Abstract
Pseudomonads are Gram-negative and ubiquitous environmental bacteria that include Pseudomonas syringae and Pseudomonas aeruginosa. P. syringae is a model plant-pathogenic bacterium, which causes huge economic lost every year in agriculture. P. aeruginosa is a predominant human opportunistic pathogen leading to outbreak in hospitals and is one of the top three causes of opportunistic human infections. Although P. syringae and P. aeruginosa have been widely studied individually for decades, the mechanisms of pathogenicity remain largely unknown. The virulence is regulated by many transcriptional factors (TFs) and several TFs associated virulence have been identified in both two strains, but a complete picture of binding profiles of all TFs and their precise targets in genome are still largely unknown.Here, we systematically performed a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach to dissect the binding motifs and preference of all 301 TFs in P. savastanoi and 371 TFs in P. aeruginosa, respectively. In P. syringae, robust enrichment of specific sequences was observed for 319 groups in total of SELEX patterns, representing 282 groups TFs binding profiles and 20,000 interactions screening in the genome of two strains. The position weight matrix (PWM) of each TF showed that about 90 % PWMs of TFs were of dimeric site type of which 80 % PWMs of TFs displayed the head-to-head palindromic binding preference in both P. aeruginosa and P. syringae.
To further explore the pathogenic mechanism of TFs, we mapped an intricate network showing complicated interactions among target genes and 319 TFs, many of which can be verified by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), EMSA, RT-qPCR and promoter activity detection. After screening genome, we identified 72 virulence-associated TFs in the virulence-associated pathways, including TrpI, RhpR, GacA, MetR, FleR, LexA1, KdpE, PilR, TsiR, GltR and MgrA in P. syringae, and GfnR, AlpR, EraR, CprR, and RpoN in P. aeruginosa and 29 unknown TFs. In addition, TFs involved in metabolism, such as TrpI and GntR, had binding sites in virulence genes (hopAK1, and hopAN1). Overall, the study of this part provides an integrated perspective for seeking the virulence associated TFs and its target genes in Pseudomonads.
Recently, the ubiquitous bacterial secondary messenger cyclic diguanylate (c-di-GMP) has been reported that regulates virulence in many bacteria. However, its function in the model plant bacterium-pathogen P. syringae remains largely elusive. To further explore the virulence mechanism caused by c-di-GMP, we constructed two strains with different level of c-di-GMP by overexpressed diguanylate cyclase (YedQ) and phosphodiesterase (YhjH), respectively. Via RNA sequencing, RT-qPCR and phenotypic experiments of these two strains, we identified that 9 key genes (fliN, fliE, flhA, alg8, alg44, pvdE, pvdP, pvsA and sodA) involved in 4 virulence associated pathways (flagellar assembly, exopolysaccharide biosynthesis, siderophore biosynthesis, oxidative stress resistance) were influenced by c-di-GMP level. Especially, we identified and constructed three reporters that were sensitive to elevated levels of c-di-GMP in Pseudomonas syringae. The three reporters can be useful in the measurement of intracellular c-di-GMP level in real time. In sum, the results of this part demonstrated that c-di-GMP regulates the virulence associated pathways in Pseudomonas syringae, suggesting that tuning its level could be an economic and efficient strategy to protect plants from attacks by it.
Overall, the study of these two parts provides a comprehensive perspective for probing the profiling DNA-binding specificities of TFs and roles of c-di-GMP. We expect that these results will significantly facilitate future studies on the overall regulatory of virulence, which will help to protect hosts from attacks by this two pathogens and other pathogens.
| Date of Award | 2 Sept 2020 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Xin DENG (Supervisor) |