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1.
Microbiology (Reading) ; 168(12)2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36748557

RESUMEN

Rhizobium adhering proteins or 'Raps' are secreted proteins identified in a very restricted group of rhizobial strains, specifically those belonging to R. leguminosarum and R. etli. The distinctive feature of members of the Rap family is the presence of one or two cadherin-like domains or CHDLs that are also present in numerous extracellular bacterial and archaeal proteins and were proposed to confer carbohydrate binding ability. We have previously made an in-depth characterization of RapA2, a calcium-binding lectin, composed by two CHDLs, involved in biofilm matrix remodelling in R. leguminosarum bv. viciae 3841. In this study, CHDLs derived from RapA2 were analysed in detail, finding significant structural and functional differences despite their considerable sequence similarity. Only the carboxy-terminal CHDL retained properties similar to those displayed by RapA2. Our findings were used to obtain a novel fluorescent probe to study biofilm matrix development by confocal laser scanning microscopy, and also to shed some light on the role of the ubiquitous CHDL domains in bacterial secreted proteins.


Asunto(s)
Rhizobium leguminosarum , Rhizobium , Rhizobium/metabolismo , Cadherinas/metabolismo , Proteínas Fluorescentes Verdes , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Proteínas Bacterianas/metabolismo
2.
Nucleic Acids Res ; 45(10): 5757-5769, 2017 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-28334833

RESUMEN

LuxR-type transcription factors control diverse physiological functions necessary for bacterial adaptation to environmental changes. In the intracellular pathogen Brucella, the LuxR homolog VjbR has been shown to regulate the expression of virulence factors acting at early stages of the intracellular infection and, directly or indirectly, hundreds of additional genes. However, the precise determination of VjbR direct targets has so far proved elusive. Here, we performed chromatin immunoprecipitation of VjbR followed by next-generation sequencing (ChIP-seq). We detected a large amount of VjbR-binding sites distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif, an unusual feature among LuxR-type regulators. RNA-seq analysis performed under conditions mimicking the eukaryotic intracellular environment revealed that, among all loci associated to VjbR-binding, this regulator directly modulated the expression of only a subset of genes encoding functions consistent with an intracellular adaptation strategy for survival during the initial stages of the host cell infection. Other VjbR-binding events, however, showed to be dissociated from transcription and may require different environmental signals to produce a transcriptional output. Taken together, our results bring new insights into the extent and functionality of LuxR-type-related transcriptional networks.


Asunto(s)
Proteínas Bacterianas/genética , Brucella abortus/genética , Brucella abortus/patogenicidad , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Represoras/genética , Transactivadores/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Brucella abortus/metabolismo , Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Motivos de Nucleótidos , Unión Proteica , Percepción de Quorum/genética , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Virulencia
3.
Molecules ; 24(12)2019 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-31234313

RESUMEN

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.


Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Oligonucleótidos/química , Etilenos/química
4.
Mol Plant Microbe Interact ; 31(10): 1075-1082, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30136892

RESUMEN

Bacterial surface molecules are crucial for the establishment of a successful rhizobia-legume symbiosis, and, in most bacteria, are also critical for adherence properties, surface colonization, and as a barrier for defense. Rhizobial mutants defective in the production of exopolysaccharides (EPSs), lipopolysaccharides (LPSs), or capsular polysaccharides are usually affected in symbiosis with their plant hosts. In the present study, we evaluated the role of the combined effects of LPS and EPS II in cell-to-cell and cell-to-surface interactions in Sinorhizobium meliloti by studying planktonic cell autoaggregation, biofilm formation, and symbiosis with the host plant Medicago sativa. The lpsB mutant, which has a defective core portion of LPS, exhibited a reduction in biofilm formation on abiotic surfaces as well as altered biofilm architecture compared with the wild-type Rm8530 strain. Atomic force microscopy and confocal laser microscopy revealed an increase in polar cell-to-cell interactions in the lpsB mutant, which might account for the biofilm deficiency. However, a certain level of biofilm development was observed in the lpsB strain compared with the EPS II-defective mutant strains. Autoaggregation experiments carried out with LPS and EPS mutant strains showed that both polysaccharides have an impact on the cell-to-cell adhesive interactions of planktonic bacteria. Although the lpsB mutation and the loss of EPS II production strongly stimulated early attachment to alfalfa roots, the number of nodules induced in M. sativa was not increased. Taken together, this work demonstrates that S. meliloti interactions with biotic and abiotic surfaces depend on the interplay between LPS and EPS II.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/fisiología , Manosiltransferasas/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/genética , Manosiltransferasas/genética , Mutación
5.
Mol Microbiol ; 103(3): 553-565, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27862467

RESUMEN

Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host.


Asunto(s)
Brucella abortus/genética , Brucella abortus/metabolismo , Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases/genética , Sitios de Unión/genética , Brucella abortus/fisiología , Biología Computacional/métodos , Matriz Extracelular/microbiología , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Sistemas de Secreción Tipo V/metabolismo , Virulencia/fisiología
6.
Int J Syst Evol Microbiol ; 68(1): 14-20, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29095137

RESUMEN

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.


Asunto(s)
Complejo Burkholderia cepacia/clasificación , Fibrosis Quística/microbiología , Filogenia , Microbiología del Suelo , Agricultura , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Humanos , Tipificación de Secuencias Multilocus , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Esputo
7.
Environ Microbiol ; 18(10): 3522-3534, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27198923

RESUMEN

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.


Asunto(s)
Inoculantes Agrícolas/fisiología , Productos Agrícolas/microbiología , Fijación del Nitrógeno , Nitrógeno/metabolismo , Pseudomonas/fisiología , Inoculantes Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Pseudomonas/genética , Triticum/crecimiento & desarrollo , Triticum/metabolismo , Triticum/microbiología , Zea mays/crecimiento & desarrollo , Zea mays/metabolismo , Zea mays/microbiología
8.
Appl Environ Microbiol ; 81(3): 1013-23, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25416773

RESUMEN

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.


Asunto(s)
Biopelículas/crecimiento & desarrollo , Lipopolisacáridos/metabolismo , Antígenos O/metabolismo , Raíces de Plantas/microbiología , Rhizobium leguminosarum/fisiología , Lipopolisacáridos/genética , Datos de Secuencia Molecular , Antígenos O/genética , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/crecimiento & desarrollo , Rhizobium leguminosarum/metabolismo , Análisis de Secuencia de ADN
9.
Proc Natl Acad Sci U S A ; 109(30): 12135-40, 2012 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-22773814

RESUMEN

Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum.


Asunto(s)
Adhesión Bacteriana/fisiología , Luz , Fotorreceptores Microbianos/metabolismo , Pisum sativum/microbiología , Nodulación de la Raíz de la Planta/fisiología , Rhizobium leguminosarum/fisiología , Simbiosis , Secuencia de Aminoácidos , Adhesión Bacteriana/efectos de la radiación , Secuencia de Bases , Biopelículas/crecimiento & desarrollo , Western Blotting , Flagelos/metabolismo , Violeta de Genciana , Histidina Quinasa , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Nodulación de la Raíz de la Planta/efectos de la radiación , Polisacáridos Bacterianos/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Rhizobium leguminosarum/efectos de la radiación , Rhizobium leguminosarum/ultraestructura , Alineación de Secuencia , Análisis de Secuencia de ADN , Estadísticas no Paramétricas
10.
J Biol Chem ; 288(4): 2893-904, 2013 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-23235153

RESUMEN

In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like ß-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins.


Asunto(s)
Proteínas Bacterianas/química , Cadherinas/química , Proteínas de Unión al Calcio/metabolismo , Lectinas/química , Lectinas/metabolismo , Polisacáridos/metabolismo , Rhizobium leguminosarum/metabolismo , Secuencia de Aminoácidos , Calcio/química , Proteínas de Unión al Calcio/química , Calorimetría/métodos , Datos de Secuencia Molecular , Polisacáridos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Solventes/química
11.
Int J Syst Evol Microbiol ; 64(Pt 6): 2003-2008, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24623656

RESUMEN

Two Gram-negative, rod-shaped bacteria were isolated from agricultural soils in Córdoba province in central Argentina. Their 16S rRNA gene sequences demonstrated that they belong to the genus Burkholderia, with Burkholderia zhejiangensis as most closely related formally named species; this relationship was confirmed through comparative gyrB sequence analysis. Whole-cell fatty acid analysis supported their assignment to the genus Burkholderia. Burkholderia sp. strain YI23, for which a whole-genome sequence is available, represents the same taxon, as demonstrated by its highly similar 16S rRNA (100% similarity) and gyrB (99.1-99.7%) gene sequences. The results of DNA-DNA hybridization experiments and physiological and biochemical characterization further substantiated the genotypic and phenotypic distinctiveness of the Argentinian soil isolates, for which the name Burkholderia cordobensis sp. nov. is proposed, with strain MMP81(T) ( = LMG 27620(T) = CCUG 64368(T)) as the type strain.


Asunto(s)
Burkholderia/clasificación , Filogenia , Microbiología del Suelo , Agricultura , Argentina , Técnicas de Tipificación Bacteriana , Composición de Base , Burkholderia/genética , Burkholderia/aislamiento & purificación , Girasa de ADN/genética , ADN Bacteriano/genética , Ácidos Grasos/química , Genotipo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
12.
J Bacteriol ; 195(20): 4611-9, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23935051

RESUMEN

Rhizobia are symbiotic bacteria able to invade and colonize the roots of legume plants, inducing the formation of nodules, where bacteria reduce atmospheric nitrogen (N2) to ammonia (NH3). Riboflavin availability influences the capacity of rhizobia to survive in the rhizosphere and to colonize roots. In this study, we identified the RL1692 gene of Rhizobium leguminosarum downstream of a flavin mononucleotide (FMN) riboswitch. RL1692 encodes a putative transmembrane permease with two EamA domains. The presence of an FMN riboswitch regulating a transmembrane protein is usually observed in riboflavin transporters, suggesting that RL1692 may be involved in riboflavin uptake. The product of RL1692, which we named RibN, is conserved in members of the alpha-, beta-, and gammaproteobacteria and shares no significant identity with any riboflavin transporter previously identified. In this work, we show that RibN is localized in the membrane cellular fraction and its expression is downregulated by riboflavin. By heterologous expression in a Brucella abortus mutant auxotrophic for riboflavin, we demonstrate that RibN possesses flavin transport activity. Similarly, we also demonstrate that RibN orthologues from Ochrobactrum anthropi and Vibrio cholerae (which lacks the FMN riboswitch) are able to transport riboflavin. An R. leguminosarum ribN null mutant exhibited lower nodule occupancy levels in pea plants during symbiosis assays. Thus, we propose that RibN and its homologues belong to a novel family of riboflavin transporters. This work provides the first experimental description of riboflavin transporters in Gram-negative bacteria.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Rhizobium leguminosarum/metabolismo , Riboflavina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Filogenia , Rhizobium leguminosarum/genética
13.
Infect Immun ; 81(3): 996-1007, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23319562

RESUMEN

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Brucella suis/metabolismo , Brucella suis/patogenicidad , Brucelosis/microbiología , Proteínas Portadoras/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Adhesinas Bacterianas/genética , Animales , Anticuerpos Antibacterianos , Adhesión Bacteriana/fisiología , Brucella suis/genética , Proteínas Portadoras/genética , Polaridad Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Ratones , Ratones Endogámicos BALB C , Familia de Multigenes , Virulencia
14.
Antimicrob Agents Chemother ; 57(6): 2467-75, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23478955

RESUMEN

We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxA- and oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6')-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6')-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6')-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ß-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6')-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ß-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/efectos de los fármacos , Plásmidos/genética , Quinolonas/farmacología , Argentina , Proteínas Bacterianas/metabolismo , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Humanos , Integrones/genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADN
15.
Cell Microbiol ; 14(6): 965-82, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22321605

RESUMEN

Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.


Asunto(s)
Adhesinas Bacterianas/fisiología , Adhesión Bacteriana , Brucella suis/fisiología , Interacciones Huésped-Patógeno , Proteínas de Transporte de Membrana/fisiología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Brucella suis/crecimiento & desarrollo , Brucella suis/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Proteínas Inmovilizadas/química , Macrófagos/microbiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Viabilidad Microbiana , Biblioteca de Péptidos , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN
16.
J Bacteriol ; 194(23): 6431-40, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23002224

RESUMEN

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals.


Asunto(s)
Brucella abortus/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Virulencia/biosíntesis , Sitios de Unión , Brucella abortus/patogenicidad , Huella de ADN , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Regiones Promotoras Genéticas , Unión Proteica
17.
Antimicrob Agents Chemother ; 56(4): 1821-7, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22290975

RESUMEN

Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.


Asunto(s)
Escherichia coli/genética , Plásmidos/genética , Salmonella/genética , Argentina , Secuencia de Bases , Evolución Biológica , ADN Bacteriano/genética , Infecciones por Escherichia coli/microbiología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Recombinación Genética , Infecciones por Salmonella/microbiología
18.
Appl Environ Microbiol ; 78(12): 4092-101, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22492433

RESUMEN

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.


Asunto(s)
Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Medicago sativa/microbiología , Sinorhizobium meliloti/aislamiento & purificación , Sinorhizobium meliloti/fisiología , Argentina , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Datos de Secuencia Molecular , Filogenia , Raíces de Plantas/microbiología , Polisacáridos Bacterianos/metabolismo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Sinorhizobium meliloti/clasificación , Sinorhizobium meliloti/genética
19.
J Immunol ; 184(11): 6386-95, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20421641

RESUMEN

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.


Asunto(s)
Biopelículas/crecimiento & desarrollo , ADN Bacteriano/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Pseudomonas aeruginosa/fisiología , Citocinas/biosíntesis , Líquido Extracelular/química , Líquido Extracelular/microbiología , Humanos , Microscopía Confocal , Neutrófilos/metabolismo
20.
Proc Natl Acad Sci U S A ; 106(32): 13230-5, 2009 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-19666539

RESUMEN

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.


Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Amicacina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Ribonucleasa P/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Bases , ADN/metabolismo , Endocitosis/efectos de los fármacos , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo
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