Skip to main content
Fig. 3. | BMC Biology

Fig. 3.

From: Competition for nutritional resources masks the true frequency of bacterial mutants

Fig. 3.

a Inhibition of Δpst colony growth by single gene knockouts from Keio collection (BW25113 background). One hundred Δpst cells were mixed with 109 bacteria carrying individual deletions in each of the following genes: cyaA, crp, crr, glpA, glpB, glpC, glpD, glpF, glpK, glpX, glpQ, glpT, and glpR. The plates were incubated for 2–3 days at which time the PCM colonies were counted. “Control” represents the Δpst strain plated in the absence of other bacteria and phoA serves as a positive control. Each bar represents the mean ± S.E.M. of at least 3 independent cultures. b Screening the E. coli collection of knockouts for Δpst growth inhibition. Δpst cells and the library knockouts (Keio collection plate no. 53) were grown overnight in TGP medium. The Δpst culture was diluted a hundredfold and 30 μ l of this dilution was used to create a linear patch on a TG2PP plate supplemented with XP. Once the Δpst patch was dry, 2 μ l of each knockout strain was dropped over the patches. The plates were incubated for 48 h at 37 C. In the vast majority of cases, a halo was formed inside the patch where the knockout strain was applied, indicating that Δpst growth was inhibited by this particular strain. Only a few knockouts described in the main text allowed the growth of Δpst, characterized by a bluish color inside the drop. These strains were further tested in a conventional inhibition assay (as in Fig. 3a) to confirm this phenotype. The strong blue color inside the phoU drop is because this mutant is a PCM that grows on TG2PP. Of those that did not inhibit Δpst, the majority was formed by auxotrophic strains. For instance, in plate 53, the knockouts of pyrE, purA, purD, purL, and purM did not inhibit Δpst growth, but they do not grow or grow very poorly in minimal medium. Halos marked with an X correspond to bacteria that are not part of the Keio collection (see the Keio collection documentation at https://shigen.nig.ac.jp/ecoli/strain/resource/keioCollection/ about). c Formation of PCM colonies from apparent cell clusters in the presence of 109 inhibitors. Approximately 100 Δpst cells were plated on TG2PP and immediately incubated at 37 C. Top-agar carrying 109phoA cells was poured over the Δpst bacteria at time 0, 3, 6, 12, or 24 h following Δpst plating. The plates were then further incubated, such that the total incubation time for each plate was 48 h. The CFU number was counted at the end of the 48-h incubation. The control plates did not contain phoA cells. Each bar represents the mean ± S.E.M. of 5 independent cultures

Back to article page