Project description:Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively.
Project description:Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively. LC-MS/MS analysis of DNA methylation was employed to verify the MTase activities. Transcriptomic analysis showed that ΔM.PaeTBCFII knockout mutant significantly downregulated nitric oxide reductase (NOR) regulating and coding gene expression such as nosR and norB, which contain methylated motifs in their promoters or coding regions. These predicted target motifs in nosR and norB were not methylated in the ΔM.PaeTBCFII knockout mutant. ΔM.PaeTBCFII exhibited reduced intercellular survival capacity in NO-producing RAW 264.7 macrophages and attenuated virulence in Galleria mellonella infection model. While the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII is frequently exists in P. aeruginosa sp as well as other bacteria species. Our work therefore provided new insights on the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa.
Project description:To further determine the origin of the increased virulence of Pseudomonas aeruginosa PA14 compared to Pseudomonas aeruginosa PAO1, we report a transcriptomic approach through RNA sequencing. Next-generation sequencing (NGS) has revolutioned sistems-based analsis of transcriptomic pathways. The goals of this study are to compare the transcriptomic profile of all 5263 orthologous genes of these nearly two strains of Pseudomonas aeruginosa.
Project description:Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4), which is positioned within the cell by ParA. Remarkably, ParB of P. aeruginosa binds to multiple heptanucleotides (half-parSs) scattered in the genome. In this work we analysed the transcriptome of P. aeruginosa with mutated 25 half-parSs forming the strongest ParB ChIP-seq peaks. Inactivation of ParB binding to even a small fraction of these sites modulated the gene expression, however this effect is most likely indirect. Overall this work suggests complex relation between ParB binding to genome and P. aeruginosa transcriptome.
Project description:To further understand the gene expression characteristics of originating biocontrol strain Pseudomonas aeruginosa M18, we have applied whole genome microarray expression profiling as a discovery platform to to specify the PCA-dependent expression of M18 genome. We constructed a series of PCA-producing mutant strains (high PCA: M18MSU1; low PCA: M18MS; and no PCA: M18MSP1P2). The comparison analysis of the M18 mutants genome expressional profiles indicated that the expression of PCA in both M18MSU1 and M18MS alters the expression of a total of 545 different genes; however, the higher level of PCA in M18MSU1 altered more genes (489) as compared to M18MS (129).
Project description:Analysis of Pseudomonas aeruginosa PAO1 treated with 200 µM sphingomyelin. Results provide insight into the response to sphingomyelin in P. aeruginosa.