RNA polymerase (RNAP) transcribes DNA into RNA and is the only transcriptional enzyme in bacteria. This key enzyme responds to external and internal signals from the cell, resolves the intensity of transcription of individual genes and thus regulates gene expression. RNAP is not only affected by its own subunits, but also protein factors, small molecules or small RNAs (sRNAs). The aim of this Thesis was to contribute to the understanding of the regulation of the RNAP and to add missing fragments to this broad topic. The first part of this Thesis is focused on the influence of selected proteins (δ, YdeB, GreA) on the sensitivity of RNAP to the concentration of the initiating nucleoside triphosphate ([iNTP]) during transcription initiation in Bacillus subtilis. We showed that δ affects the sensitivity of RNAP to [iNTP] at [iNTP]-sensitive promoters, but not at [iNTP]-insensitive promoters neither in vitro nor in vivo. The δ subunit is essential for cell survival during competition with other strains, because it enables the cell to react immediately to changing conditions. Further we showed that YdeB protein does not bind to RNAP in B. subtilis, and has not shown any effect on transcription in vitro. We found that both, GreA and YdeB proteins (unlike δ subunit) were unable to affect RNAP by [iNTP] at...

Résumé : Chez toutes les cellules, une modulation précise de l’expression des gènes est essentielle afin de réguler adéquatement leur métabolisme et de s’adapter aux changements environnementaux. En effet, c’est l’expression des gènes, plutôt que la séquence d’ADN, qui détermine en grande partie la diversité et la complexité des organismes. Celle-ci dépend principalement des changements dans les niveaux d’ARNs cellulaires résultant de la modification de l’équilibre entre leurs taux relatifs de synthèse et de dégradation. Alors que la régulation transcriptionnelle a été largement étudiée par le passé, des études récentes révèlent que la stabilité de l’ARN joue aussi un rôle important dans le modelage du transcriptome. Toutefois, les mécanismes qui assurent la dégradation précise et sélective des ARNs sont globalement mal compris. Au cours de cette thèse, j’ai utilisé la ribonucléase III de levure Saccharomyces cerevisiae (Rnt1p) comme modèle pour étudier comment des transcrits spécifiques sont ciblés pour la dégradation et évaluer sa contribution à la régulation de l’expression génique. Les résultats indiquent que Rnt1p régule l’expression des gènes en utilisant une spécificité élargie pour des structures tige-boucles d’ARN. En effet, un nouveau motif structurel de Rnt1p permet la discrimination des tige-boucles ayant une séquence spécifique tout en bloquant la liaison à des hélices génériques d’ARN double-brin. D’un autre côté, l’identification des signaux de dégradation de Rnt1p à l’échelle du transcriptome a permis de révéler plus de 384 transcrits clivés par Rnt1p, dont la majorité sont des ARN messagers. En outre, l’impact de la délétion de RNT1 sur l’expression de ces gènes est influencé par les conditions de culture des cellules, ce qui suggère que Rnt1p est un important régulateur conditionnel de l’expression génique. Somme toute, les résultats présentés dans cette thèse démontrent comment des ARNs sont spécifiquement choisis pour la dégradation et soulignent l’importance de la dégradation nucléaire dans la régulation de l’expression génique en réponse à des changements environnementaux. // Abstract : Precise modulation of gene expression is essential for any cell in order to regulate its metabolism and adapt to environmental changes. In fact, it is gene expression, rather than DNA sequence alone, which mostly explains the functional diversity and complexity between the different cell types. As such, gene expression mainly results from changes in the levels of cellular RNAs which are, in turn, dependent on the equilibrium between their relative rates of synthesis and degradation. While transcriptional control has been largely studied in the past, recent publications reveal that changes in RNA stability also play an important role in shaping the transcriptome. Unfortunately though, the mechanisms ensuring precise and selective RNA degradation remains poorly understood. In this thesis, I have used the yeast Saccharomyces cerevisiae ribonuclease III (Rnt1p) as a model to study how specific transcripts are targeted for degradation and evaluate its contribution to the regulation of gene expression. The results indicate that Rnt1p regulates gene expression using a broad specificity for structured RNA stem loops. Indeed, a new structural motif of Rnt1p permits discrimination of hairpins with specific sequence while blocking the binding of the generic RNA duplexes recognized by other members of the RNase III family. This highly specific mode of substrate recognition was found to be easily modulated by a flexible network of protein RNA interactions. On the other hand, transcriptome-wide identification of Rnt1p degradation signals uncovered more than 384 transcripts, including 291 mRNAs. Interestingly, the impact of RNT1 deletion on mRNA expression is modulated by changes in the growth conditions of the cell, indicating that Rnt1p is an important regulator of conditional gene expression. Overall, the results presented in this thesis demonstrate how specific RNAs are selected for degradation and highlight the importance of nuclear RNA decay for fine tuning gene expression in response to changes in growth conditions.

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La mucoviscidose (CF, Cystic Fibrosis), maladie autosomique récessive la plus fréquente dans la population caucasienne, se manifeste par l’atteinte de plusieurs organes due à l’absence ou au dysfonctionnement du canal CFTR. Au niveau des poumons, la perte de l’activité du canal se traduit par une déshydration du mucus, des cycles d’infections et d’inflammation aboutissant à terme à la destruction du parenchyme pulmonaire. Les thérapeutiques proposées à l’heure actuelle, sont principalement symptomatiques et l’exploration de nouvelles pistes reste nécessaire. De récents travaux émanant de notre équipe ont révélé qu’une intervention au niveau de la stabilisation des transcrits pourrait être bénéfique pour améliorer la quantité de protéines nécessaires pour restaurer une activité suffisante du canal CFTR. Une autre piste qui pourrait être explorée serait de cibler, en plus de l’expression du gène CFTR, des régulateurs clés qui participent aux processus de réparation cellulaire ou à la réponse inflammatoire. Dans cet objectif, nous nous sommes focalisés sur des régulateurs clés comme les miARN et les ARE-BP (protéines de liaison à l’ARN) qui sont dérégulés dans les tissus CF et qui peuvent agir ensemble dans le contrôle de l’expression de nombreux gènes.Les travaux que nous avons réalisés nous ont permis d’une part, de montrer la dérégulation de miARN dans des échantillons CF ainsi que des isoformes de certains miARN. Deux miARN ont retenu notre attention, le miR-101 dont la distribution de ses séquences isomiR varient dans des cultures CF et le miR-181a-5p qui présente une dérégulation de son expression dans trois modèles ex-vivo CF que nous avons utilisés. Ces miARN ont la particularité de moduler l’expression de gènes clés dans les voies de signalisation PI3K-Akt/MAPK-Erk et Wnt. D’autre part, nos travaux ont révélé la dérégulation de la protéine ARE-BP TTP (Tristétraproline ou ZFP36) en contexte CF. Cette protéine est d’autant plus intéressante qu’elle est connue pour être un régulateur clé de l’inflammation. La suite de ce travail a donc été d’étudier sa régulation et son impact sur ses ARNm cibles. L’activité de cette ARE-BP a été principalement décrite pour être contrôlée par la voie p38-MAPK. Nous montrons l’implication de la voie p42/p44-MAPK (Erk1/2) dans la régulation de la phosphorylation de TTP dans des cultures bronchiques. La recherche des interactions entre TTP et ses cibles a également révélé que cette protéine agissait sur ses propres régulateurs ainsi que sur l’ARNm CFTR. Nous montrons ainsi pour la première fois que TTP se fixe au niveau de la région 3’UTR du transcrit CFTR et joue un rôle de stabilisateur sur ce transcrit.Identifier de nouveaux acteurs de la régulation du gène CFTR mais également des régulateurs centraux des processus altérés en contexte CF offre de nouvelles opportunités pour concevoir des outils ciblés pour combattre cette maladie. / Cystic fibrosis (CF), the most common life-shortening genetic disorder in Caucasians, affects many organs but chronic lung disease is the main cause of morbidity and mortality. This disease, characterized by CFTR gene alterations, results in ions transport dysfunctions that contribute to impair mucociliary clearance, favoring bacterial colonization and inflammation, and ultimately leading to lung destruction. Nowadays, therapeutic drugs have limited benefit, so development of new alternatives or complementary molecules remains essential. Recently, our team demonstrated that the intervention on CFTR mRNA stability leads to an increase in CFTR protein level and an improvement of the CFTR channel activity. An alternative way would be to target key actors such as miRNA and ARE-BP (RNA binding protein), deregulated in CF tissues, that display a pleiotropic activity and act together to control expression of several genes.Our findings led to the identification of dysregulated miRNA in CF samples and revealed multiple isoforms relative to miRNA of reference (i.e. isomiRs). We focused on two miRNA, miR-101, that displays a modification in isomiR distribution and miR-181a-5p that is highly deregulated, in three CF airways models. These miRNA modulate expression of key genes or related genes in PI3K-Akt/MAPK-Erk and Wnt signaling pathways.Our work also revealed the deregulation of an ARE-BP protein, TTP (Tristetraprolin, ZFP36), in CF context. This protein is a master regulator in inflammatory resolution. We next investigated the mechanism whereby this ARE-BP is regulated in bronchial cells and showed that TTP phosphorylation is regulated by MK2 through ERK activation. We also determined for the first time that TTP binds to the 3’UTR of CFTR mRNA where TTP binding stabilizes CFTR mRNA level.These data bring new insights into CF physiopathology and open new research opportunities in CF.

Em bactérias, os fatores sigma alternativos permitem a rápida adaptação da célula a alterações no ambiente. Dentre estes, os fatores sigma ECF (função extra-itoplasmática) caracterizam-se pelo envolvimento na resposta a sinais da região extra-citoplasmática da célula. O sequenciamento do genoma de Caulobacter crescentus indicou a presença de 13 ORFs codificando fatores ECF. Este trabalho descreve a caracterização de cepas mutantes em cinco genes que codificam fatores sigma ECF de C. crescentus, sendo eles sigL, sigM, sigN, sigU e sigF. A regulação da expressão destes genes em respostas a diferentes estresses foi também analisada, pelo uso de fusões de transcrição de suas regiões promotoras ao gene reporter lacZ. Todos os mutantes mostraram-se viáveis, sendo também tão resistentes quanto a cepa parental a uma série de estresses ambientais testados, indicando que estes genes não são essenciais. Verificou-se, porém, que os mutantes nos genes sigL e sigM são mais sensíveis ao choque térmico extremo (48°C). A caracterização da cepa mutante em sigF mostrou que este gene é essencial para a resistência da célula a estresse oxidativo durante a fase estacionária. A análise da expressão de sigF indicou um controle pós-transcricional, com o acúmulo da proteína SigF durante esta fase do crescimento, sem um aumento na transcrição do gene. Oito genes regulados por &#963;F durante a fase estacionária foram identificados em experimentos de \"microarray\", incluindo os genes de resposta a estresse oxidativo, sodA e msrA. A análise da atividade do promotor de sigU mostrou sua indução na entrada na fase estacionária e após estresse salino e osmótico. No entanto, o mutante nulo em sigU não se apresentou mais sensível que a cepa parental a esses estresses. Os resultados aqui descritos permitiram identificar a importância de alguns dos fatores ECF de C. crescentus na resposta a estresses nesta bactéria. / Alternative sigma factors permit the rapid adaptation to environmental changes in bacteria. Among them, members of the ECF subfamily (extrac</i<ytoplasmic function) are characterized by their involvement in responses to changes in the extracytoplasmic compartment of the cell. Analysis of the complete genome sequence of Caulobacter crescentus has led to the identification of 13 ORFs encoding putative sigma factors of the ECF subclass. The present work describes the characterization of mutant strains in five genes encoding ECF sigma factors from C. crescentus, named sigL, sigM, sigN, sigU and sigF. The expression of these genes in response to distinct stress conditions was also investigated, using transcriptional fusions of their promoter regions to the lacZ reporter gene. The five mutants strains obtained were viable and did not show increased sensitivity, when compared to the parental strain, to a series of environmental stress conditions, indicating that these genes are not essential. However, the sigL and sigM mutant strains were shown to be more sensitive to extreme heat shock (48°C). Furthermore, the characterization of the sigF mutant strain demonstrated that this gene is essential for oxidative stress survival during stationary phase. Analysis of sigF expression indicated a post-transcriptional control, with an increase in the levels of SigF protein during this growth phase, without changes in the transcription rate of the gene. Eight genes regulated by &#963;F during stationary phase were identified in microarray experiments, including the oxidative stress response genes sodA and msrA. Analysis of sigU promoter activity in response to distinct stress conditions showed induction upon entry into stationary phase and during saline and osmotic stress. Nevertheless, the sigU null mutant did not show increased sensitivity to these stresses. The results described here identified the importance of some of the C. crescentus ECF sigma factors in the response to stresses in this bacterium.

P. aeruginosa PA14 é uma linhagem isolada de queimadura que apresenta vários fatores de patogenicidade comuns no quadro de infecção de hospedeiros filogeneticamente distintos (plantas, mamíferos ou invertebrados). O gene kerV foi revelado numa busca por mutantes atenuados em virulência em uma biblioteca de mutantes por transposons da linhagem PA14 (Rahme et al., 1997). A caracterização da linhagem D12, mutante em kerV, confirmou sua virulência atenuada (Apidianakis et al., 2005 e An et al., 2009) e resultados do transcriptoma mostraram alteração na expressão de mais de 500 genes, sendo alguns relacionados com o sistema de \"quorum sensing\" (Rahme et al, dados não publicados). O gene kerV está próximo à montante ao gene gloB, envolvido em detoxificação de metilglioxal, e à jusante aos genes rnhA e dnaQ, que codificam proteínas envolvidas na replicação e reparo do DNA. Este trabalho teve como objetivo estudar a função molecular do produto de kerV e a expressão dos genes do lócus kerV-rnhA-dnaQ. Análises de bioinformática indicam que a proteína KerV é uma metiltransferase dependente de S-adenosil-metionina (SAM), apresentando um domínio conservado de ligação a SAM e uma arquitetura de domínio compatível com a organização em fitas-beta e hélices-alfa alternadas descritas para a família das metiltransferases dependentes de SAM. Ela não apresenta outros domínios conservados que indiquem seu substrato de metilação. A expressão heteróloga desta proteína em E. coli, mostrou que ela é expressa de maneira parcialmente solúvel quando co-expressa com as chaperoninas GroEL/GroES em baixas temperaturas ou quando fusionada a MBP ou GST. A purificação desta proteína mostra que ela é co-eluída com a chaperonina GroEL sugerindo que para atingir sua conformação nativa ela necessita dessas proteínas acessórias. MBP-KerV purificado foi usado para ensaios \"in vitro\" de atividade de metiltransferase e ligação a SAM, que não foram conclusivos, pois não há certeza do seu correto estado de enovelamento. Ensaios de duplo-híbrido mostraram que KerV não interage com os produtos de rnhA e dnaQ, sugerindo que KerV não está diretamente relacionado com suas funções. A freqüência de mutação na linhagem D12 está levemente aumentada (aproximadamente quatro vezes), o que sugere que KerV não está diretamente envolvida no reparo de DNA do tipo ´mismatch repair`. Os ensaios usados para detectar metilação do DNA, proteínas e rRNAs não revelaram que KerV estaria envolvido com a metilação destes substratos. Os inícios de transcrição dos genes kerV, rnhA e dnaQ foram determinados. A deleção de kerV causa um efeito polar na transcrição do gene rnhA, que não se reflete nos níveis da proteína. A deleção também afeta a expressão de dnaQ, sugerindo que KerV seja importante para sua regulação. Os ensaios de complementação da virulência em modelos invertebrados e de células epiteliais de pulmão mostram que apenas a presença dos três genes e seus produtos em níveis normais são capazes de reverter a maioria dos fenótipos atenuados. KerV se mostrou essencial para a inibição da translocação de NF-kB para o núcleo das células, comprovando que esta proteína é relevante para a virulência de PA14, contribuindo com o silenciamento da resposta imune do hospedeiro. O conjunto dos resultados indicam uma complexa inter-relação entre a expressão dos genes kerV, rnhA e dnaQ e seu papel na biologia de P. aeruginosa. / P. aeruginosa PA14 is a burn isolate multi-host pathogen strain. The screening for virulence attenuated mutants in a PA14 transposon mutant library revealed the kerV gene (Rahme et al., 1997). The characterization of D12 strain, a kerV mutant, confirmed the attenuated virulence phenotype (Apidianakis et al., 2005 and An et al., 2009) and transcriptome analysis showed the expression of more than 500 genes are affected in D12, some of these genes are related with quorum sensing (Rahme et al, unpublished data). kerV is upstream of the gloB gene, related with methylglioxal detoxification and downstream of the rnhA and dnaQ genes, both related with DNA replication and repair. The purpose of this work was to study the molecular function of KerV product and the expression of kerV-rnhA-dnaQ locus. Bioinformatics analysis indicated that KerV is a SAM dependent methyltransferase that have a conserved SAM binding domain with architecture compatible with classic alternating &#946;-stranded and &#945;-helical regions. KerV does not show any other conserved motif that could indicate its methylation substrate. Heterologous expression in E. coli showed that KerV is partially soluble only when co-expressed with GroeL/GroES chaperones at low temperatures or when KerV is in fusion with MBP or GST tag. During the purification process KerV was copurified with GroEL chaperone suggesting that this association may be required for the correct folding of KerV. Methyltransferase activity and SAM binding assays were done with purified MBPKerV and the results were not conclusive since the proper conformation of MBP-KerV cannot be verified. Yeast two-hybrid assays indicated that RNaseH and DnaQ are not interaction partners of KerV, suggesting that their functions are not directly related. The mutation frequency of D12 strain increased only about four times in relation to PA14, suggesting that KerV is not directly involved with DNA mismatch repair. The assays to detect methylation in DNA, RNAs and proteins do not show that KerV is involved with methylation of these substrates. The transcription start sites of kerV, rnhA and dnaQ genes were mapped through 5\'-RACE- and primer extension experiments. The kerV deletion causes a polar effect on the transcription of rnhA gene, which is not reflected on RNaseH protein levels. The kerV deletion also affects dnaQ expression, suggesting that KerV is important for its regulation.The virulence complementation assays in flies and lung epithelial cells showed that the fully rescue of the wild type phenotype was achieved only when the entire locus is present. KerV was essential to inhibit the NF-kB nucleus translocation, demonstrating that KerV is relevant to PA14 virulence, contributing for the silencing of host immune system. Altogether, these data showed a complex inter-relation among kerV, rnhA and dnaQ genes and its role in P. aeruginosa biology

L’ostréiculture française repose essentiellement sur l’élevage de l’huître creuse, Crassostrea gigas confronté cependant à des épisodes de mortalités anormales, touchant les différents stades de vie de l'huître. Plusieurs études ont démontré l’implication d’agents infectieux comme des bactéries du genre Vibrio dans ces mortalités. En France, V. aestuarianus est une bactérie connue depuis les années 2000 pour impacter la survie des huîtres. Sa fréquence de détection dans les cas de mortalités d’huîtres adultes analysés par le réseau REPAMO (REseau de PAthologie des Mollusques) est cependant en augmentation depuis 2011. Dans ce contexte, afin d’étudier le développement de la maladie induite par V. aestuarianus chez C. gigas, un modèle d’expérimentation par balnéation dans de l’eau de mer contenant des bactéries fraichement excrétées, au plus proche des modes de contaminations naturelles, a été développé. Le suivi de la présence de la souche 12/016 (souche virulente) et son mutant 12/016ΔvarS (souche non-virulente) dans l’eau de mer, dans les différents tissus et dans l’hémolymphe des animaux vivants et moribonds a montré que le cycle infectieux est constitué de I) une phase de pénétration rapide de la bactérie dans l’hôte (moins de 24h) et de colonisation initiale de l’hémolymphe et des branchies, II) une phase d’incubation de 3-4 jours au cours de laquelle la souche virulente se multiplie dans l'ensemble des tissus d'huître et III) une phase de mortalités aiguës (mort de l'animal par septicémie). A ce stade, le recrutement et la lyse hémocytaire ainsi que différentes lésions tissulaires comme la lyse du tissu conjonctif sous-épithélial au niveau du manteau et l’atrophie de diverticules digestives ont été observés. D'autre part, l'étude d’expression relative de 18 gènes de virulence connus chez d’autres Vibrion a montré que l’expression des facteurs de virulence de V. aestuarianus est régulée différemment au cours de différentes étapes de l'infection et nous avons observé que la métalloprotéase vam est significativement sur-exprimée dans l’hémolymphe des animaux contaminés à j4 post infection (étape intermédiaire de l’infection) par rapport à son niveau d’expression au premier jour de l’infection (étape précoce). / Oyster-farming in France is mainly based on pacific cupped oysters, Crassostrea gigas culture. Currently, oyster culture is confronted by several abnormal episodes of mass mortality affecting all life stages. These outbeaks involve, among other factors, infectious agents including bacteria of the genus Vibrio. In France, since 2000, V. aestuarianus is known as a bacterium that impacts the survival of C. gigas. Since 2011, its detection frequency in adult oyster mortalities cases reported by REPAMO network (REseau de PAthologie des Mollusques), is constantly increasing. In this context, to study V. aestuarianus disease development in C. gigas, an experimental infection model based on immersion in sea water containing freshly shed bacteria was developed. By monitoring the presence of strain 12/016 (virulent strain) and its mutant 12/016 ΔvarS (non-virulent strain) in the seawater, in the different tissues and in the haemolymph of live and moribund animals, we showed that the infectious cycle consists of several successive phases: I) rapid penetration of the bacterium into the host (less than 24 hours) and initial colonization of the haemolymph and gills, II) 3-4 days of incubation during which the virulent strain multiplies in whole oyster tissues and III) acute mortalities (animal death due to septicemia). At this stage, recruitment and haemocyte lysis as well as different tissue lesions such as lysis of the sub-epithelial connective tissue in the mantle and atrophy of digestive diverticula were observed. On the other hand, relative expression of 18 virulence genes (known in other Vibrion) were analyzed by RT-QPCR. Virulence factors are regulated differently during different stages of infection and vam metalloprotease is significantly over-expressed in the haemolymph of infected animals at day 4 post infection (intermediate stage of infection) compared to its level of expression at day 1 post infection (early stage).

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Em bactérias, os fatores sigma alternativos permitem a rápida adaptação da célula a alterações no ambiente. Dentre estes, os fatores sigma ECF (função extra-itoplasmática) caracterizam-se pelo envolvimento na resposta a sinais da região extra-citoplasmática da célula. O sequenciamento do genoma de Caulobacter crescentus indicou a presença de 13 ORFs codificando fatores ECF. Este trabalho descreve a caracterização de cepas mutantes em cinco genes que codificam fatores sigma ECF de C. crescentus, sendo eles sigL, sigM, sigN, sigU e sigF. A regulação da expressão destes genes em respostas a diferentes estresses foi também analisada, pelo uso de fusões de transcrição de suas regiões promotoras ao gene reporter lacZ. Todos os mutantes mostraram-se viáveis, sendo também tão resistentes quanto a cepa parental a uma série de estresses ambientais testados, indicando que estes genes não são essenciais. Verificou-se, porém, que os mutantes nos genes sigL e sigM são mais sensíveis ao choque térmico extremo (48°C). A caracterização da cepa mutante em sigF mostrou que este gene é essencial para a resistência da célula a estresse oxidativo durante a fase estacionária. A análise da expressão de sigF indicou um controle pós-transcricional, com o acúmulo da proteína SigF durante esta fase do crescimento, sem um aumento na transcrição do gene. Oito genes regulados por &#963;F durante a fase estacionária foram identificados em experimentos de \"microarray\", incluindo os genes de resposta a estresse oxidativo, sodA e msrA. A análise da atividade do promotor de sigU mostrou sua indução na entrada na fase estacionária e após estresse salino e osmótico. No entanto, o mutante nulo em sigU não se apresentou mais sensível que a cepa parental a esses estresses. Os resultados aqui descritos permitiram identificar a importância de alguns dos fatores ECF de C. crescentus na resposta a estresses nesta bactéria. / Alternative sigma factors permit the rapid adaptation to environmental changes in bacteria. Among them, members of the ECF subfamily (extrac</i<ytoplasmic function) are characterized by their involvement in responses to changes in the extracytoplasmic compartment of the cell. Analysis of the complete genome sequence of Caulobacter crescentus has led to the identification of 13 ORFs encoding putative sigma factors of the ECF subclass. The present work describes the characterization of mutant strains in five genes encoding ECF sigma factors from C. crescentus, named sigL, sigM, sigN, sigU and sigF. The expression of these genes in response to distinct stress conditions was also investigated, using transcriptional fusions of their promoter regions to the lacZ reporter gene. The five mutants strains obtained were viable and did not show increased sensitivity, when compared to the parental strain, to a series of environmental stress conditions, indicating that these genes are not essential. However, the sigL and sigM mutant strains were shown to be more sensitive to extreme heat shock (48°C). Furthermore, the characterization of the sigF mutant strain demonstrated that this gene is essential for oxidative stress survival during stationary phase. Analysis of sigF expression indicated a post-transcriptional control, with an increase in the levels of SigF protein during this growth phase, without changes in the transcription rate of the gene. Eight genes regulated by &#963;F during stationary phase were identified in microarray experiments, including the oxidative stress response genes sodA and msrA. Analysis of sigU promoter activity in response to distinct stress conditions showed induction upon entry into stationary phase and during saline and osmotic stress. Nevertheless, the sigU null mutant did not show increased sensitivity to these stresses. The results described here identified the importance of some of the C. crescentus ECF sigma factors in the response to stresses in this bacterium.