title: Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.
abstract: BACKGROUND
  The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.
  
  
  RESULTS
  In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.
  
  
  CONCLUSIONS
  In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. vaniapobre@itqb.unl.pt.
authors.forename: Vânia
authors.initials: V
authors.lastname: Pobre
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. cecilia@itqb.unl.pt.
authors.forename: Cecília M
authors.initials: CM
authors.lastname: Arraiano
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D002465:Cell Movement / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D005095:Exoribonucleases / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C489683:rnr protein, E coli; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C022535:exoribonuclease II