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Multiple transcription-activating sequences regulate the RsmZ regulatory small RNA of Pseudomonas brassicacearum.

ABSTRACT: The mutS-rpoS region is known to be a highly polymorphic segment of the chromosome owing to horizontal gene transfer and evolutionary processes. In Pseudomonas, mutS-fdxA-rsmZ-rpoS organization is highly conserved, as well as the promoter region of the RsmZ small RNA (sRNA)-encoding gene. One exception to this conservation is in Pseudomonas brassicacearum, where a 308-nucleotide (nt) sequence, predicted to form a hairpin structure in single-stranded DNA (ssDNA), is inserted between the rpoS and rsmZ genes. Using MEME software, we identified nine consensus motifs in the rsmZ promoter region of 16 sequenced Pseudomonas genomes. We observed that an upstream activation sequence (UAS) and an M1 motif (located between the -10 promoter element and the UAS) are shared among examined Pseudomonas genomes. A third motif, the M2 motif, is localized within the coding sequence of the rpoS gene. Constructs fusing the different identified motifs to the lacZ reporter were produced. Our in vivo analysis of the rsmZ-activating elements indicates that the palindromic UAS located 180 bp upstream of the rsmZ transcriptional start in P. brassicacearum NFM 421 is essential, but not sufficient, for full rsmZ expression. Here, we demonstrate a role for the three motifs in the activation of the rsmZ gene, and we hypothesize the role of additional transcriptional factors, along with the DNA structuring role of the hairpin in the complex network controlling the expression of rsmZ.

SUBMITTER: Lalaouna D

PROVIDER: S-EPMC3430305 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Multiple transcription-activating sequences regulate the RsmZ regulatory small RNA of Pseudomonas brassicacearum.

Lalaouna D D Fochesato S S Barakat M M Ortet P P Achouak W W

Journal of bacteriology 20120706 18

The mutS-rpoS region is known to be a highly polymorphic segment of the chromosome owing to horizontal gene transfer and evolutionary processes. In Pseudomonas, mutS-fdxA-rsmZ-rpoS organization is highly conserved, as well as the promoter region of the RsmZ small RNA (sRNA)-encoding gene. One exception to this conservation is in Pseudomonas brassicacearum, where a 308-nucleotide (nt) sequence, predicted to form a hairpin structure in single-stranded DNA (ssDNA), is inserted between the rpoS and ...[more]

PMID: 22773788

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Project description:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

Dataset Information

Multiple transcription-activating sequences regulate the RsmZ regulatory small RNA of Pseudomonas brassicacearum.

Publications

Multiple transcription-activating sequences regulate the RsmZ regulatory small RNA of Pseudomonas brassicacearum.

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