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Caracterización del sistema toxina-antitoxina reibe2 de spectrococcus pneumoniae
|Advisor:||Espinosa, Manuel ; Nieto, Concha|
Toxinas-Antitoxinas de tipo II
|Publisher:||CSIC - Centro de Investigaciones Biológicas (CIB)|
Universidad Complutense de Madrid
|Abstract:||Type II (proteic) toxin-antitoxin (TA) loci are ubiquitous in bacteria and are composed by two genes that are organized in an operon. The genes encode a stable toxin and an unstable antitoxin. In general, the antitoxin gene precedes, and frequently overlaps, the toxin gene. Both proteins bind to each other, generating a harmless complex, which regulate their own synthesis. These type II TA systems (TAS) were initially discovered in plasmids, but subsequently were identified in the chromosome of different bacteria and archaea. The chromosomally encoded TAS seem to be involved in different cellular processes, such as response to stress, programmed cell death, persistence and biofilm formation.
This Thesis’ work is focused on the chromosomal RelBE2 system of the pathogenic Gram-positive bacterium Streptococcus pneumoniae.|
The objectives of this Thesis based on the available data were: 1) The structural characterization of the RelBE2Spn protein complex and of the RelB2Spn antitoxin, 2) The functional characterization of the RelBE2Spn proteincomplex and RelB2Spn antitoxin, and 3) The searching for the role of pneumococcal RelBE2Spn and YefMYoeBSpn systems in the bacteria lifestyle. Concerning the structural characterization of the proteins, whereas the antitoxin protein alone, and the antitoxin-toxin proteins were readily purified, we could not get the purification of the toxin alone. Purified proteins samples were used to perform several biochemical and biophysical approaches. Firstly, circular dichroism experiments revealed features of the secondary structure of the RelB2Spn antitoxin and the RelBE2Spn complex, indicating that both proteins show a high average of secondary structure, although the antitoxin was partially unfolded. Secondly, analytical ultracentrifugation and native mass spectrometry were used to study theoligomerization state and the stoichiometry of the RelB2Spn and RelBE2Spn complex. The antitoxin behaved as a dimer in solution, whereas, the RelBE2Spn complex resulted to be a heterohexamer formed by the association of two heterotrimers. Each heterotrimer includes one antitoxin dimer and one toxin monomer. Finally, the crystal structure of the RelBE2Spn complex was resolved. Heterotetrameric and heterohexameric species were detected in the RelBE2Spn crystal. In these two molecular species, the toxin displays topological similarity to others toxins of the family and to the RNases. The antitoxins in heterotetramers and heterohexamers species exhibit, in their N-terminal region, a ribbon-helix-helix DNA-binding motif (RHH), similar to other RelB antitoxins. However each RelB2Spn monomer in these oligomeric structures shows a different fold. The existence of two oligomeric structures, heterotetramers and heterohexamers, might be related to transcriptional repression or to conditional cooperativity.
In the crystal structure, the pneumococcal antitoxin RelB dimerized through a RHH motif, present in several transcriptional repressors, and it is involved in the binding of the protein to its DNA target. By means of band-shift and footprinting assays, we found that the RelB2Spn antitoxin as well as the RelBE2Spn complex were able to bind to a 26-bp DNA operator sequence which spans the -10 promoter sequence, the transcription- initiation site and two inversely-repeated sequences of 3 and 6 bp. Transcriptional regulation assays indicated that RelBE2Spn regulates the expression of the relBE operon, the RelB2Spn antitoxin acting as the repressor and the RelE2Spn toxin as the co-repressor. Analytical ultracentrifugation and native mass spectrometry were performed with the DNA target and the RelBE2Spn protein complex. These techniques allowed us to determine that the two proteins bound to their DNA operator forms a heterohexamer. Considering the structural data, the hydroxyl radical protections and the hydrodynamic properties of the two proteins, a structural model is proposed to explain the interaction of the RelBE2Spn complex with its DNA operator. The RelBE2Spn heterohexamer makes contacts with the DNA-backbone within the relBE operator on the same side of the DNA helix covering two consecutive major and minor grooves. The heterohexamer could contact with the DNA through the RHH motifs of each antitoxin dimer. The third part of this Thesis deals with the role of the pneumococcal TAS RelBE2Spn and YefMYoeBSpn in the bacteria lifestyle.
. To this end, mutant strains deleted in relBE2Spn, in yefMyoeBSpn, and in both operons were constructed. The wild type and the mutant strains were tested for a number of physiological (response to stress, morphology of the cells, growth, and phase variation) parameters, and no major differences were found between them. However, differences were found in two important processes of the pneumococcal lifestyle, namely biofilm formation, and competence for transformation with chromosomal DNAs. Our results indicate that both, relBE2Spn and yefMyoeBSpn, participate directly or indirectly in genetic transformation, whereas only yefMyoeBSpn participate in biofilm formation. The main general conclusions derived from the work presented in this Thesis are: - The antitoxin RelB2Spn has a relatively high occurrence of α-helices and it is partially folded. RelB2Spn shows a relatively high heat stability being less prone to thermal aggregation than the TA complex. - The RelBE2Spn protein complex has ahighaverage of secondary structure, exhibiting a relatively high content in α-helices. - The RelB2Spn antitoxin behaves as a dimer in solution whereas the RelBE2Spn complex is a heterohexamer. - The three-dimensional structure of the RelBE2Spn protein complex has been solved by X-ray crystallography. The crystals included the two species, heterotetramers and heterohexamers. - The RelB2Spn antitoxin is the transcriptional regulator of the relBESpn operon. RelB2Spn interacts weakly with its DNA target in a dimeric form; however, RelB2Spn in complex with RelE2Spn binds more efficiently to its DNA operatorsequence. - The RelBE2Spn operator site consists of a 26 bp region that includes the -10 region of the promoter, the transcription initiation site, and two inverted repeats of 3 and 6 bp. - The RelBE2Spn complex binds as a heterohexamer to the operator sequence. Each heterohexamer is composed of two heterotrimers, and the RelBE2Spn heterotrimer contains one RelB2Spn dimer and one molecule of RelE2Spn.
- The relBE2Spn and yefMyoeBSpn operons are not essential, at least under the conditions tested. Deletion of the operons does not affect cell viability, cell morphology or phase variation. - The RelBE2Spn and YefMYoeBSpn proteins do not seem to play a role on growth regulation under acidic stress and high zinc concentration. - The relBE2Spn and yefMyoeBSpn operons could participate in the induction of the competence state for genetic transformation but only the YefMYoeBSpn system participates in biofilm formation of S. pneumoniae.
|Description:||204 p.-68 fig.-15 tab.|
|Appears in Collections:||(CIB) Tesis|
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