MuA Transposase Host Range: Bacterial Species Suitable for Mutagenesis and Library Construction

Beyond the Published Mu Literature

The practical applicability of MuA transposition is likely much broader than the currently published literature illustrates.

Many researchers are familiar with the Tn5 transposome systems, which use a conceptually similar workflow: a purified transposase is assembled onto DNA in vitro to form a stable nucleoprotein complex that is subsequently introduced into cells. Commercial Tn5 technologies have been successfully applied to a wide range of Gram-negative and Gram-positive bacteria.

Because MuA transposase can likewise be delivered as a preassembled transposition complex, bacterial species that have proven amenable to Tn5 mutagenesis are excellent candidates for MuA-mediated mutagenesis as well.

While each organism should be evaluated experimentally, published results suggest that transposome-based mutagenesis can be applied to a remarkably broad spectrum of microorganisms spanning industrial, environmental, agricultural, and medically important species.

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Applications of MuA Across Diverse Bacterial Species

MuA transposase is suitable for a wide variety of applications, including:

• Random insertion mutagenesis
• Genome-wide mutant library construction
• Functional genomics studies
• Gene discovery and pathway analysis
• Identification of essential genes
• Metabolic engineering
• Strain improvement
• Screening for antibiotic resistance mechanisms
• Mapping genotype-phenotype relationships
• Stable chromosomal integration of heterologous DNA
• Development of high-diversity insertion libraries

Because MuA insertions exhibit minimal sequence bias and can generate highly diverse insertion populations, the system is particularly valuable for large-scale screening applications.

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Is MuA Suitable for Your Organism?

MuA transposase is often a strong candidate when any of the following apply:

• The organism can be transformed or electroporated.
• Tn5-based mutagenesis has previously been reported.
• The species belongs to a bacterial genus related to published Mu hosts.
• Large-scale mutant libraries are required.
• Stable genomic integration is desired.
• Existing genetic engineering tools are limited or unavailable.

Even if your organism has not yet been reported in the Mu literature, successful Tn5 mutagenesis is often a strong indicator that MuA-based approaches may also be feasible. Compared to wild type MuA, the hyperactive version of MuA (v.3 MuA, in vivo integrator) is ten times more efficient in its genomic integration capacity. Thus, it is always adviced to use v.3 MuA when new bacterial strains are being engineered or when highly diverse genomic insertion mutant libraries are being generated.

Bacterial/Archaeal Species Reported for MuA or Tn5-Based Mutagenesis

The table below summarizes microbial species reported in the published Mu literature and/or commonly used with Tn5-based transposome systems. Together, these examples illustrate the broad applicability of transposome-based mutagenesis across various microorganisms.

 

Species	Type	MuA / Mu Reported	Tn5 Reported	Reference(s)
Haloferax volcanii	Archaeon	✓	–	[1]
Escherichia coli	Gram-negative	✓	✓	[2],[3],[9]
Salmonella enterica	Gram-negative	✓	✓	[2],[10]
Yersinia enterocolitica	Gram-negative	✓	✓	[2],[10]
Erwinia carotovora	Gram-negative	✓	✓	[4],[9]
Klebsiella aerogenes	Gram-negative	✓	✓	[9],[10]
Klebsiella pneumoniae	Gram-negative	✓	✓	[9],[10]
Klebsiella oxytoca	Gram-negative	✓	✓	[9],[10]
Citrobacter freundii	Gram-negative	–	✓	[10]
Serratia marcescens	Gram-negative	✓	✓	[9],[10]
Proteus spp.	Gram-negative	✓	✓	[9],[10]
Pantoea agglomerans	Gram-negative	–	✓	[10]
Pseudomonas aeruginosa	Gram-negative	✓	✓	[9],[10]
Pseudomonas fluorescens	Gram-negative	✓	✓	[9],[10]
Pseudomonas putida	Gram-negative	✓	✓	[9],[10]
Acinetobacter calcoaceticus	Gram-negative	✓	✓	[9],[10]
Acinetobacter baumannii	Gram-negative	–	✓	[10]
Burkholderia spp.	Gram-negative	–	✓	[10]
Shewanella oneidensis	Gram-negative	–	✓	[10]
Vibrio cholerae	Gram-negative	–	✓	[10]
Aeromonas hydrophila	Gram-negative	–	✓	[10]
Agrobacterium tumefaciens	Gram-negative	✓	✓	[9],[10]
Rhizobium trifolii	Gram-negative	✓	✓	[9]
Rhizobium japonicum	Gram-negative	✓	✓	[9]
Sinorhizobium meliloti	Gram-negative	–	✓	[10]
Methylophilus methylotrophus	Gram-negative	✓	–	[8]
Bacillus subtilis	Gram-positive	✓	✓	[9],[10]
Bacillus cereus	Gram-positive	✓	✓	[9]
Bacillus sphaericus	Gram-positive	✓	✓	[9]
Clostridium perfringens	Gram-positive	✓	–	[7]
Listeria monocytogenes	Gram-positive	✓	✓	[5],[10]
Staphylococcus aureus	Gram-positive	✓	✓	[6],[10]
Staphylococcus epidermidis	Gram-positive	–	✓	[10]
Enterococcus faecalis	Gram-positive	–	✓	[10]
Streptococcus pneumoniae	Gram-positive	–	✓	[10]
Streptococcus mutans	Gram-positive	–	✓	[10]
Lactococcus lactis	Gram-positive	✓	–	[11]
Corynebacterium glutamicum	Gram-positive	✓	✓	[8],[9]
Streptomyces coelicolor	Gram-positive	✓	✓	[9]
Arthrobacter simplex	Gram-positive	✓	–	[9]
Brevibacterium ammoniagenes	Gram-positive	✓	–	[9]
Micrococcus spp.	Gram-positive	✓	–	[9]

Are You Not Seeing Your Favourite Organism on the List?

The species listed above likely represent only a subset of microorganisms that have been successfully modified using Mu- or Tn5-based transposition systems.

Because MuA transposition relies on externally supplied transposition machinery, many additional bacterial species may be suitable candidates for MuA-mediated mutagenesis, insertion library construction, and genome engineering.

If your organism can be transformed or electroporated, we would be happy to discuss whether MuA transposase may be suitable for your project.

Partner with Domus Biotechnologies

Domus Biotechnologies provides high-quality MuA transposases optimized for random mutagenesis, insertion library construction, and genome engineering applications.

Whether you are working with a model organism, an industrial production strain, a plant-associated bacterium, or a challenging environmental isolate, our team can help evaluate the suitability of MuA transposition for your application and assist in designing an efficient mutagenesis strategy.

Interested in testing MuA in your organism? Contact us to discuss your project and library construction goals.

 

References

[1] Kiljunen S, Pajunen MI, Dilks K, Storf S, Pohlschroder M, Savilahti H. Generation of a comprehensive transposon insertion mutant library for the model archaeon Haloferax volcanii and its use for gene discovery. BMC Biology. 2014;12:103.

[2] Lamberg A, Nieminen S, Qiao M, Savilahti H. An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction. Applied and Environmental Microbiology. 2002;68:705-712.

[3] Rasila TS, Pajunen MI, Savilahti H. Mu transpososome activity-profiling yields hyperactive MuA variants for genome engineering applications. Nucleic Acids Research. 2018;46:2755-2771.

[4] Laasik E, Ojarand M, Pajunen MI, Savilahti H, Mäe A. Novel mutants of Erwinia carotovora defective in plant cell wall degrading enzyme production generated by Mu transpososome-mediated insertion mutagenesis. FEMS Microbiology Letters. 2005;243:93-99.

[5] Pajunen MI, Pulliainen AT, Finne J, Savilahti H. Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes. Microbiology. 2005;151:1205-1214.

[6] Tu Quoc PH, Genevaux P, Pajunen MI, Savilahti H, Georgopoulos C, Schrenzel J, Kelley WL. Isolation and characterization of biofilm formation-defective mutants of Staphylococcus aureus. Infection and Immunity. 2007;75:1079-1088.

[7] Lanckriet A, Timbermont L, Happonen LJ, Pajunen MI, Pasmans F, Haesebrouck F, Ducatelle R, Savilahti H, Van Immerseel F. Generation of a single-insertion mutant library in Clostridium perfringens using a phage Mu-derived transposon. Applied and Environmental Microbiology. 2009;75:2638-2642.

[8] Akhverdyan VZ et al. Application of the bacteriophage Mu-driven system for integration and amplification of target genes in chromosomes of engineered Gram-negative bacteria. Applied Microbiology and Biotechnology. 2011.

[9] Murooka Y, Takizawa N, Harada T. Expansion of the host range of bacteriophage Mu. Journal of Bacteriology. 1981;145:358-368.

[10] Epicentre Biotechnologies. EZ-Tn5™ Transposome™ Technology Guide.

[11] Wu Z, Xuanyuan Z, Li R, Jiang D, Li C, Xu H, Bai Y, Zhang X, Turakainen H, Saris PEJ, Savilahti H, Qiao M. Mu transposition complex mutagenesis in Lactococcus lactis: identification of genes affecting nisin production. Journal of Applied Microbiology. 2009;106:41-48.

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