dMvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.
Understanding the interplay between a plasmid and its host system is a bottleneck towards prediction of the fate of plasmid-harbouring strains in the natural environments. Here, we studied the impact of the conjugative plasmid pCAR1, involved in carbazole degradation, on the proteome of Pseudomonas putida KT2440 using SILAC method. Furthermore, we investigated two acyl lysine modifications (acetylation and succinylation) that respond to the metabolic status of the cell and are implicated in regulation of various cellular processes. The total proteome analysis revealed that the abundance of key proteins involved in metabolism, signal transduction and motility was affected by pCAR1 carriage. In total, we identified 1359 unique acetylation sites on 637 proteins and 567 unique succinylation sites on 259 proteins. Changes in the acylation status of proteins involved in metabolism and translation by pCAR1 carriage were detected. Remarkably, acylation was identified on proteins involved in important plasmid functions, including partitioning and carbazole degradation, and on nucleoid-associated proteins that play a key role in the functional interaction with the chromosome. This study provides a novel insight on the functional consequences of plasmid carriage and improves our understanding of the plasmid-host cross-talk.
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