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. 2019 Jun 4;112(2):632–648. doi: 10.1111/mmi.14279

Figure 1.

Figure 1

Impact of c‐d‐GMP on Tde1, Tae and Hcp levels. A. The T6SS cluster composed of the divergently transcribed imp (Green) and hcp (Red) operons encoding 14 and 9 genes, respectively, and the remote island starting from atu3642 (vgrG‐2) encoded by A. tumefaciens strain C58. B. Expression of wild‐type SadC and a catalytically inactive SadC (psadC*) in wild‐type A. tumefaciens grown to early exponential phase in 523 medium. C. Western blots using Tde1, Tae, Hcp and RpoA (Loading Control) antibodies on whole lysates of ΔtssL cells (No secretion control) or wild‐type cells transformed with either pBBRMCS4 (vector control), psadC, psadC*, pwspR and pPA2133 grown to early exponential phase in 523 medium. D. C‐di‐GMP levels were quantified via LC‐MS/MS. Transformed cells were grown for 16 h in 523 medium and cells equivalent to an OD600 5 were collected. Samples of interest were compared to a standard curve derived from measurements of known concentrations of pure c‐di‐GMP to determine the concentration (in nM) of c‐di‐GMP in the samples. All experiments are the mean of two independent biological experiments with standard deviation error bars. Statistical significance was determined using students t‐test with p < 0.05*, p < 0.01 **, p < 0.001***.