Table 1.
Bacterial strains used in this study.
Table 2.
DNA primers used in this study.
Figure 1.
Effects of growth conditions on virulence gene transcriptions.
V. cholerae toxT::lacZ (A and B), ctx::phoA (C) and tcpA::phoA (D) transcription in different growth conditions were measured. Bacteria were inoculated into LB (pH 6.5) and shaken at 30°C for 6 hrs (A and B) or 16 hrs (C and D). Malonate was added to LB (pH 6.5) as indicated (A) or at 40 mM (C and D). NaCl was added to LB (pH 6.5) as indicated (B). Autoagglutination of the V. cholerae O395N1 cells was evaluated visually after 16–24 hrs of incubation (“+” means autoagglutination, “−” means no autoagglutination) (D). All experiments were repeated more than three times. The error bars indicate standard deviations. P values were calculated by one-way ANOVA followed by post hoc Tukey test (A and B), or Student’s t test (C and D).
Figure 2.
Effect of malonate on virulence gene expressions.
V. cholerae O395N1 were inoculated into LB (pH 6.5) with or without 40 mM malonate and shaken at 30°C for 6 hrs. Total RNA was extracted and analyzed by qRT-PCR. Gene expression levels were normalized between the samples by using 16S ribosomal RNA. All experiments were repeated three times. The error bars indicate standard deviations. P values were calculated by Student’s t test and *indicates P≤0.05.
Table 3.
CT production of different V. cholerae strains under various growth conditions.
Figure 3.
Effects of malonate, glycerol and glucose on V.cholerae growth.
Bacteria were inoculated into M9 medium supplemented with different carbon sources (f.c. 0.4%). Bacterial growth was measured after 24 hrs shaking. 40 mM malonate was added to the M9-glycerol and M9-glucose media as indicated. All experiments were repeated more than three times. The error bars indicate standard deviations. P values were calculated by Student’s t test.
Table 4.
V. cholerae predicted acyl-CoA ligases that show similarity to malonyl-CoA synthetase from Rhizobium leguminosarum.
Figure 4.
Current working model for the link between malonate and virulence gene expression in V. cholerae.
Malonate is possibly imported by one of the Auxin Efflux Carrier (AEC) Family proteins (VC1229 and VCA0024). We propose that increased levels of malonyl-CoA decrease the available intracellular CoA levels which results in decreased acetyl-CoA (AcCoA) levels. When TCA cycle activity is low, AcCoA levels should be increased and V. cholerae produce acetate from AcCoA and excrete it into the external medium most likely via the PTA and ACK system (see text for more details). Such a link between AcCoA levels and toxT transcription, as depicted by the red arrow, has previously been observed [12].