Escherichia coli est le commensal aérobie le plus fréquent du tube digestif de l’homme et des animaux à sang chaud et le bacille à Gram négatif pathogène opportuniste le plus souvent impliqué dans les infections intestinales et extra intestinales de l’homme. C’est une espèce clonale chez laquelle 4 groupes phylogénétiques principaux, A, B1, B2 et D ont été décrits. L’objectif de cette thèse est d’étudier l’adaptation de E. coli et les rapports de cette adaptation avec la structure génétique de l’espèce caractérisée par les 4 groupes phylogénétiques dans deux circonstances : le commensalisme intestinal de l’homme et de plusieurs espèces animales sauvages et domestiques, herbivores et omnivores d’une part et la virulence extra-intestinale mesurée par l’expression des gènes codants pour un sidérophore, la yersiniabactine, dont les gènes sont situés au sein de l’ilot de pathogénicité HPI (PAI IV). La répartition dans les 4 groupes phylogénétiques des souches commensales du tube digestif de 100 hommes et de 137 animaux a été étudiée par une technique de PCR en temps réel originale. Trois principaux entérocolitypes, correspondant à des associations préférentielles de phylogroupes ont été ainsi décrits comme plus fréquents en fonction de la nature des hôtes.Chez l’homme, les souches du groupe B2 ont été retrouvés exclusives chez 15 % des individus et ont été clairement distinctes des souches B2 des animaux sauvages par la plus grande fréquence de leurs facteurs de virulences (sfa/foc et pks). L’effet du fond génétique des sous groupes II, III et IX du groupe B2 sur l’expression de la virulence liée au HPI a été étudié dans un modèle murin de virulence extra-intestinale et dans un modèle d’amibe sociale Dictyostelium discoideum, pouvant être assimilé à un macrophage. Le HPI chez E. coli interagît avec la clonalité de l’espèce qui s’exprime par l’existence des sous-groupes de B2. Dans les modèles de virulence que nous avons développés, les mêmes gènes ont, en fonction du fond génétique des différents isolats naturels, des effets différents. / Escherichia coli is the most abundant aerobic bacteria of the human microbiota, and a major opportunistic pathogen in humans. It is the clonal species for wich main phylogenetic groups have been described. The aim of this thesis is to study E. coli adaptation through the genetic structure of the specis in two circumstances : the intestinal comensalism, and the extra-intestinal virulence estimated via expression of genes encoding for yersiniabactin, a major siderophore, located on a high patogenicity island (HPI). The repartition of the 4 phylogroups has been studied in faecal microbiota of 100 humans and 137 animals thanks to an original quantitative PCR assay. Three main enterocolitypes, corresponding to associations of phylogroups, have been described. In humans, B2 phylogroup strains were exclusive in 15% of individuals and were shown to be clearly distinct from animal B2 strains on the base of the presence of two virulence factors (sfa/foc and pks). The impact of the genetic background of the B2 sub-groups II, III an IX on the virulence based on HPI was studied in a mice model and in an amoeba model Dictyostelium discoideum. The HPI interacts with the clonality of the species represented by the existence of the B2 subgroups.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, prodigiosin and a carbapenem, and the plant cell wall degrading exoenzymes, pectate lyase and cellulase. A complex regulatory network controls production of prodigiosin, including a quorum sensing (QS) system, and the role of post-transcriptional regulation was investigated. It was hypothesized that Hfq-dependent small regulatory RNAs (sRNAs) might also play a role. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in other bacterial species. An S39006 ∆hfq mutant was constructed and in the mutants production of prodigiosin and carbapenem was abolished, while production of the QS molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, it was found that Hfq regulated the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in the ∆hfq mutant, and virulence was attenuated in potato and C. elegans. It was also shown that the phenotype of an hfq mutant is independent of its role in regulating the stationary phase sigma factor, rpoS. In order to define the complete regulon of Hfq and identify relevant potential sRNAs, deep sequencing of strand-specific cDNAs (RNA-seq) was used to analyse the whole transcriptome of S39006 WT and the ∆hfq mutant. The regulon of another post-transcriptional regulator, RsmA, also involved in regulating prodigiosin production, was investigated by performing RNA-seq on an rsmA mutant. Moreover, global changes in the proteome of the hfq mutant was analysed using an LC-MS/MS approach with isobaric tags for relative and absolute quantification (iTRAQ). This study confirms a role for Hfq in pathogenesis and the regulation of antibiotic production in S39006, and begins to provide a systems-level understanding of Hfq and RsmA regulation using a combination of transcriptomics and proteomics.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Secretion is a fundamental process of bacterial microorganisms. It is responsible for diverse functions such as cell-to-cell communication, nutritional up-take, environmental adaptation, physiological responses, and evasion of the immune system of a host. To accomplish the task of secretion, bacteria have evolved multi-protein complexes, known as secretion apparatuses, which span the bacterial membranes serving as a conduit between the interior of bacteria and the extracellular milieu. Francisella tularensis is a Gram negative bacterium capable of growth inside macrophages. Francisella tularensis causes a rare but severe disease known as tularemia. The Francisella pathogenicity island (FPI) is a circa 30-kb genetic region that harbours genes of unknown function implicated in virulence of this organism. Although many of the FPI-encoded protein products do not appear to have any known homologues, some of the FPI proteins show similarity to proteins involved in type VI secretion (T6S) of other bacteria. T6S systems are newly described bacterial virulence factors evolutionarily related to bacteriophages. We have tested the hypothesis the FPI encodes a secretion system. The FPI-encoded secretion system secretes a novel protein, IglC, into the extracellular milieu during broth growth. Systematic deletion mutagenesis determined the contribution of individual FPI genes to intramacrophage growth and secretion. We further characterized the secretion system by determining the subcellular localization of each FPI protein in the bacterial cell. An interaction between two inner membrane proteins, PdpB and DotU, was observed by co-immunoprecipitation, and the stability of PdpB requires DotU. Similarly, an interaction of IglA and IglB was demonstrated. Biochemical and fluorescence microscopy evidence suggest IglC is secreted into macrophages during intracellular localization of bacteria. Finally, a model of the FPI-encoded secretion system is presented. Our experiments provide biochemical, genetic and microscopy evidence that the FPI encodes a secretion system. The analysis of FPI-encoded secretion provides novel insights that may help us understand the role of FPI-encoded secretion in Francisella intracellular growth and virulence.

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Several genes contained in the FPI encode proteins needed for the intracellular growth and virulence of Francisella tularensis. Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. The experiments outlined in this dissertation examine the properties of PdpA protein expression and localization as well as the phenotypes of non-polar F. novicida pdpA mutants. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands. Experiments were designed to mutagenize a putative F-box domain within the amino terminus of PdpA. Deletion of amino acids 112-227 created a strain which was impaired in intracellular replication and exhibited severely reduced virulence. However, alanine mutagenesis of key conserved leucine residues required for the interaction of F-box domains with host proteins had no observed effect on bacterial growth in macrophages and did not affect virulence in chicken embryos or mice. Mono and polyubiquitinated proteins associated with both the wild type F. novicida and ΔpdpA bacterial strains early during the infection of J774A.1 macrophages. After 1 hour of infection the wild type strain developed a more intimate association with mono and polyubiquitinated proteins whereas the ΔpdpA strain did not. Inhibition of the host cell proteasome during infection did not affect the intracellular growth of wild type F. novicida. PdpA research concludes by examining the secretion patterns of F. novicida. PdpA was not detected as a surface exposed protein using biotinylation whereas IglA, IglB and IglC were found to be surface exposed in both wild type and ΔpdpA backgrounds. These observations suggest that PdpA is not involved in the assembly or function of the Francisella secretion system. FLAG tagged PdpA protein could not be detected in the TCA precipitated supernatant of broth grown cultures or in the immunoprecipitated cytosol of infected macrophages suggesting that PdpA is not a secreted protein.

This diploma thesis focused on screening for bacterial pathogens and antagonists suppressing common scab mainly caused by Streptomyces scabiei. Common scab affects some agricultural crops causing significant economical losses. Bacterial strains, mostly streptomycetes, were isolated from potato rhizosphere because they belong to most important producents of secondary metabolites and the causative agents of the disease are also members of this genus. The isolated bacteria were characterised by PCR amplification and sequencing of 16S rRNA gene to reveal their phylogenetic relationships. The ability of isolated strains to suppress growth of Streptomyces scabiei was tested by a simple co-cultivation experiment. The strains were tested by PCR for presence of specific genes for biosynthesis of thaxtomin A, a common virulence factor found in all described pathogens causing symptoms of this disease on the surface of affected tubers. Genes for synthesis of thaxtomin belong to pathogenicity island. Standard of phytotoxin thaxtomin A was used to optimize its analysis by mass spectroscopy for further in vivo and in vitro experiments. Phylogenetic analysis of strains harboring one of the genes necessary for thaxtomin A biosynthesis supported the hypothesis of sharing the pathogenicity island by horizontal gene...

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de β-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a γ-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.