Plasmid transformation and expression of the firefly luciferase in Microbacterium testaceum type and endophytic colonizing field strains

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Abstract
Microbacterium testaceum is a predominant endophytic bacterial species isolated from corn and sorghum in the midwestern United States. The development of genetic transfer systems for M. testaceum may enable its use for biocontrol and other applications. The type strain (IFO 12675) and field isolates (SE017, SE034, and CE648) were grown to mid-exponential phase, concentrated (1.0 × 1011CFU·mL–1), electroporated ( Escherichia coli – Clavibacter shuttle plasmid pDM302), and plated on TSA with 10 µg·mL–1chloramphenicol. Transformation efficiencies averaged 140 CFU·µg–1of DNA. Restriction endonuclease analysis showed that pDM302 was not altered after extraction from transformants and re-introduction into E. coli. Transformants with pDM302 were also subjected to nonselective growth conditions, with the frequency of loss after one passage being 84% for IFO 12675 and 88% for SE034. We inserted the green fluorescent protein and the firefly luciferase (FFlux) reporter genes into pDM302, confirming the expression of FFlux in IFO 12675 and SE034. The SE034 FFlux strain was recovered from inoculated corn in greenhouse studies and found to fluoresce by luminometry. These results in M. testaceum demonstrate for the first time its transformability, pDM302 replication, FFlux gene expression, and the recovery of the FFlux recombinant strain from inoculated corn.

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