Fig 1.
Analysis of cdiAPA2462 and cdiAPA0041 gene expression profile.
Expression of cdiAPA2462 and cdiAPA0041 in WT and ΔrsmA strains grown 7 h under agitation (planktonic) or without agitation (static) in LB medium. For each gene, expression was normalized to 16S expression and is shown relative to the WT planktonic condition level. Error bars represent the standard error of the mean from three independent experiments. Data were analyzed for significance using a two-tailed Student’s t-test. ** 0.025<p-value <0.01; *** p-value<0.01; ns: non-significant.
Fig 2.
The growth of cdiBAIPA2462 and cdiBAIPA0041 mutants is inhibited by contact with a wild-type strain.
Gentamycin-resistant wild-type and mutant target bacteria were mixed with or without wild-type or ΔrsmA attackers and the number of viable target cells was calculated as the number of CFU/ml after contact (t24h) divided by the number of CFU/ml before contact (t0h). Error bars represent the standard error of the mean from three independent experiments.
Fig 3.
Roles of CdiAPA2462, CdiAPA0041 in growth competition.
All experiments were performed in a ΔrsmA background. A) ΔBAIPA2462 and B) ΔBAIPA0041 target strains were mixed with different attacker cells. Error bars represent the standard error of the mean from three independent experiments.
Fig 4.
Roles of adhesion and biofilm formation in competition assays.
(A). CLSM of adhesive structures formed by WT, cdiAPA2462 and cdiAPA0041 mutants (ΔAPA2462 and ΔAPA0041 respectively) at 8 h post-inoculation. All strains carry a gfp gene inserted at the glmS locus on the P. aeruginosa PAO1 chromosome. White scale bar = 10 μm. (B) and (C). After staining with crystal violet, biofilm was quantified over time by measuring the optical density at 550 nm (OD550). Each strain was tested in triplicate experiment. Data were examined for significance using a two-tailed Student’s t-test. *** p-value<0.01; ns: non-significant. D. Competition experiments were performed in a ΔrsmA background. WT or ΔpilAΔfliC attacker strains were mixed with ΔBAIPA2462 or BAIPA0041 target bacteria. Error bars represent the standard error of the mean from three independent experiments.
Fig 5.
CdiIPA2462 and CdiIPA0041 play a protective role.
All experiments were carried out in a ΔrsmA background. Wild-type (WT) attacker was mixed with different gentamycin-resistant mutant targets: (A) ΔBAIPA2462 carrying or not the pJN107 plasmid encoding the CdiIPA2462 protein or the BAPA2462 mutant. No arabinose was added to produce CdiIPA2462 from pJN107-IPA2462; (B) BAIPA0041 carrying or not the pJN107 plasmid encoding the CdiIPA0041 protein or the BAPA0041 mutant. As control, ΔBAIPA0041 expressing cdiIPA0041 was tested alone in inter-bacterial competition (grey bar). 0.5% arabinose was used to induce CdiIPA0041.
Fig 6.
Intracellular toxicity of CdiA-CTPA2462 and CdiA-CTPA0041 in E. coli cells and protection by their cognate CdiI immunity proteins.
Production of (A) CdiA-CTPA2462 and (B) CdiA-CTPA0041 were repressed with 0.5% glucose (grey bars) or induced with 1% arabinose (black bars). Error bars represent the standard error of the mean from three independent experiments. C. Purified CdiA-CTPA0041 and CdiI-His6 proteins were mixed in vitro with Ni2+-NTA resin. Fractions were analyzed by SDS-PAGE and Coomassie blue staining. Control experiments with purified proteins incubated alone are shown on the left panel while CdiA-CT/CdiI-His6 mixtures are shown on the right panel. Marker lane (kDa) is shown on the left.
Table 1.
Predicted Pseudomonas CDI-encoding locus.
Fig 7.
Representative CdiA proteins of each class of pre-toxin motif.
The signal peptide (SP) is a sequence potentially recognized by the sec machinery and cleaved during the export through the inner membrane. TPS domain found in N-terminal part of TpsA allows targeting to the TpsB transporter and subsequent translocation through the outer membrane. Haemagglutinin repeats are highly divergent repeats found in FHA-like proteins. DUF637 is a conserved region found in bacterial haemagglutinins or hemolysins. WVHN, VENN, LYVT, DAMV, NEALV are the pre-toxin motifs delimiting the conserved N-terminal and the variable CT domains.
Fig 8.
CdiA-CT encoded by P. aeruginosa PA7, PA14 and P. syringae pv. tomato DC3000 inhibit E. coli cell growth.
Production of (A) CdiA-CTPSPA7_2777, (B) CdiA-CTPA14_32790 and (C) CdiA-CTPSPTO_3229 was repressed with 0.5% glucose (grey bars) and induced with 1% arabinose (black bars). CdiI-His6 proteins were produced from pTrc99a with 1 mM IPTG. The number of CFU/ml was calculated after 6 h of culture and the graphs show the mean of three independent experiments.