Deletion in the C-terminal domain of ClpX delayed entry of Salmonella enterica into a viable but non-culturable state

Under stressful conditions, bacteria enter a viable but non-culturable (VBNC) state in which they are alive but fail to grow on conventional media. The molecular basis underlying this state is unknown. To identify the key gene responsible for the VBNC state in Salmonella spp., we examined a S. Typhimurium LT2 VBNC mutant, which shows a characteristic delay in entering the VBNC state. The mutant showed a higher level of expression of general stress sigma factor RpoS than wild-type LT2. The mutant carried a 99-bp in-frame deletion in the clpX gene (clpXΔ323–355). ClpX is known to form a ClpXP protease complex with ClpP, which plays a role in the degradation of RpoS. To investigate the effect of clpXΔ323–355 on VBNC induction, ΔclpX and clpXΔ323–355 strains were generated from LT2 cells. Compared to LT2, the ΔclpX and clpXΔ323–355 strains showed greater amounts of RpoS and required a longer incubation time for induction into the VBNC state. These results suggest that residues 323–355 of ClpX play a major role in the hexameric formation or function of ClpX and in the rate of induction of the VBNC state.