Fig. 1. Tse5-CT has a bacteriolytic effect when expressed in P. putida.

a Monitoring growth (OD₆₀₀) in LB medium of P. putida cells harbouring pS238D1 empty vector (EV) as a negative control or plasmids directing the expression of Tse5-CT, or Tse5-CT and Tsi5 together. Expression of Tse5-CT and Tsi5 was induced after 8 h of bacterial growth (see the arrow) with 1 mM m-Toluic acid (TA) and 0.1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG), respectively. Co-expression of Tse5-CT and Tsi5 was induced by adding to the media both TA and IPTG. To stop the induction, the recovered cells (recov.) were washed and resuspended in fresh LB without inductors (see the arrow). Expression of Tse5-CT caused inhibition of bacterial growth, and cells did not recover the capacity to duplicate after washing. Tsi5 confers cell protection from Tse5 expression. b Monitoring growth on solid medium. Samples of P. putida growing cells were taken every 2 h, and dilutions were spotted on LB agar plates. Colony-forming units (CFU) were counted 24 h later. All measurements were made in triplicate (n = 3 biological replicates). The graphs show the mean values and ± standard deviations (SD). Some error bars are not visible due to overlaps with symbols.