RNA-seq and ChIP-seq for Lon

Lon protease plays vital roles in many biological processes in Pseudomonas syringae, including type III secretion systems (T3SS), transcription regulation, protein synthesis and energy metabolism. Lon also functions as a transcriptional regulator in other bacterial species (e.g., Escherichia coli and Brevibacillus thermoruber). Therefore, we hypothesise that Lon has dual functions in P. syringae. To reveal the molecular mechanisms of Lon as a transcriptional regulator and protease under different environmental conditions, we used a combination of transcriptome sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the genes or proteins regulated by Lon. As a transcriptional regulator, Lon bound to the promoter regions of PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA and consequently regulated 1-dodecanol oxidation activity, motility, pyoverdine production, gluconokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase (SHMT) activity in King's B medium (KB). In minimal medium (MM), Lon regulated SHMT activity and lon expression by binding to the promoter regions of glyA and lon, respectively. As a protease, Lon regulated the T3SS and metabolic pathways (e.g., amino acid metabolism). In MM, Lon regulated the polysaccharide metabolic process by controlling PSPPH_0514, AlgA, CysD and PSPPH_4991. Taken together, these data demonstrate that Lon acts as a transcriptional regulator or protease in different environments and tunes its virulence and metabolic functions accordingly. Overall design: In order to identify the direct binding loci in P. syringae, ChIP-seq assay was employed in KB or MM in vivo, by expressing lon with its native promoter fused with HA-tag in the pHM2 plasmid in P. syringae pv. phaseolicola (also named P. savastanoi pv. phaseolicola). Four sets of ChIP-seq samples were performed: (1) the pHM2-Lon-HA plasmid in the wild-type (WT) strain in KB, (2) the empty pHM2 plasmid in WT strain in KB, (3) the pHM2-Lon-HA plasmid in WT strain in MM, and (4) the empty pHM2 plasmid in WT strain in MM. By comparing with the control strains carrying the empty plasmid, the present work identified a total of 926 and 545 Lon-enriched loci in KB and MM, respectively. These results indicated that Lon functions as a transcriptional regulator in a nutritional-dependent manner in P. syrinage. Notably, nearly half of the Lon-binding loci were located in the upstream or the overlapping the start regions, suggesting the potential regulatory functions of Lon on these genes. The lon locus was deleted by homologous recombination with its upstream and downstream flank DNA fragments. Compared to the WT, 1260 and 354 genes were up-regulated in ?lon in KB (= 2-fold and p < 0.05) and MM, respectively. Meanwhile, the expression level of 1334 and 389 genes was down-regulated (= 2-fold and p <0.05) in ?lon in KB and MM, respectively. Taken together, the Venn diagram illuminated that 2187 KB-dependent genes and 336 MM-dependent genes in the Lon regulon, suggesting a more crucial role of Lon in KB than MM. RNA-seq and ChIP-seq for Lon