Recent Advances in the Genetic Manipulation of Methylosinus trichosporium OB3b

Abstract

Methanotrophic bacteria utilize methane as their sole carbon and energy source. Studies of the model Type II methanotroph Methylosinus trichosporium OB3b have provided insight into multiple aspects of methanotrophy, including methane assimilation, copper accumulation, and metal-dependent gene expression. Development of genetic tools for chromosomal editing was crucial for advancing these studies. Recent interest in methanotroph metabolic engineering has led to new protocols for genetic manipulation of methanotrophs that are effective and simple to use. We have incorporated these newer molecular tools into existing protocols for Ms. trichosporium OB3b. The modifications include additional shuttle and replicative plasmids as well as improved gene delivery and genotyping. The methods described here render gene editing in Ms. trichosporium OB3b efficient and accessible.

Keywords: Conjugation; Electroporation; Gene editing; Genetic manipulation; Methanotrophs; Methylosinus trichosporium OB3b.

Fig. 1

Plasmid maps of pk18mobsacB_p15a and pAWP89_p15a.

Fig. 2

Ms. trichosporium OB3b transformed with pAWP89_p15a (A) compared to wild-type Ms. trichosporium OB3b (B) on 25 µg/mL kanamycin NMS agar selection plates.

Fig. 3

Gene deletion via conjugation in Ms. trichosporium OB3b. Following conjugation, the introduced plasmid recombines into the targeted site in the chromosome during the first crossover. Transconjugant selection followed by sucrose counterselection induces a second crossover event to remove the plasmid backbone from the chromosome and produce the mutant with the gentamicin resistance cassette (gen^R) disrupting gene A.

Fig. 4

Construction of a gene-disrupting linear DNA fragment using fusion PCR. (1) PCR amplification of gen^R and the 5′ and 3′ DNA regions flanking gene targeted for disruption. (2) The 5′ DNA fragment and gen^R are used as templates for the first round of fusion PCR. (3) The resulting PCR product and the 3′ DNA fragment are used as templates for the second round of PCR to produce the final gene-disrupting linear DNA construct. The linear DNA construct is (4) electroporated into wild-type Ms. trichosporium OB3b followed by (5) selection for the Ms. trichosporium OB3b Δgene A mutant. Primer binding sites are represented by half arrows.

Fig. 5

Amount of Ms. trichosporium OB3b cells required for DNA extraction shown on a 1-µL inoculating loop.