Cells of the Δ 17125 mutant displayed translucent morphology when plated on agar plates, and ectopic expression of AT00_ 17125 via the plasmid pBBR1MCS- 17125 under the control of its own promoter in the Δ 17125 mutant restored the wild-type opaque morphology (Fig. 6c).
While both single and double mutants form colonies by day 2 when plated alone, only double mutants can do so in the presence of a tester lawn.
It is unlikely that this delay was simply due to a subpopulation of spores that were able to rapidly germinate, as wild-type and mutant spores had similar titers when plated on rich media (data not shown).
In reconstruction experiments, doubly mutant cells formed colonies by day 2 when plated with or without a tester lawn.
This enhanced conditional phenotype is ELG1-dependent given that both wildtype and ctf7 mutant cells devoid of plasmid exhibited equivalent growth when plated on galactose (data not shown).
Indeed, the expression level of the individual virB genes was more than thirty times lower in the ptsP mutant than in the wild-type strain (Fig. 6C) When plated on 2YT rich medium, the ΔptsP, ΔptsO, ΔptsN and, to a lesser extent, the ΔhprK mutant displayed a heterogeneity in colony size compared to the wild-type strain B. melitensis 16M (Fig. 7A).
Alternatively, or in addition, some auxotrophic mutants might be leaky and allow some growth, especially when plated using the RoToR.
In the other single substitution mutant, magmas Y93G cells grew normally upto 34°C and slower at 37°C when plated in rich media, while cells were Ts at 37°C in minimal media (Fig. 3A and B).
The growth of the ipl1 -321 mutant is improved upon deletion of RAS2 or overexpression of BCY1 but not when plated on nonfermentable carbon sources.
While no differences were observed between growth of the four mutants and the wild-type strain when cultivated in liquid 2YT medium (Fig. 7C), the ΔptsO, ΔptsP and to a lesser extent the ΔptsN and ΔhprK mutants displayed a heterogeneity in colony size compared to the wild-type strain when plated on solid 2YT rich medium (Fig.7A and C).
