RESUMO
Polyamines bind to various cellular components, such as nucleic acids, phospholipids, proteins and nucleotides. They are involved in the virulence and protection against physiological stresses of several bacterial species. Streptococcus agalactiae is able to colonize the vaginal tract of asymptomatic pregnant women and to resist, by an as yet poorly characterized mechanism, pH 4.0, the low physiological pH of this environment. We identified a transporter of the amino acid/polyamine antiporter family (SAK_1604 in strain A909) that shares 39.8â% similar amino acids with CadB and 34.7â% with PotE, two transporters implicated in acid resistance in Escherichia coli. We found that sak_1604 is overexpressed in the presence of spermidine and during citric acid stress at the vaginal pH, but not during lactic acid or HCl stresses at the same pH or during a sodium citrate stress at pH 7.4. Dihydrogen citrate is the predominant form of citric acid at pH 4.0. Using a deletion mutant, we proved that SAK_1604 is involved in the survival of S. agalactiae during citric acid stress at pH 4.0 in the presence of spermidine, and we showed by TLC analysis that it is involved in spermidine transport in these conditions. Our data open new perspectives on the comprehension of the molecular mechanisms allowing S. agalactiae to survive at the physiological pH of the vagina and on the unsuspected role of an ionic form of citric acid.
Assuntos
Antiporters , Espermidina , Aminoácidos/metabolismo , Antiporters/genética , Antiporters/metabolismo , Ácido Cítrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Gravidez , Espermidina/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismoRESUMO
Polyamines constitute a group of organic polycations positively charged at physiological pH. They are involved in a large variety of biological processes, including the protection against physiological stress. In this study, we show that the genome of Streptococcus agalactiae, a commensal bacterium of the intestine and the vagina and one of the most common agents responsible of neonate infections, does not encode proteins homologous to the specific enzymes involved in the known polyamine synthetic pathways. This lack of biosynthetic capability was verified experimentally by TLC analysis of the intracellular content of S. agalactiae grown in the absence of polyamines. However, similar analyses showed that the polyamines spermidine, spermine and putrescine can be imported from the growth media into the bacteria. We found that all strains of S. agalactiae possess the genes encoding the polyamine ABC transporter PotABCD. We demonstrated that these genes form an operon with folK, a gene involved in folate biosynthesis, murB, a gene involved in peptidoglycan biosynthesis, and with clc, a gene encoding a Cl-/H+ antiporter involved in resistance to acid stress in Escherichia coli. Transcription of the potABCD operon is induced by peroxide-induced oxidative stress but not by acidic stress. Spermidine and spermine were found to be inducers of potABCD transcription at pH 7.4 whereas putrescine induces this expression only during peroxide-induced oxidative stress. Using a deletion mutant of potABCD, we were nevertheless unable to associate phenotypic traits to the PotABCD transporter, probably due to the existence of one or more as yet identified transporters with a redundant action.
Assuntos
Poliaminas , Streptococcus agalactiae , Transporte Biológico , Humanos , Recém-Nascido , Proteínas de Membrana Transportadoras/genética , Poliaminas/metabolismo , Putrescina/metabolismo , Espermidina/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismoRESUMO
Competence for natural transformation is a widespread developmental process of streptococci. By allowing the uptake and recombination of exogenous naked DNA into the genome, natural transformation, as transposable elements, plays a key role in the plasticity of bacterial genomes. We previously analysed the insertion sites of IS1548, an insertion sequence present in Streptococcus agalactiae and S. pyogenes, and showed that some targeted loci are involved in competence induction. In this work, we investigated on a large scale if loci coding for early competence factors (ComX and the two pheromone-dependent signalling systems ComCDE and ComRS) of streptococci are especially targeted by transposable elements. The transposable elements inserted in regions surrounding these genes and housekeeping genes used for Multilocus Sequence Typing (MLST) were systematically searched for. We found numerous insertion events in the close vicinity of early competence genes, but only very few into the MLST loci. The incidence of transposable elements, mainly insertion sequences, is particularly high in the intergenic regions surrounding comX alleles in numerous species belonging to most streptococcal groups. The identification of scarce disruptive insertions inside early competence genes indicates that the maintenance of competence is essential for streptococci. The specific association of transposable elements with intergenic regions bordering the main regulatory genes of competence may impact on the induction of transformability and so, on the genome plasticity and adaptive evolution of streptococci. This widespread phenomenon brings new perspectives on our understanding of competence regulation and its role in the bacterial life cycle.
Assuntos
Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Genoma Bacteriano/genética , Streptococcus/genética , Sítios de Ligação/genética , DNA Intergênico/genética , Regulação Bacteriana da Expressão Gênica , Tipagem de Sequências Multilocus , Mutagênese Insercional , Filogenia , Especificidade da Espécie , Streptococcus/classificaçãoRESUMO
The fru2 metabolic operon of Streptococcus agalactiae encodes the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) enzyme II complex Fru2 (EIIBFru2 , EIIAFru2 , and EIICFru2 ); Fru2 R, a transcriptional activator with PTS regulatory domains (PRDs); a d-allulose-6-phosphate 3-epimerase; a transaldolase; and a transketolase. We showed that the transcription of fru2 is induced during the stationary phase of growth in complex media and during incubation in human cerebrospinal or amniotic fluids. d-allose and d-ribose are environmental signals governing this induction. PTSFru2 is involved in the activation of the fru2 promoter, and the histidine-67 of EIIAFru2 and the cysteine-9 of EIIBFru2 are important for this function. The activation of fru2 is also controlled by Fru2 R. The histidine-243 in the PRD1 domain, the histidine-323 in the PRD2 domain, the cysteine-400 in the EIIB-like domain, and the histidine-549 in the EIIA-like domain are important for the function of Fru2 R. Fru2 R binds to a DNA region containing palindromic sequences upstream of the identified transcriptional start site. EIIBFru2 interacts physically with the C-terminal part of Fru2 R (expressing the EIIB-like and EIIA-like motifs) and with EIIAFru2 . We propose a model of regulation of fru2 depending on the presence of an activatory carbohydrate in the growth medium.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores/genética , Glucose/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Ribose/metabolismo , Streptococcus agalactiae , Líquido Amniótico/metabolismo , Líquido Cefalorraquidiano/metabolismo , Meios de Cultura/metabolismo , Ilhas Genômicas/genética , Humanos , Óperon/genética , Regiões Promotoras Genéticas/genética , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/metabolismo , Ativação Transcricional/genéticaRESUMO
We have referenced and described Streptococcus agalactiae transposable elements encoding DDE transposases. These elements belonged to nine families of insertion sequences (ISs) and to a family of conjugative transposons (TnGBSs). An overview of the physiological impact of the insertion of all these elements is provided. DDE-transposable elements affect S. agalactiae in a number of aspects of its capability to adapt to various environments and modulate the expression of several virulence genes, the scpB-lmB genomic region and the genes involved in capsule expression and haemolysin transport being the targets of several different mobile elements. The referenced mobile elements modify S. agalactiae behaviour by transferring new gene(s) to its genome, by modifying the expression of neighbouring genes at the integration site or by promoting genomic rearrangements. Transposition of some of these elements occurs in vivo, suggesting that by dynamically regulating some adaptation and/or virulence genes, they improve the ability of S. agalactiae to reach different niches within its host and ensure the 'success' of the infectious process.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis/genética , Genoma Bacteriano/genética , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/fisiologia , Proteínas de Bactérias/metabolismo , Humanos , Família Multigênica , Streptococcus agalactiae/enzimologia , Streptococcus agalactiae/genética , Transposases/genética , Transposases/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
IS1548, a 1316-bp element of the ISAs1 family affects the expression of several genes of the opportunistic pathogen Streptococcus agalactiae. Furthermore, certain lineages of S. agalactiae are more frequently associated to particular diseases than other [1, 2]. We took advantage of the release of the genome sequences of a huge number of epidemiologically unrelated S. agalactiae strains of various origin to analyze the prevalence of IS1548 among S. agalactiae strains. To this end, S. agalactiae genome available at the National Center for Biotechnology Information (NCBI) database were blasted with IS1548 DNA sequences. A sequence type (ST), based on the allelic profile of seven housekeeping genes, was assigned to each strain possessing IS1548. These strains were then grouped into clonal complexes (CCs). The data obtained will give the opportunity to compare the sequenced genomes of S. agalactiae based on their lineage and/or possession of IS1548, and to select the corresponding strains for comparative experimental studies. The data is related to the research article « Dual and divergent transcriptional impact of IS1548 insertion upstream of the peptidoglycan biosynthesis murB gene of Streptococcus agalactiae" [2].
RESUMO
Prebiotics such as fructooligosaccharides (FOS) are increasingly being used in some countries for improving human and animal health and as an alternative to antibiotic growth promoters in animals, with various degrees of success. It has been observed that FOS stimulate the proliferation of probiotic bacteria and, at the same time, decrease the population of bacteria associated with disease. This observation assumes that pathogenic bacteria do not metabolize FOS and, therefore, lose their competitive advantage over beneficial bacteria. Here we present evidence that some pathogenic Escherichia coli strains can metabolize FOS and show that this property helps the bacterium colonize the intestine. These findings highlight the potential risk that a high level of prebiotic usage could lead to the emergence of well-adapted pathogenic strains that metabolize prebiotic substances.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Frutose/metabolismo , Trato Gastrointestinal/microbiologia , Genoma Bacteriano , Intestinos/microbiologia , Oligossacarídeos/metabolismo , Animais , Mapeamento Cromossômico , Escherichia coli/metabolismo , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/genética , Genes fos , Glicosídeos/metabolismo , Humanos , Oligossacarídeos/uso terapêutico , Plasmídeos , Infecções Urinárias/microbiologiaRESUMO
We identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenic Escherichia coli (ExPEC) strains. The frz operon codes for three subunits of a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) transporter of the fructose subfamily, for a transcriptional activator of PTSs of the MgA family, for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase (repressor, open reading frame, kinase family [ROK]), and for a protein of the cupin superfamily. We proved that the frz operon promotes bacterial fitness under stressful conditions, such as oxygen restriction, late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed that frz is involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of the fim operon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PTS activator and the metabolic enzymes encoded by the frz operon are involved in these phenotypes.
Assuntos
Escherichia coli/genética , Escherichia coli/patogenicidade , Óperon/fisiologia , Virulência/genética , Animais , Linhagem Celular , Galinhas , Cricetinae , Infecções por Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Óperon/genética , Estresse Oxidativo/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.
Assuntos
Proteínas de Bactérias/genética , DNA Intergênico , Regulação Bacteriana da Expressão Gênica , Sequências Repetitivas Dispersas , Mutagênese Insercional , Peptidoglicano/biossíntese , Streptococcus agalactiae/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/metabolismoRESUMO
IbeA in extraintestinal pathogenic Escherichia coli (ExPEC) strains was previously described for its role in invasion. Here we investigated the role of IbeA and IbeT, encoded by a gene located downstream of ibeA, in the adhesion of the avian ExPEC strain BEN2908 to human brain microvascular endothelial cells (HBMEC). The DeltaibeA mutant was less adhesive to HBMEC than the wild-type strain BEN2908 was. Because strain BEN2908 also expresses type 1 fimbriae, we measured the adhesion specifically due to IbeA by comparing the adhesive properties of a Deltafim derivative of strain BEN2908 to those of a double Deltafim DeltaibeA mutant. No differences were observed, indicating that the reduction of adhesion in BEN2908 DeltaibeA could be due to a decrease in type 1 fimbria expression. We indeed showed that the decreased adhesion of BEN2908 DeltaibeA was correlated with a decrease in type 1 fimbria expression. Accordingly, more bacteria had a fim promoter orientated in the off position in a culture of BEN2908 DeltaibeA than in a culture of BEN2908. Expression of fimB and fimE, two genes encoding recombinases participating in controlling the orientation of the fim promoter, was decreased in BEN2908 DeltaibeA. A reduction of type 1 fimbria expression due to a preferential orientation of the fim promoter in the off position was also seen in an ibeT mutant of strain BEN2908. We finally suggest a role for IbeA and IbeT in modulating the expression of type 1 fimbriae through an as yet unknown mechanism.
Assuntos
Aderência Bacteriana , Proteínas de Escherichia coli/fisiologia , Escherichia coli/patogenicidade , Fímbrias Bacterianas/metabolismo , Proteínas de Membrana/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Fatores de Virulência/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Células Endoteliais/microbiologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Deleção de Genes , Humanos , Integrases/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Trocadores de Sódio-Hidrogênio/biossíntese , Trocadores de Sódio-Hidrogênio/genética , Fatores de Virulência/genéticaRESUMO
Extraintestinal pathogenic Escherichia coli strains are responsible for a number of infections in humans and animals. Several ExPEC virulence genes have already been described such as iutA involved in iron acquisition and ibeA required for invasion of eukaryotic cells. In this study we used the chicken model to study the expression of iutA and ibeA by two ExPEC strains during growth of bacteria in LB medium and during the infection. Expression of iutA and ibeA were shown to be higher in stationary phase than in exponential phase in vitro. During infection, iutA expression was increased at least 50-fold in the airsac and in the lung 3, 6 and 24h. p.i. compared to in vitro grown bacteria. Expression of ibeA was increased 2.5-9-fold in the airsac in the early stages of the infection only. This is the first report analyzing quantitatively the expression of ExPEC virulence genes during the course of the infection. The model described could be useful to study the expression of other ExPEC virulence genes.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Fatores de Virulência/biossíntese , Sacos Aéreos/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/biossíntese , Galinhas , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/biossíntese , Pulmão/microbiologia , Proteínas de Membrana/biossíntese , Regulação para CimaRESUMO
We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos/genética , Ilhas Genômicas/genética , Proteínas de Transporte de Monossacarídeos/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Streptococcus agalactiae/genética , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Humanos , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase Multiplex , Óperon/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although human herpesvirus (HHV)-6 is now recognized as a frequent pathogen after transplantation, the real impact of this infection in patients undergoing transplantation remains unclear. METHODS: During 27 months, 30 consecutive heart-lung- and lung-transplant recipients were included on the day of transplantation and prospectively followed during 100 days for HHV-6 infection. RESULTS: HHV-6 infection occurred in 20 (66%) patients after a median delay of 18 days after transplantation. The virus was detected by polymerase chain reaction or culture, or both, in 15.7 % of blood specimens, in 14.5% of bronchoalveolar lavage fluids, and in many organs at postmortem examination; it was found by culture in eight patients. No clinical manifestations could clearly be associated with HHV-6 alone. However, patients with HHV-6 infection had a higher mortality rate than patients without HHV-6 infection (7 of 20 vs. 0 of 10; P=0.04), and all the deceased patients died during periods of HHV-6 infection. We did not observe higher incidence of infectious or graft-rejection episodes in HHV-6-positive patients. However, eight of nine viral or fungal infections occurred during HHV-6 infection and three were directly responsible for death. CONCLUSION: Although frequently detected after transplantation, HHV-6 was not associated with any specific clinical manifestation. The higher mortality rate observed in patients with HHV-6 infection was not related to a higher incidence of bacterial infections or graft rejection but might be associated with more viral and fungal infections.
Assuntos
Transplante de Coração/efeitos adversos , Transplante de Coração-Pulmão/efeitos adversos , Herpesvirus Humano 6 , Complicações Pós-Operatórias/virologia , Infecções por Roseolovirus/epidemiologia , Adulto , Causas de Morte , DNA Viral/sangue , Quimioterapia Combinada , Feminino , Transplante de Coração/mortalidade , Transplante de Coração-Pulmão/mortalidade , Herpesvirus Humano 6/isolamento & purificação , Humanos , Imunossupressores/uso terapêutico , Incidência , Estudos Longitudinais , Masculino , Reação em Cadeia da Polimerase , Complicações Pós-Operatórias/mortalidade , Estudos Prospectivos , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/mortalidade , Taxa de Sobrevida , Fatores de TempoRESUMO
Listeria monocytogenes is a gram-positive, non-sporulating food-borne pathogen of man and animals that is able to invade many eukaryotic cells. L. monocytogenes possesses several proteins that bind fibronectin. In this study, an L. monocytogenes DNA library in pUC19 was screened with fibronectin and a gene encoding a 24.6-kDa fibronectin-binding protein (Fbp) was isolated and sequenced. Transcripts of the fbp gene were found in wild-type, in deltaprfA, and PrfA-S183A strains, despite the presence of a 'PrfA-like' box around its ribosome-binding site. The fbp gene was found to be present in all tested isolates of the species L. monocytogenes and a homologous DNA fragment was amplified in L. welshimeri. No homologies between the fbp gene and its translation product with any other DNA or proteins deposited in databanks were found. Restriction endonuclease-PCR (RE-PCR) showed that the fbp gene displays a degree of allelic variation among isolates of L. monocytogenes, whereas the corresponding amplified fragment of L. welshimeri seems to be monomorphic among isolates of this species. RE-PCR with Hha I, Dde I or Taq I produced DNA banding profiles specific for each of these two species, allowing their identification.
Assuntos
Adesinas Bacterianas , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Fibronectinas/metabolismo , Listeria monocytogenes/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Western Blotting , Proteínas de Transporte/química , Clonagem Molecular , Primers do DNA/química , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Fibronectinas/química , Humanos , Listeria monocytogenes/química , Listeriose/microbiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
We developed a PCR method with outward insertion sequence-specific and -unrelated primers to identify IS1548 targets in the genome of unsequenced Streptococcus agalactiae strains. Our rapid and easy method allowed the identification of previously known but also of yet unnoticed integration sites in the three clinical isolates tested.
Assuntos
Primers do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genética Microbiana/métodos , Reação em Cadeia da Polimerase/métodos , Streptococcus agalactiae/genética , DNA Bacteriano/química , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
The prevalence of the insertion sequence IS1548 is strongly linked to clonal complex 19 Streptococcus agalactiae strains associated with neonatal meningitis and endocarditis. We previously reported that IS1548 insertion upstream of lmb is involved in stronger binding of a S. agalactiae meningitic strain to laminin. A few other IS1548 insertion sites were also identified by others. In this study, we analyzed IS1548 described target sites in S. agalactiae and showed that most of them are linked to zinc-responsive genes. Moreover, we identified two not yet described IS1548 insertion sites in the adcRCB operon encoding the main regulator of zinc homeostasis and subunits of a zinc ABC transporter. We also identified two conserved motifs of 8 and 10 bp close to IS1548 insertion sites. These motifs representing potential IS1548 targets were found upstream of several S. agalactiae ORFs. One of these predicted IS1548 targets was validated experimentally, allowing the identification of an IS1548 insertion site upstream of murB in all of the clonal complex 19 strains tested. The possible effects of these insertions on the virulence of the strains are discussed.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Streptococcus agalactiae/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Humanos , Recém-Nascido , Dados de Sequência Molecular , Gravidez , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/patogenicidade , VirulênciaRESUMO
The yicJI operon of the common genetic backbone of Escherichia coli codes an α-xylosidase and a transporter of the galactosides--pentoses--hexuronides:cation symporter family. In the extraintestinal pathogenic E. coli strain BEN2908, a metabolic operon (frz) of seven genes is found downstream of the yicI gene. It was proved that frz promotes bacterial fitness under stressful conditions. During this work, we identified a motif containing a palindromic sequence in the promoter region of both the frz and the yicJI operons. We then showed that these two operons are cotranscribed, suggesting a functional relationship. The phenotypes of frz and yicJI deletion mutants were compared. Our results showed that although the yicJI operon is not essential for the life of E. coli, it is necessary for its fitness under all the growth conditions tested.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , Óperon , Sequência de Bases , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Regiões Promotoras GenéticasRESUMO
Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a wide range of diseases in humans and animals. Using in vitro invasion assays and transmission electron microscopy, we showed that BEN2908, an ExPEC strain of avian origin (also termed APEC for Avian Pathogenic E. coli), is able to usurp cellular endocytic pathways to invade A549 human type II pneumocytes and LMH avian hepatocytes where it is able to survive over several days. Although type 1 fimbriae are the major adhesin of BEN2908, proportions of adherent fimbriated or afimbriated bacteria that entered cells were comparable. Internalization of BEN2908 into human pneumocytes reinforces previous studies indicating that APEC strains could represent a zoonotic risk.
Assuntos
Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Viabilidade Microbiana , Células Epiteliais Alveolares/microbiologia , Animais , Antibacterianos/farmacologia , Aves , Linhagem Celular , Clorpromazina/farmacologia , Citocalasina D/farmacologia , Endocitose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Filipina/farmacologia , Fímbrias Bacterianas/metabolismo , Hepatócitos/microbiologia , Humanos , Microscopia Eletrônica de Transmissão , Nistatina/farmacologia , Fatores de TempoRESUMO
The complete nucleotide sequence and genetic organization of a new genomic island (AGI-3) isolated from the extraintestinal avian pathogenic Escherichia coli strain BEN2908 is reported. This 49,600-bp island is inserted at the selC locus and contains putative mobile genetic elements such as a phage-related integrase gene, transposase genes, and direct repeats. AGI-3 shows a mosaic structure of five modules. Some of these modules are present in other E. coli strains and in other pathogenic bacterial species. The gene cluster aec-35 to aec-37 of module 1 encodes proteins associated with carbohydrates assimilation such as a major facilitator superfamily transporter (Aec-36), a glycosidase (Aec-37), and a putative transcriptional regulator of the LacI family (Aec-35). The aec-35 to aec-37 cluster was found in 11.6% of the tested pathogenic and nonpathogenic E. coli strains. When present, the aec-35 to aec-37 cluster is strongly associated with the selC locus (97%). Deletion of the aec-35-aec-37 region affects the assimilation of seven carbohydrates, decreases the growth rate of the strain in minimal medium containing galacturonate or trehalose, and attenuates the virulence of E. coli BEN2908 for chickens.