Introduction ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium ( S. typhimurium ). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated. Objectives The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism. Method We constructed a clpP rpoS double deletion mutant TT-19 (Δ clpP Δ rpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (Δ rpoS TT-1), clpP deletion mutant TT-16 (Δ clpP TT-1), and clpP rpoS double deletion mutant TT-19 (Δ clpP Δ rpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS). Results Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l -5-oxoproline, l -glutamic acid, and l -tryptophan) and intracellular concentrations of 10 compounds ( l -isoleucine, glycine, serine, l -methionine, l -phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted. Conclusion ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.
Motility is finely regulated and is crucial to bacterial processes including colonization and biofilm formation. There is a trade-off between motility and growth in bacteria with molecular mechanisms not fully understood. Hypermotile Escherichia coli could be isolated by evolving non-motile cells on soft agar plates. Most of the isolates carried mutations located upstream of the flhDC promoter region, which upregulate the transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. Here, we identified that spontaneous mutations in clpX boosted the motility of E. coli largely, inducing several folds of changes in swimming speed. Among the mutations identified, we further elucidated the molecular mechanism underlying the ClpXV78F mutation on the regulation of E. coli motility. We found that the V78F mutation affected ATP binding to ClpX, resulting in the inability of the mutated ClpXP protease to degrade FlhD as indicated by both structure modeling and in vitro protein degradation assays. Moreover, our proteomic data indicated that the ClpXV78F mutation elevated the stability of known ClpXP targets to various degrees with FlhD as one of the most affected. In addition, the specific tag at the C-terminus of FlhD being recognized for ClpXP degradation was identified. Finally, our transcriptome data characterized that the enhanced expression of the motility genes in the ClpXV78F mutations was intrinsically accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of the hypermotile strains. A similar pattern was observed for previously isolated hypermotile E. coli strains showing high expression of flhDC at the transcriptional level. Hence, clpX appears to be a hot locus comparable to the upstream of the flhDC promoter region evolved to boost bacterial motility, and our finding provides insight into the reduced fitness of the hypermotile bacteria.
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