Characterization of the cheB2 Mutant 
We chose to further characterize the TB0173s mutant (cheB2), as this isolate was affected in all phenotypes tested and thus exhibited a general motility and virulence defect (Table 1).
To ensure that the cheB2 gene in the TB strain is located in a cluster similar to the che2 cluster described for the PAO1 or PA14 strains, we further analyzed the corresponding TB chromosomal region.
A series of 8 PCR reactions were performed using oligonucleotides based on the nucleotide sequence obtained from the PA14 genome sequence (Table S1).
The 8 PCR products were meant to cover the totality of the che2 cluster (Figure 1 and Figure S2) starting from the mcpA gene to the PA0171 gene located downstream cheB2 (PA0173).
The reactions were performed simultaneously using PA14 or TB genomic DNA as matrix.
Each paired reaction set (TB/PA14) resulted in products running at the same migratory rate within the 1.5% agarose gel (data not shown).
These results show that there is little difference between the two isolates, TB and PA14, with regards the overall organization of the che2 chromosomal region.
To validate that the loss of virulence and other phenotypic changes observed with the TB strain were a result of the transposon disruption of the cheB2 gene, we generated a new mutant by interrupting the cheB2 gene (see Materials and Methods).
As shown in Figure 3A, the newly engineered cheB2 mutant, called TB0173i (p<0.005), showed a delayed C. elegans killing comparable to the original STM cheB2 mutant TB0173s (p<0.004).
Both cheB2 mutants, TB0173s and TB0173i, required at least one additional day, as compared to TB, to kill 50% of exposed nematodes.
In addition, we engineered a similar cheB2 mutation in the P. aeruginosa PA14 strain, yielding PA140173i (see Materials and Methods), and observed similar virulence attenuation on C. elegans (Figure S3).
This further confirmed the virulence phenotype of a cheB2 mutant and indicated that it is not a TB strain-specific trait.
Finally, a complementation assay was performed.
We reintroduced, in the cheB2 mutant, the wild-type cheB2 gene using an integration-proficient vector, mini-CTX1 [47].
This vector contains the phiCTX attachment site allowing integration of exogenous DNA fragments at the attB site within the genome of P. aeruginosa.
The cheB2 gene was cloned into the mini-CTX1, yielding CTXp2B2 (see Materials and Methods).
This construct was introduced into the cheB2 mutant, TB0173s, generating the TB0173sp2B2 strain.
The empty vector, mini-CTX1, was also introduced into the TB0173s cheB2 mutant, generating TB0173sCTX.
As shown in Figure 3B the virulence was restored in the complemented cheB2 mutant TB0173sp2B2 (p<0.009), whereas the cheB2 mutant strain TB0173sCTX (p<0.005) remained fully attenuated.
