496 research outputs found

    In silico identification of a multi-functional regulatory protein involved in Holliday junction resolution in bacteria

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    BACKGROUND: Homologous recombination is a fundamental cellular process that is most widely used by cells to rearrange genes and accurately repair DNA double-strand breaks. It may result in the formation of a critical intermediate named Holliday junction, which is a four-way DNA junction and needs to be resolved to allow chromosome segregation. Different Holliday junction resolution systems and enzymes have been characterized from all three domains of life. In bacteria, the RuvABC complex is the most important resolution system. RESULTS: In this study, we conducted comparative genomics studies to identify a novel DNA-binding protein, YebC, which may serve as a key transcriptional regulator that mainly regulates the gene expression of RuvABC resolvasome in bacteria. On the other hand, the presence of YebC orthologs in some organisms lacking RuvC implied that it might participate in other biological processes. Further phylogenetic analysis of YebC protein sequences revealed two functionally different subtypes: YebC_I and YebC_II. Distribution of YebC_I is much wider than YebC_II. Only YebC_I proteins may play an important role in regulating RuvABC gene expression in bacteria. Investigation of YebC-like proteins in eukaryotes suggested that they may have originated from YebC_II proteins and evolved a new function as a specific translational activator in mitochondria. Finally, additional phylum-specific genes associated with Holliday junction resolution were predicted. CONCLUSIONS: Overall, our data provide new insights into the basic mechanism of Holliday junction resolution and homologous recombination in bacteria

    Interactions of insertion sequences targetting integron associated recombination sites

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    Bacteria of genus Pseudomonas are known for crossing ecological barriers and establishing in clinical settings. Their ability to withstand antibiotic selection in the clinical environment is largely due to interactions between different mobile genetic elements (MGEs) and the presence of multidrug resistance integrons (MRIs). It is difficult to predict resistance gene maintenance within a bacterial population, as the source of these genes is unknown, and the biological processes governing their flow is difficult to quantify in vivo. This study explores the IS1111-attC subgroup of insertion sequences as a model for this process in Pseudomonads. In this study, IS1111-attC elements were found to be overrepresented within non-clinical Pseudomonas isolates relative to clinical Pseudomonads, as well as an enteric outgroup. The observed IS1111-attC distribution suggests that all instances of IS1111-attC elements in class 1 integrons represent recent invasions of attC sites occurring when class 1 integrons were present in the same cells as chromosomal integrons. Target site preferences and transposition mechanisms of the IS1111-attC elements distribution patterns were investigated using in vitro and in vivo models. These elements were shown to specifically recognize the attC recombination sites of integrons in binding assays and to specifically target the attC in mobility assays. Factors affecting the rate of movement between environmental and clinical Pseudomonads were also examined. Significantly, the IS1111-attC transposase binds preferentially to the single strand forms of the top strand of the attC site, rather than the bottom strand attC site which is the target of the integron integrase. This is the first evidence for IS1111-attC mobility in Pseudomonas cells occurring via a similar mechanism to integron gene mobilization, illustrating a way for these elements both to move between chromosomal and plasmid borne integrons, and to facilitate interactions between them

    DNA Repair Mechanisms as Drug Targets in Prokaryotes

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    Nowadays, a great amount of pathogenic bacteria has been identified such as Mycobacterium sp. and Helicobacter pylori and have become a serious health problem around the world. These bacteria have developed several DNA repair mechanisms as a strategy to neutralize the effect of the exposure to endogenous and exogenous agents that will lead to two different kinds of DNA damage: single strand breaks (SSBs) and double strand breaks (DSBs). For SSBs repair, bacteria use the base excision repair (BER) and nucleotide excision repair (NER) mechanisms, which fix the damaged strand replacing the damaged base or nucleotide. DSBs repair in bacteria is performed by homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). HRR uses the homologous sequence to fix the two damaged strand, while NHEJ repair does not require the use of its homologous sequence. The use of unspecific antibiotics to treat bacterial infections has caused a great deal of multiple resistant strains making less effective the current therapies with antibiotics. In this review, we emphasized the mechanisms mentioned above to identify molecular targets that can be used to develop novel and more efficient drugs in future.Nowadays, a great amount of pathogenic bacteria has been identified such as Mycobacterium sp. and Helicobacter pylori and have become a serious health problem around the world. These bacteria have developed several DNA repair mechanisms as a strategy to neutralize the effect of the exposure to endogenous and exogenous agents that will lead to two different kinds of DNA damage: single strand breaks (SSBs) and double strand breaks (DSBs). For SSBs repair, bacteria use the base excision repair (BER) and nucleotide excision repair (NER) mechanisms, which fix the damaged strand replacing the damaged base or nucleotide. DSBs repair in bacteria is performed by homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). HRR uses the homologous sequence to fix the two damaged strand, while NHEJ repair does not require the use of its homologous sequence. The use of unspecific antibiotics to treat bacterial infections has caused a great deal of multiple resistant strains making less effective the current therapies with antibiotics. In this review, we emphasized the mechanisms mentioned above to identify molecular targets that can be used to develop novel and more efficient drugs in future

    Binding studies of the FOXP2 forkhead domain and its cognate DNA sequences

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    A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2015.FOXP2 is the gene product of the so-called “language gene” and is the only protein known to be involved in a monogenetic autosomally inherited language disorder. This disorder has been termed Speech-Language Disorder 1. In addition to the role it plays in language, FOXP2 is thought to be involved in cancer, autism and schizophrenia. FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the forkhead domain. The aim of this work was to investigate the binding mechanism of the FOXP2 forkhead domain and various DNA sequences in order to assess affinity and specificity. It was shown by surface plasmon resonance that the FOXP2 forkhead domain can recognise a variety of DNA sequences, including a novel sequence, identified by systematic evolution of ligands by exponential enrichment. This motif has not previously been reported as a binding motif of the FOXP2 forkhead domain. Kinetic analysis by surface plasmon resonance showed that the novel sequence, as well as other published cognate sequences, each binds to the FOXP2 forkhead domain with different rates and affinities. Molecular docking of the DNA sequences to the FOXP2 forkhead domain revealed that electrostatic interactions between positively charged amino acids and the DNA backbone, as well as basespecific interactions between His554 and the DNA appear to be key in determining rates and affinities of binding interactions of the FOXP2 forkhead domain and DNA. Based on these findings, three types of DNA-binding are proposed for the FOXP2 forkhead domain. These types are: low affinity, nonfunctional binding; moderate affinity, non-functional binding and high affinity, functional binding. It is probable that each type of binding serves to control the vii spatial location of the protein within the nucleus, as well as the local concentration of protein. The proposed mechanism of binding for the forkhead domain of FOXP2 may have a future impact on the binding and function of full length FOXP2

    Etudes phylogénomiques et moléculaires des hélicases de type Lhr chez les Archaea et les bactéries

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    Les hĂ©licases, classĂ©es en six superfamilles (SF1-6), sont des protĂ©ines qui utilisent l'Ă©nergie de l'ATP pour dĂ©rouler les acides nuclĂ©iques et pour remodeler les complexes protĂ©ines-acides nuclĂ©iques. Elles sont impliquĂ©es dans presque tous les aspects du mĂ©tabolisme de l'ADN et de l'ARN en participant Ă  de nombreux mĂ©canismes de maintien de l'intĂ©gritĂ© cellulaire. Les protĂ©ines de type Lhr sont des hĂ©licases SF2 qui sont pour la plupart non caractĂ©risĂ©es. RĂ©cemment, par des approches phylogĂ©nomiques, Dr H. Chamieh et ses collaborateurs ont classĂ© toutes les hĂ©licases SF1 et SF2 prĂ©sentes dans les gĂ©nomes d'Archaea et ont montrĂ© que les protĂ©ines de type Lhr sont ubiquitaires (Chamieh et al. 2016). De plus, en dĂ©terminant les rĂ©seaux d'interaction des protĂ©ines impliquĂ©es dans le mĂ©tabolisme de l'ARN, comme la ribonuclĂ©ase aRNase J et l'hĂ©licase ASH-Ski2, Dr B. Clouet-d'Orval et ses collaborateurs ont identifiĂ© un lien entre les machines de traduction, de dĂ©gradation de l'ARN et de transcription chez les Thermococcales -archaea hyperthermophile- avec, au sein de ces rĂ©seaux, une protĂ©ine annotĂ©e comme une hĂ©licase de type Lhr (Phung et al. 2020). Dans ce contexte, les travaux de ma thĂšse ont pour objectif d'effectuer des analyses phylogĂ©nomiques approfondies des hĂ©licases de type Lhr chez les archĂ©es et les bactĂ©ries, de dissĂ©quer la fonction molĂ©culaire de aLhr2 chez Thermococcus barophilus, organisme modĂšle pour les Ă©tudes biochimiques et gĂ©nĂ©tiques chez les Thermococcales, et d'Ă©tudier le rĂŽle de l'hĂ©licase Lhr de E. coli oĂč le gĂšne lhr est en opĂ©ron avec le gĂšne codant pour la RNase T. Dans une premiĂšre partie, une Ă©tude bibliographique (publication d'un chapitre de livre ; Hajj et al, 2019) et des analyses phylogĂ©nomiques ont permis de dĂ©finir les protĂ©ines de type Lhr comme ubiquitaire chez les archĂ©es et d'identifier cinq groupes d'orthologues. Ces analyses permettent de proposer un chemin Ă©volutif pour les protĂ©ines Lhr d'archĂ©es et de bactĂ©ries et d'Ă©mettre des hypothĂšses sur leurs fonctions dans la cellule (Hajj et al, Manuscrit en prĂ©paration). Dans une deuxiĂšme partie, nous nous sommes focalisĂ©s sur l'Ă©tude molĂ©culaire de aLhr2 de Thermococcus barophilus (Tbar). Pour Ă©tudier les activitĂ©s enzymatiques de Tbar-aLhr2, le gĂšne alhr2 a Ă©tĂ© clonĂ© et la protĂ©ine recombinante Tbar-aLhr2 exprimĂ©e Ă  l'aide du systĂšme d'expression pET chez E. coli. Nous avons dĂ©montrĂ© que Tbar-aLhr2 est une ATPase avec une affinitĂ© similaire pour l'ADN et l'ARN simple brin qui peut spĂ©cifiquement former et dĂ©rouler des duplex ADN/ARN avec une extrĂ©mitĂ© 3' sortante. Enfin, nous avons effectuĂ© des analyses protĂ©omiques et transcriptomiques pour identifier le rĂ©seau de protĂ©ines associĂ©es Ă  Tbar-aLhr2 et pour dĂ©terminer l'impact de la dĂ©lĂ©tion du gĂšne alhr2 (Δalhr2) sur l'expression gĂ©nique chez T. barophilus. Au regard de ces rĂ©sultats, nous proposons que Tbar-aLhr2 est impliquĂ© au niveau de la transcription et/ou de la rĂ©paration de l'ADN chez les Thermococcales en agissant au niveau des "R-loop" (duplex ARN/ADN) (Hajj et al, Manuscrit en prĂ©paration). Dans une troisiĂšme partie, nous avons initiĂ© une Ă©tude fonctionnelle de l'hĂ©licase bLhr de E. coli (Eco). Pour tester l'interaction entre Eco-bLhr et la RNase T qui sont exprimĂ©es au sein d'un mĂȘme opĂ©ron, le gĂšne Eco-blhr a Ă©tĂ© clonĂ© et la protĂ©ine recombinante Eco-bLhr exprimĂ©e. La RNase T est une ribonuclĂ©ase connue pour ĂȘtre impliquĂ©e dans la rĂ©paration de l'ADN et le mĂ©tabolisme des ARNt et ARNr. Finalement, une discussion permet de comparer le(s) rĂŽle(s) proposĂ©(s) pour les hĂ©licases de type Lhr chez les bactĂ©ries et les archĂ©es en dĂ©gageant l'ARN comme acteur clĂ© dans la rĂ©paration des dommages de l'ADN.Helicases are proteins that use ATP energy to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of the DNA and RNA metabolism and participate in numerous repair mechanisms maintaining cellular integrity. Helicases are classified into six superfamilies (SF1-6). The Lhr-type proteins belong to SF2 helicases that are poorly characterized to date. A phylogenomic study performed by Chamieh et al classified SF1 and SF2 helicases from archaeal sequenced genomes and showed that Lhr-type proteins are ubiquitous in Archaea (Chamieh et al. 2016). Another study conducted by Clouet D'orval et al, determined the interaction networks of proteins involved in RNA metabolism, such as the ribonuclease aRNase J and the helicase ASH-Ski2, they identified a cross-talk between the translation, RNA degradation and transcription machineries in Thermococcales a group of -hyperthermophilic Archaea- and remarkably Lhr-type helicase was found to be a partner in these networks (Phung et al. 2020). In this context, my PhD thesis aim to perform in-depth phylogenomic analyses of Lhr-type helicases in Archaea and to extend this work further to bacteria, to dissect the molecular function of aLhr2 in Thermococcus barophilus, a model organism used for biochemical and genetic studies in Thermococcales. Further, we aim to investigate the role of the bacterial Lhr (bLhr) helicase in E.coli where the gene is co-transcribed with the RNase T in proteobacteria. The first part of our work was initiated with a bibliographic survey (published book chapter; Hajj et al, 2019)/ followed by phylogenomic studies on the Lhr-type proteins. We were able to define the Lhr-type proteins as ubiquitous enzyme in Archaea and identify five orthologous groups. Based on these analyses, we proposed an evolution route for the five archaeal and bacterial Lhr groups and hypothesize on their functions in the cell (Hajj et al, Manuscript in preparation). In a second part, we focused on the molecular study of aLhr2 from Thermococcus barophilus (Tbar). To investigate the enzymatic activities of Tbar-aLhr2, alhr2 gene was cloned and the recombinant protein recombinant Tbar-aLhr2 protein produced using the pET expression system in E. coli. We demonstrated that Tbar-aLhr2 is an ATPase that has the same affinity for single stranded DNA and RNA and can specifically anneal and unwind DNA/RNA duplex with a 3' overhang. Finally, proteomic and transcriptomic analyses were performed to identify Tbar-aLhr2 protein network and to determine the effect of lhr2 deletion (Δlhr2) on gene expressions in T. barophilus. In light of all these results, we propose that Tbar-aLhr2 is involved in transcription or/and DNA repair in Thermococcales and acts on R-loops (RNA/DNA duplex) (Hajj et al, Manuscript in preparation). In the third part of the work, we initiated a functional study of the bLhr helicase of E. coli (Eco). To examine a putative interaction between Eco-bLhr and RNase T that are expressed as an operon, the Eco-blhr gene was cloned and the recombinant Eco-bLhr protein produced. RNase T is a ribonuclease known to be involved in DNA repair and tRNA/rRNA metabolism. Finally, we discussed and compared the putative role(s) of Lhr helicases in Bacteria and Archaea in RNA metabolism and DNA repair in eliciting RNA as a key player in the repair of DNA damage

    Mitokondriaalne DNA hargnemisi mobiliseeriv ensĂŒĂŒm Irc3

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    VĂ€itekirja elektrooniline versioon ei sisalda publikatsiooneMitokonder (mt) on raku organell, mida tihtipeale seostatakse oksĂŒdatiivse fosforĂŒleerimisega ehk energia tootmisega ATP kujul. Mitokondris on oma genoom, mis kodeerib mĂ”ningaid valgulisi komponente oksĂŒdatiivse fosforĂŒĂŒlimise lĂ€biviimiseks. SeetĂ”ttu on mitokondri genoom hĂ€davajalik enamike eukarĂŒootsete organismide ellujÀÀmiseks. Kuna mt genoom ei kodeeri kĂ”iki mitokondri funktsioneerimiseks vajalikke valke, siis on enamik mitokondriaalseid valke tuuma pĂ€ritolu. Kuidas mitokondriaalset DNAd sĂ€ilitatakse ja millised valgud selles osalevad pole lĂ”plikult teada. Seda protsessi aitab paremini mĂ”ista uute mtDNA sĂ€ilitamises osalevate valguliste faktorite vĂ€ljaselgitamine. PagaripĂ€rm S. cerevisiae on eukarĂŒootne mudelorganism, mille eripĂ€raks on vĂ”ime eksisteerida ilma mtDNA-ta. See teeb antud mudelorganismi mugavaks mudelobjektiks mtDNA sĂ€ilitamisega seotud protsesside uurimisel. Antud töö eesmĂ€rk oli uurida valgu Irc3 bioloogilist rolli pagaripĂ€rims. Senini oli teada, et see valk sisaldab konserveerunud helikaasseid motiive ning tundmatu funktsiooniga C-terminaalset domeeni. Lisaks arvati, et Irc3 omab funktsiooni mitokondris. Leidsime, et Irc3 on tĂ”epoolest mitokondriaalne valk, mis on vajalik mtDNA sĂ€ilitamiseks. Lisaks nĂ€itasid biokeemilised katsed, et Irc3 on spetsiifiline DNA helikaas, mis töötab hargnenuid DNA struktuuridega nagu replikatioonikahvlid ja Holliday ĂŒhendused. Uurides valku Irc3 lĂ€hemalt selgus, et ensĂŒĂŒmi C-terminaalne domeen tagab valgu spetsifilisuse nimetatud struktuuride suhtes ja on vajalik enamuste ensĂŒĂŒmi aktiivsuste sĂ€ilimiseks. Minu doktoritöö tulemusel paigutub Irc3 spetsiifiliste helikaaside hulka. Need valgud funktsioneerivad homoloogilise rekombinatsiooni valdkonnas. Arvatavasti omab Irc3 pĂ€rmi mitokondris mitu funktsiooni olles nii spetsiifilise mtDNA reparatsiooni raja, kĂ€ivitaja, homoloogilise rekombinatsiooni katalĂŒĂŒsija kui ka rekombinatsiooni kontrollija. Rekombinatsioon on sage protsess pĂ€rmi mitokondris ja hĂ€davajalik mtDNA sĂ€ilitamiseks. Antud töö nĂ€itas veenvalt, et helikaasse aktiivsusega valk, mis töötab hargnenud DNA molekulide peal, on vajalik pĂ€rmi mitokondriaalse sĂ€ilitamise DNA jaoks.Mitochondria are cellular organelles required for production of energy via oxidative phosphorylation. Mitochondria contain their own reduced genome that encodes some protein components of the respiratory chain complexes. Therefore, the mitochondrial genome is essential for survival of the most eukaryotic organisms. The process of mitochondrial DNA maintenance is largely uncharacterized. The vast majority of mitochondrial proteins have nuclear origin and the total number of proteins implicated in mitochondrial DNA maintenance remains unknown. BakerÂŽs yeast S. cerevisiae is eukaryotic model organism that can survive without mitochondrial DNA and by thus is very suitable for studying processes involved in mitochondrial DNA maintenance. The aim of this work was to understand the biological role of Irc3 in S. cerevisiae. In silico analysis predicted that Irc3 is a helicase as its sequence contained conserved helicase motifs. However, the protein also contained a large C-terminal domain of unknown function. Irc3 gained our attention as the protein was predicted to be targeted to mitochondria. Our analysis demonstrated that Irc3 is indeed a mitochondrial protein that is required for stable mitochondrial DNA maintenance. Our biochemical experiments defined Irc3 as specific DNA helicase that can remodel branched DNA structures such as replication forks and Holliday junctions. The analysis of Irc3 mutants demonstrated that the C-terminal domain of the protein is required for the binding of branched DNA structures and is indispensable for almost any enzymatic function. The results of my studies place Irc3 into the group of specific helicases functioning in homologous recombination. Most likely, Irc3 is a multifunctional protein in yeast mitochondria. The protein could promote the specific DNA repair pathway, catalyze homologous recombination and have a role in recombination surveillance. Recombination is a frequent event in yeast mitochondria important for the maintenance of mitochondrial DNA. This work has convincingly shown that a DNA helicase involved in the processing of branched DNA molecules is required for yeast mitochondrial DNA maintenance

    Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

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    Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed

    Expanding the role of FurA as essential global regulator in cyanobacteria

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    In the nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA plays a global regulatory role. Failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we developed a selectively regulated furA expression system by the replacement of furA promoter in the Anabaena sp. chromosomes with the Co2+/Zn2+ inducible coaT promoter from Synechocystis sp. PCC 6803. By removing Co2+ and Zn2+ from the medium and shutting off furA expression, we showed that FurA was absolutely required for cyanobacterial growth. RNA-seq based comparative transcriptome analyses of the furA-turning off strain and its parental wild-type in conjunction with subsequent electrophoretic mobility shift assays and semi-quantitative RT-PCR were carried out in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approaches led us to identify 15 novel direct iron-dependent transcriptional targets belonging to different functional categories including detoxification and defences against oxidative stress, phycobilisome degradation, chlorophyll catabolism and programmed cell death, light sensing and response, heterocyst differentiation, exopolysaccharide biosynthesis, among others. Our analyses evidence novel interactions in the complex regulatory network orchestrated by FurA in cyanobacteria

    Reconstruction of Transcription Control Networks in Mollicutes by High-Throughput Identification of Promoters

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    Bacteria of the class Mollicutes have significantly reduced genomes and gene expression control systems. They are also efficient pathogens that can colonize a broad range of hosts including plants and animals. Despite their simplicity, Mollicutes demonstrate complex transcriptional responses to various conditions, which contradicts their reduction in gene expression regulation mechanisms. We analyzed the conservation and distribution of transcription regulators across the 50 Mollicutes species. The majority of the transcription factors regulate transport and metabolism, and there are four transcription factors that demonstrate significant conservation across the analyzed bacteria. These factors include repressors of chaperone HrcA, cell cycle regulator MraZ and two regulators with unclear function from the WhiA and YebC/PmpR families. We then used three representative species of the major clades of Mollicutes (Acholeplasma laidlawii, Spiroplasma melliferum and Mycoplasma gallisepticum) to perform promoters mapping and activity quantitation. We revealed that Mollicutes evolved towards a promoter architecture simplification that correlates with a diminishing role of transcription regulation and an increase in transcriptional noise. Using the identified operons structure and a comparative genomics approach, we reconstructed the transcription control networks for these three species. The organization of the networks reflects the adaptation of bacteria to specific conditions and hosts
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