accumulating the carbon as poly-3-hydroxybutyrate, a biodegradable plastic polymer. Here we present the isolation and characterisation of two novel Methylocystis strains with the ability to produce up to 55.7 ± 1.9% poly-3-hydroxybutyrate of cell dry weight when grown on methane from different waste sources such as landfill and anaerobic digester gas. Methylocystis rosea BRCS1 isolated from a recreational lake and Methylocystis parvus BRCS2 isolated from a bog were whole genome sequenced using PacBio and Illumina genome sequencing technologies. In addition to potassium nitrate, these strains were also shown to grow on ammonium chloride, glutamine and ornithine as nitrogen source. Growth of Methylocystis parvus BRCS2 on Nitrate Mineral Salt (NMS) media with 0.1% methanol vapor as carbon source was demonstrated. The genetic tractability by conjugation was also determined with conjugation efficiencies up to 2.8 × 10-2 and 1.8 × 10-2 for Methylocystis rosea BRCS1 and Methylocystis parvus BRCS2 respectively using a plasmid with ColE1 origin of replication. Finally, we show that Methylocystis species can produce considerable amounts of poly-3-hydroxybutyrate on waste methane sources without impaired growth, a proof of concept which opens doors to their use in integrated bio-facilities like landfills and anaerobic digesters.