Journal: PLoS Genetics
Article Title: CbtA toxin of Escherichia coli inhibits cell division and cell elongation via direct and independent interactions with FtsZ and MreB
Figure Lengend Snippet: CbtA homologs YpjF and YkfI inhibit cell elongation and cell division in a conserved manner. (A) Spot dilution analysis shows that overproduction of His 6 -YpjF-GFP and His 6 -YkfI-GFP from pCA24N-derived plasmids pMT138 and p3-37, respectively, is toxic. Overnight cultures of BW27785/pMT136 (directing the synthesis of His 6 -GFP), BW27785/pMT138, and BW27785/p3-37 were back diluted to a starting OD600 of 0.05 in fresh LB (Cm) and grown until late-log phase at 37°C. Cultures were normalized, serially diluted, and spotted on LB (Cm) with or without 100 μM IPTG. Plates were incubated at 37°C overnight. Dilutions 10 −1 to 10 −5 are shown. (B) Cell morphology phenotypes. Strains BW27785/pMT138 ( his 6 -ypjF-gfp) , BW27785/pMT188 ( his 6 -ypjF-F65S-gfp ), BW27785/p3-37 ( his 6 -ykfI-gfp) , or BW27785/pMT144 ( his 6 -ykfI-F65S-gfp ) were imaged after 1.5 h induction at 37°C with 100 μM IPTG. (C, D) Bacterial two-hybrid assay detects interactions of YpjF and YkfI with FtsZ or MreB. Results of β-galactosidase assays performed with reporter strain cells containing compatible plasmids encoding the indicated λCI fusion protein and the indicated α fusion protein (α-FtsZ in (C) and α-MreB in (D)) or wild-type α. Cells were grown in the presence of 25 μM IPTG (C) and 100 μM IPTG (D). (E) Two-hybrid analysis shows that the Bsu FtsZ chimera containing the Eco H6/H7 loop (fused to λCI) can interact with both YkfI and YpjF (fused to α). Cells were grown in the presence of 100 μM IPTG. (F) Effect of MreB substitutions E262G and S269F on the two-hybrid interaction between MreB and YpjF. Cells were grown in the presence of 100 μM IPTG. In (C-F), bars represent averages of triplicate values, error bars represent standard deviation, and dashed lines designate highest basal lacZ expression. (G) Strains DH118/pFB149 and DH118/pDH278 (see legend to Fig 9D ) were transformed with either the empty vector pBAD33 control plasmid or pBAD33- ypjF-F65S (pDH289). Transformants were selected on M9 maltose (0.2% maltose, 0.2% casamino acids, 1 mM MgSO 4 ) plates supplemented with appropriate antibiotics and 250 μM IPTG at 30°C. Overnight cultures were grown in M9 maltose + 250 μM IPTG at 30°C. The next morning, cultures were back diluted to a starting OD600 of 0.03 in LB (Cm + Carb) supplemented with 250 μM IPTG, and grown for 1 h at 30°C (reaching an OD600 ~0.08). Cultures were induced by addition of 0.2% arabinose and grown for an additional 2 h at 30°C, at which point microscopic analysis was performed. Scale bars represent 5 μm in (B) and (G).
Article Snippet: Growth was monitored every 30–60 min by transferring 200 μL to a sterile microtitre plate and taking OD600 measurements on a plate reader (Molecular Devices).
Techniques: Derivative Assay, Incubation, Two Hybrid Assay, Standard Deviation, Expressing, Transformation Assay, Plasmid Preparation