5.5.1. Transformation of Paenibacillus larvae

For this purpose plasmid pAD43-25 (BGSC, Bacillus Genetic Stock Center), carrying the gene sequence for a GFP variant (gfpmut3a), was transformed into wild type P. larvae strains ATCC9545 and 04-309 representing both relevant genotypes ERIC I and ERIC II, respectively (Genersch et al., 2005, 2006). Plasmid pAD43-25 functions as an E. coli/gram-positive shuttle vector and enables bacteria to constitutively express the GFP variant gfpmut3a (Dunn and Handelsman, 1999). High level constitutive expression of mutant GFP in vegetative cells is facilitated by the Bacillus cereus UW85 Pupp promoter upstream of gfpmut3a. Mutant gfpmut3a has an optimal excitation wavelength of 498 nm and the plasmid contains a chloramphenicol resistance cassette.

The first critical step of the molecular manipulation of P. larvae is the uptake of foreign DNA, e. g. plasmid DNA. Because P. larvae is a gram-positive bacterium, transformation needs a high voltage electric pulse for a successful uptake of foreign plasmid DNA. For this purpose electrocompetent bacterial cells need to be prepared as described by Murray and Aronstein (2008):

  1. Grow P. larvae cultures to early exponential phase (OD600 0.3).
  2. Harvest by centrifugation.
  3. Wash bacterial pellets three times using 0.625 M sucrose.
  4. Resuspend the final bacterial pellet in 1/500 of the initial culture volume.
  5. Store at -80°C in 40 µl aliquots.

       Electrocompetent P. larvae cells can then be transformed with a plasmid containing a GFP-gene like pAD43-25 using the following protocol:

  1. For pAD43-25 transformation, thaw competent cells at 4°C.
  2. Add 500 ng of pAD43-25 in a maximum volume of 10 µl.
  3. During an incubation time of 15-20 min gently mix each transformation tube every 5 min.
  4. Pulse probes in ice cold 1 mm electroporation cuvettes (Eppendorf).
  5. The best transformation results can be achieved with 9 kV/cm for representatives of the genotype ERIC I and 10 kV/cm for representatives of the genotype ERIC II. An average of 1.8E+04 and 1.1E+06 transformants per 0.5 µg DNA (3.6E+04 and 2.2E+06 transformants per µg DNA) for ERIC I and ERIC II, is obtained using these conditions.
  6. Immediately transfer transformation tubes containing shocked cells to 960 µl pre-warmed MYPGP broth.
  7. Incubated at 37°C for 16 hours with shaking (350 x g).
  8. Dilute regenerated transformation mixes.
  9. Plate on MYPGP agar supplemented with 5 µg/ml chloramphenicol.
  10. Incubate agar plates at 37°C.
  11. Determine the number of colony forming units (cfu) after 3 days.


For pAD43-25 transformation, thaw competent cells at 4°C.

Add 500 ng of pAD43-25 in a maximum volume of 10 µl.

During an incubation time of 15-20 min gently mix each transformation tube every 5 min.

Pulse probes in ice cold 1 mm electroporation cuvettes (Eppendorf).

The best transformation results can be achieved with 9 kV/cm for representatives of the genotype ERIC I and 10 kV/cm for representatives of the genotype ERIC II. An average of 1.8E+04 and 1.1E+06 transformants per 0.5 µg DNA (3.6E+04 and 2.2E+06 transformants per µg DNA) for ERIC I and ERIC II, is obtained using these conditions.

Immediately transfer transformation tubes containing shocked cells to 960 µl pre-warmed MYPGP broth.

Incubated at 37°C for 16 hours with shaking (350 x g).

Dilute regenerated transformation mixes.

Plate on MYPGP agar supplemented with 5 µg/ml chloramphenicol.

Incubate agar plates at 37°C.

Determine the number of colony forming units (cfu) after 3 days.