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pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium
- Source :
- Frontiers in Microbiology, Frontiers in Microbiology, Vol 11 (2020)
- Publication Year :
- 2020
- Publisher :
- Frontiers Media SA, 2020.
-
Abstract
- Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheS AG , which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheS AG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The P tac promoter and the ribosome-binding site region of mmoX were employed to control pheS AG , producing the pheS AG -3 CSM. M. buryatense 5GB1C harboring pheS AG -3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheS AG -3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.
- Subjects :
- Microbiology (medical)
Operon
Zeocin
lcsh:QR1-502
pheS
Microbiology
Genome
lcsh:Microbiology
03 medical and health sciences
chemistry.chemical_compound
electroporation-based
Methods
Methylotuvimicrobium
Gene
030304 developmental biology
0303 health sciences
biology
030306 microbiology
Fast Growth Rate
counterselection
biology.organism_classification
Molecular biology
markerless mutagenesis
chemistry
Tac-Promoter
Bacteria
Subjects
Details
- ISSN :
- 1664302X
- Volume :
- 11
- Database :
- OpenAIRE
- Journal :
- Frontiers in Microbiology
- Accession number :
- edsair.doi.dedup.....dc739770a04ee028ce9fcfa1045bbd96