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S P U Scuola D Post-Tran UNIVER I Dottor Nscriptio SITÀ DE Rato in S X UNIVERSITÀ DEGLI STUDI DI MILANNO Scuola di Dottorato in Scienze Biologiche e Molecolari XXV Ciclo Post-transcriptional regulation in Gram-negative bacteria Franccesco Delvillani PhD Thesis Scientific tutor: Dr. Federica Briani Academic year: 2012 SSD: BIO/18; BIO/19 Thesis performed at Bioscience Department Contents Contents ................................................................................................................................................ 3 ABSTRACT ......................................................................................................................................... 1 PART I ................................................................................................................................................. 2 mRNA decay in Bacteria ...................................................................................................................... 4 E. coli enzymes involved in mRNA decay .......................................................................................... 6 Endoribonucleases ............................................................................................................................ 6 Exoribonucleases ............................................................................................................................ 10 RNase R ...................................................................................................................................... 11 OligoRNase. ............................................................................................................................... 13 Other activities involved in mRNA degradation ............................................................................ 13 PAPI ........................................................................................................................................... 13 Translation initiation regulation in Gram-negative bacteria .............................................................. 13 The bacterial ribosome ................................................................................................................... 13 Translation initiation ...................................................................................................................... 14 Altered translation specificity of modified ribosomes ................................................................... 15 Interplay between translation and mRNA decay ................................................................................ 16 S1 ribosomal protein ...................................................................................................................... 17 RNA-based mechanisms of post-transcriptional regulation ............................................................... 18 Regulation by small non coding RNAs .......................................................................................... 18 Approaches to the identification of sRNA targets ......................................................................... 20 P. aeruginosa sRNAs ...................................................................................................................... 21 Translation modulation by cis-acting structures ............................................................................ 22 New P. aeruginosa RNATs identified by Tet-Trap ........................................................................ 25 References .......................................................................................................................................... 27 PART II .............................................................................................................................................. 35 Content: .............................................................................................................................................. 35 PART III………………………………………………………………………….…………………36 Content………………………………………………………………………….…………………..36 RNA-based regulation in bacteria ABSTRACT Post-transcriptional regulation plays a pivotal role in gene expression control as it ensures a fast response to environmental changes. This is particularly important in unicellular organisms that are exposed to environmental changes. During my PhD, I studied different aspects of this type of regulation in two Gram-negative bacteria: Escherichia coli and the opportunistic pathogen Pseudomonas aeruginosa. In E. coli I investigated the role of the ribosomal protein S1 in the interplay between translation and mRNA decay. In particular we showed that S1 not associated to 30S can sequester and protect mRNA from RNase E, the main endonuclease in E. coli. Another aspect that I considered in my research has been regulation by RNA molecules in P. aeruginosa. In the last decade, it has become clear how RNA-based regulatory mechanisms are important in controlling bacterial gene expression. However, little is known about it in this relevant human pathogen. By RNA deep sequencing we identified more than 150 novel candidate sRNAs in the P. aeruginosa strains PAO1 and PA14, which differ in virulence degree. We confirmed by Northern blotting the expression of 52 new sRNAs, substantially increasing the number of known sRNAs expressed by this bacterium. In this context we developed a genetic screen for the identification of genes post-transcriptionally regulated by RNA determinants and applied this system to the search of RNA thermometers (RNATs), i.e. mRNA determinants that couple translation with temperature changes. We identified four putative RNATs and validated two of them in E. coli. Interestingly, the two are located upstream of genes previously implicated in P. aeruginosa pathogenesis, namely dsbA and ptxS. ptxS RNAT was validated also in P. aeruginosa and represents the first RNAT ever described in this bacterium. 1 RNA-based regulation in bacteria PART I 2 RNA-based regulation in bacteria Bacteria are highly adaptable organisms able to survive in a wide range of environments with variable temperature, pH, availability of water, salts and nutrients. This is due to high metabolic versatility and efficient gene regulation that allow them to respond effectively to environmental changes by accordingly modulating cell physiology. Genes can be regulated at many stages, from transcription initiation to protein activity and degradation. The early elucidation of regulation mechanisms of Escherichia coli lactose operon and lambda phage lysogenic state, both based on repressors modulating transcription initiation, prompted the idea that gene expression would be essentially regulated by such mechanism. Conversely, successive research has enlarged the repertoire of bacterial regulatory strategies, showing that, albeit transcription initiation is indeed regulated at many promoters, further steps of gene expression are also targeted by regulators. Moreover, it has been found that the chemical nature of regulators is not limited to proteins, as RNAs and small molecules (i.e. modified nucleotides) control relevant aspects of bacterial physiology. My research as a PhD student has addressed different aspects of translation initiation regulation and interplay between translation and mRNA decay in two Gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa. Escherichia coli is a Gram-negative facultative anaerobic bacterium that belongs to the class of Gammaproteobacteria. It is commonly found in the lower intestine of humans and other warm- blooded organisms. The first complete genome sequence of E. coli was published in 1997; the sequenced strain (E. coli K-12 MG1655) owns a 4.6x106 bp genome with 4288 annotated open reading frames (ORFs) 1. Sequencing of other E. coli strains showed that this species has a genome ranging from 4.6x106 bp to 5.5x106 bp. E. coli is the most widely studied prokaryote and the best known model organism. It is also widely used in the field of biotechnology and as host for heterologous gene expression. However, though profusely studied, about 30% of E. coli genes have unknown function 2. Most E. coli strains are harmless, but some can cause from mild to severe infections. Eight pathovars have been described and their mechanism of pathogenesis studied. Six of them are diarrheagenic and the other two cause extraintestinal infections. The EPEC strain is the major cause of infection in developing countries. This strain acquired a particular pathogenicity island (PAI), named LEE (locus of enterocyte effacement). This element encodes a type III secretion system (T3SS) used to translocate bacterial effectors into the host cells. The genome of pathogenic strains may be 1x106 bp larger than that of commensal strains. They usually contain PAIs and plasmids 3 RNA-based regulation in bacteria coding for genes involved in the attachment, in the virulence and in the proliferation into the host cells 3. Pseudomonas aeruginosa is a highly adaptable Gram-negative bacterium, which thrives in a broad range of ecological niches. It is usually found in the soil and is able to grow also in hypoxic environment. Moreover, it can infect organisms as different as nematodes, mammalians and plants. This bacterium owns a relatively big (6-7 Mb), GC-rich genome with around 6000 predicted open reading frames. In human, P. aeruginosa behaves as an opportunistic pathogen infecting wounds, burns and medical devices. In cystic fibrosis patients P. aeruginosa is the most common pathogen that infects the respiratory tract and it is the
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