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Assessing the transcriptional landscape of Pseudomonas chlororaphis phage 201f2-1: uncovering the small regulatory details of a giant phage

ABSTRACT: The global transcriptional profile of Pseudomonas chlororaphis infecting phage 201f2-1 was obtained using the long-read RNA sequencing technique ONT-cappable-seq. this resulted in a comprehensive genome-wide map of viral transcription start and termination sites. In addition, we were able to identify different transcription units and gained new insights in the molecular mechanisms of of transcriptional regulation of members of the Phikzvirus.

ORGANISM(S): Pseudomonas phage 201phi2-1

PROVIDER: GSE245572 | GEO | 2024/10/17

REPOSITORIES: GEO

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Project description:The GacS/GacA two component regulatory system globally activates the production of secondary metabolites including phenazines in Pseudomonas chlororaphis 30-84. To better understand the regulatory role of the Gac system, we conducted RNA-seq analyses to determine the regulon of the response regulator GacA. Transcriptome analyses identified over 700 genes differentially regulated by GacA. Consistent with our previous findings, phenazine biosynthetic genes were significantly down-regulated in a gacA mutant. The expression levels of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS and pip were also decreased. Moreover, the expression of three none-coding RNAs (ncRNAs) including rsmX, rsmY and rsmZ was significantly decreased by gacA mutation consistent with the presence of GacA binding sites in their promoters. Our results also demonstrated that over-expression of rsmZ from a non-gac regulated promoter resulted in the restoration of AHL and phenazine production as well as the expression of other secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production was also investigated. Over-expression of rsmE, but not rsmA, resulted in decreased AHL production and phenazine gene expression in P. chlororaphis. Consistently, a mutation in rsmE bypassed the requirement of GacA in phenazine gene expression. On the contrary, constitutive expression of the phzI/phzR quorum sensing system was not able to rescue phenazine production in the gacA mutant indicating the direct impact of Gac system on the transcript stability of phenazine biosynthetic genes. Together, these results indicate that the Gac system regulates phenazine production at multiple levels and exerts its positive effect on AHL and phenazine biosynthesis via RsmZ and RsmE. A model is proposed to illustrate the GacA regulon in P. chlororaphis 30-84. A total of 6 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (3 replicates); Pseudomonas chlororaphis gacA mutant (3 replicates).

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Assessing the transcriptional landscape of Pseudomonas chlororaphis phage 201f2-1: uncovering the small regulatory details of a giant phage

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