Development of modular expression across phylogenetically distinct diazotrophs

Area of Science:

• Microbiology
• Synthetic Biology
• Agricultural Science

Background:

• Diazotrophic bacteria convert atmospheric nitrogen to ammonia, crucial for plant growth.
• These bacteria associate with plant roots, enhancing nitrogen availability and acting as plant growth promoters.
• Current genetic tools for diazotrophs are limited, hindering agricultural applications and crop microbiome engineering.

Purpose of the Study:

• To develop and optimize genetic tools for manipulating a diverse range of diazotrophic bacteria.
• To enable synthetic biology and genetic engineering in diazotrophs for agricultural applications.
• To expand the range of crops and root microbiomes that can be targeted for nitrogen fixation enhancement.

Main Methods:

• Utilized broad-host-range plasmids for compatibility across multiple diazotroph species.
• Tested seven promoters and eleven ribosomal binding sites for modular gene expression using fluorescent proteins.
• Evaluated four small molecule inducible systems and demonstrated genome editing in Klebsiella michiganensis M5al.

Main Results:

• Identified a specific broad-host-range plasmid (RK2 origin, kanamycin resistance) with high compatibility across tested bacteria.
• Demonstrated successful modular gene expression and validated inducible systems in three diazotroph species.
• Achieved genome editing in Klebsiella michiganensis M5al, showcasing the utility of the developed tools.

Conclusions:

• Optimized protocols and plasmids significantly advance genetic manipulation capabilities in diverse diazotrophic bacteria.
• The developed synthetic genetic parts and expression tools pave the way for engineering diazotrophs in agriculture.
• This work broadens the scope for utilizing diazotrophic bacteria in sustainable agriculture and crop improvement.