Functional Analyses of Transcription Factors in Pseudomonas

假單胞菌中轉錄因子的功能分析

Student thesis: Doctoral Thesis

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Award date10 Aug 2021

Abstract

The transcription factors (TFs) regulated gene expression by binding to a specific sequence located in both coding and intergenic DNA regions. In the intergenic region, TFs regulated gene expression by binding to promoter or enhancer regions. In the coding region (CDS), TFs bound to the cryptic promoter to regulate transcript expression. However, the mechanism of TFs regulating the activity of the cryptic promoter in the CDS in bacteria remains elusive.

We analyzed the high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) from Pseudomonas syringae (P. syringae) and Pseudomonas. aeruginosa (P. aeruginosa). We found most of the position weight matrix (PWM) motifs were distributed in CDS and had similar find individual motif occurrences (FIMO) scores and q-value to those in the intergenic regions.

To further study TF binding preference in vivo, we analyzed chromatin immunoprecipitation followed by sequencing (ChIP-seq) data from 7 different strains, including Mycobacterium tuberculosis (M. tuberculosis), P. aeruginosa, P. syringae, Salmonella enterica (S. enterica), Vibrio cholerae (V. cholerae), Bacillus subtilis (B. subtilis), and E. coli. The aforementioned ChIP-seq data showed that most of these peaks were located in CDS (93.13%). The average occupancy of peak and FIMO scores centered over consensus motifs were similar in the intergenic and coding regions. The CDS binding TFs could influence the nearby transcript expression. For example, RhpR could regulate the expression of PSPPH_4417 by binding in PSPPH_4418. The CDS binding TFs also regulated the antisense transcript expression. For instance, RhpR regulated the antisense transcript by binding to the CDS of PSPPH_2787.

Lon, a member of the AAA+ protease family, plays vital roles in type III secretion systems (T3SS), agglutination, and colony shape in the model plant pathogen Pseudomonas syringae. Lon also functions as a transcriptional regulator in other bacterial species such as Escherichia coli and Brevibacillus thermoruber. To reveal the molecular mechanisms of Lon as a dual-function protein in P. syringae, we studied Lon-regulated genes by using RNA sequencing (RNA-seq), ChIP-seq, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a transcriptional regulator, Lon directly regulated a group of genes (PSPPH_4788, gacA, fur, gntR, clpS, lon, and glyA), and consequently regulated their functions, such as 1-dodecanol oxidation activity, motility, pyoverdine production, glucokinase activity, N-end rule pathway, lon expression, and serine hydroxymethyltransferase (SHMT) activity. Mass spectrometry results revealed that the expression levels of 5 T3SS proteins (such as HrcV, HrpW1) were higher in the ∆lon strain than the wild-type strain in KB. In MM, 12 metabolic proteins (such as AcdS and NuoI) showed lower levels in the ∆lon strain than the wild-type strain. In summary, these data demonstrate that the dual-function protein Lon sophisticatedly regulates virulence and metabolism in P. syringae.

In summary, our study revealed that TFs regulated transcript expression by binding to both CDS and intergenic regions. By binding to the intergenic region, TFs activate antisense transcript expression. Besides, Lon functions as a dual-function protein that sophisticatedly regulates virulence and metabolism in P. syringae. These results provide new insights into the pathogenic mechanism in P. syringae.