RESUMEN
Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.
Asunto(s)
Agrobacterium tumefaciens/genética , Brucella abortus/patogenicidad , Brucelosis/prevención & control , Sinorhizobium meliloti/genética , Agrobacterium tumefaciens/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucelosis/genética , Brucelosis/microbiología , Brucelosis/patología , Regulación Bacteriana de la Expresión Génica/genética , Interacciones Huésped-Parásitos/genética , Humanos , Ratones , Mutación/genética , Polimixina B/farmacología , Sinorhizobium meliloti/efectos de los fármacos , Virulencia/genéticaRESUMEN
Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2-tridecanone (2-TDC), a methylketone (MK) known as a natural insecticide. Application of pure 2-TDC to the wild-type strain phenocopies the free-living and symbiotic behaviours of the fadD mutant. Structural features of the MK determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella-independent motility. Transcriptomic analyses showed that 2-TDC induces differential expression of iron uptake, redox and stress-related genes. Interestingly, this MK also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2-TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2-TDC as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions by interfering with microbial colonization of plant tissues.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cetonas/metabolismo , Cetonas/farmacología , Medicago sativa/microbiología , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/metabolismo , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Mutación , Fenotipo , Sinorhizobium meliloti/genética , SimbiosisRESUMEN
Small non-coding regulatory RNAs (sRNAs) are key players in post-transcriptional regulation of gene expression. Hundreds of sRNAs have been identified in Sinorhizobium meliloti, but their biological function remains unknown for most of them. In this study, we characterized the expression pattern of the gene encoding the 77-nt sRNA MmgR in S. meliloti strain 2011. A chromosomal transcriptional reporter fusion (PmmgR-gfp) showed that the mmgR promoter is active along different stages of the interaction with alfalfa roots. In pure cultures, PmmgR-gfp activity paralleled the sRNA abundance indicating that mmgR expression is primarily controlled at the level of transcriptional initiation. PmmgR-gfp activity was higher during growth in rhizobial defined medium (RDM) than in TY medium. Furthermore, PmmgR-gfp was induced at 60 min after shifting growing cells from TY to RDM medium, i.e. shorter than the cell doubling time. In defined RDM medium containing NO3 (-), both PmmgR-gfp and MmgR level were repressed by the addition of tryptone or single amino acids, suggesting that mmgR expression depends on the cellular nitrogen (N) status. In silico analysis failed to detect conserved motifs upstream the promoter RNA polymerase binding site, but revealed a strongly conserved motif centered at -28 that may be linked to the observed regulatory pattern by the N source.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Sinorhizobium meliloti/genética , Aminoácidos/farmacología , Medios de Cultivo/química , Medicago sativa/microbiología , Fijación del Nitrógeno , Peptonas/farmacología , Regiones Promotoras Genéticas , Rhizobium/química , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Three Klebsiella pneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all ß-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named bla BKC-1. Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a ß-lactamase of Sinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low kcat values (0.0016 to 0.031 s(-1)). In fact, oxacillin was the best substrate for BKC-1 (kcat /Km , 53,522.6 mM(-1) s(-1)). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of ß-lactamases in K. pneumoniae clinical isolates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Klebsiella pneumoniae/enzimología , beta-Lactamasas/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Brasil , Carbapenémicos/metabolismo , Carbapenémicos/farmacología , Cefalosporinas/metabolismo , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana , Electroforesis en Gel de Campo Pulsado , Pruebas de Sensibilidad Microbiana , Monobactamas/metabolismo , Monobactamas/farmacología , Penicilinas/metabolismo , Penicilinas/farmacología , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/metabolismoRESUMEN
Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented.
Asunto(s)
Burkholderia cenocepacia/enzimología , Burkholderia cenocepacia/genética , Dioxigenasas/genética , Ácidos Grasos/metabolismo , Genes Bacterianos/genética , Lípidos de la Membrana/metabolismo , Ornitina/análogos & derivados , Ácidos/farmacología , Amidas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/efectos de los fármacos , Burkholderia cenocepacia/crecimiento & desarrollo , Cardiolipinas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Dioxigenasas/metabolismo , Esterificación/efectos de los fármacos , Ácidos Grasos/química , Hidroxilación/efectos de los fármacos , Lípidos/química , Espectrometría de Masas , Lípidos de la Membrana/química , Mutación/genética , Ornitina/química , Ornitina/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/enzimología , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/metabolismoRESUMEN
The acidophilic proteobacterium Acidithiobacillus ferrooxidans is involved in the industrial biorecovery of copper. It is found in acidic environments in biofilms and is important in the biogeochemical cycling of metals and nutrients. Its genome contains a cluster of four genes, glyQ, glysS, gph, and act, that are predicted to encode the alpha and beta subunits of glycine tRNA synthetase, a phosphatase, and an acyltransferase, respectively (GenBank accession no. DQ149607). act, cloned and expressed in Escherichia coli, produces acyl homoserine lactones (AHLs) principally of chain length C14 according to gas chromatography and mass spectrometry measurements. The AHLs have biological activity as shown by in vivo studies using the reporter strain Sinorhizobium meliloti Rm41 SinI-. Reverse transcription-PCR (RT-PCR) experiments indicate that the four genes are expressed as a single transcript, demonstrating that they constitute an operon. According to semiquantitative RT-PCR results, act is expressed more highly when A. ferrooxidans is grown in medium containing iron than when it is grown in medium containing sulfur. Since AHLs are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression, this result suggests that A. ferrooxidans has two quorum-sensing systems, one based on Act, as described herein, and the other based on a Lux-like quorum-sensing system, reported previously. The latter system was shown to be upregulated in A. ferrooxidans grown in sulfur medium, suggesting that the two quorum-sensing systems respond to different environmental signals that may be related to their abilities to colonize and use different solid sulfur- and iron-containing minerals.
Asunto(s)
4-Butirolactona/análogos & derivados , Acidithiobacillus/enzimología , Acidithiobacillus/genética , Redes y Vías Metabólicas/genética , 4-Butirolactona/biosíntesis , Acidithiobacillus/metabolismo , Aciltransferasas/genética , Proteínas Bacterianas/genética , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glicina-ARNt Ligasa/genética , Hierro/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Operón , Monoéster Fosfórico Hidrolasas/genética , ARN Bacteriano/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/fisiología , Azufre/metabolismoRESUMEN
Rhizobia are non-spore-forming soil bacteria that fix atmospheric nitrogen into ammonia in a symbiosis with legume roots. However, in the absence of a legume host, rhizobia manage to survive and hence must have evolved strategies to adapt to diverse environmental conditions. The capacity to respond to variations in nutrient availability enables the persistence of rhizobial species in soil, and consequently improves their ability to colonize and to survive in the host plant. Rhizobia, like many other soil bacteria, persist in nature most likely in sessile communities known as biofilms, which are most often composed of multiple microbial species. We have been employing in vitro assays to study environmental parameters that might influence biofilm formation in the Medicago symbiont Sinorhizobium meliloti. These parameters include carbon source, amount of nitrate, phosphate, calcium and magnesium as well as the effects of osmolarity and pH. The microtiter plate assay facilitates the detection of subtle differences in rhizobial biofilms in response to these parameters, thereby providing insight into how environmental stress or nutritional status influences rhizobial survival. Nutrients such as sucrose, phosphate and calcium enhance biofilm formation as their concentrations increase, whereas extreme temperatures and pH negatively affect biofilm formation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Sinorhizobium meliloti/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Calcio/farmacología , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Microscopía Fluorescente , Nitratos/farmacología , Fosfatos/farmacología , Sinorhizobium meliloti/efectos de los fármacos , Cloruro de Sodio/farmacología , Sorbitol/farmacología , Sacarosa/farmacología , TemperaturaRESUMEN
A survey of the changes in populations of heterotrophic bacteria, coliform microorganisms and S. meliloti was conducted in samples taken from the water irrigation channels of the Neuquén River (Argentina). Fifty-six water samples were collected during the spring-summer seasons of 1997-1999 years. Both the heterotrophic plate count bacterial and the number of coliforms oscillated between 110-5050 CFU/ml and 8-1400 CFU/100 ml, respectively, during the period this study was carried out. Fecal coliforms were detected in 91.1% of the water samples investigated. Moreover, the results showed that S. meliloti capable of nodulating alfalfa (Medicago sativa L.) Cuf 101 were present in 68% of the water samples and in effectiveness studies, no isolate out of 25 evaluated could be classified as superior N fixers. That is, they did not produce plants equal in weight to nitrate-grown plants (KNO3 0.05%). All the S. meliloti strains were resistant to novobiocin and bacitracin, while 72% of the microsymbionts demonstrated resistance to between seven and ten antibiotics. Results presented in this study showed that irrigation waters of the Neuquén river could act as dispersal agents of both ineffective S. meliloti strains and thermotolerant coliform bacteria.
Asunto(s)
Enterobacteriaceae/aislamiento & purificación , Sinorhizobium meliloti/aislamiento & purificación , Microbiología del Agua , Contaminación del Agua/análisis , Argentina , Bacitracina/farmacología , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Calor , Medicago sativa/microbiología , Fijación del Nitrógeno , Novobiocina/farmacología , Estaciones del Año , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/fisiologíaRESUMEN
A survey of the changes in populations of heterotrophic bacteria, coliform microorganisms and S. meliloti was conducted in samples taken from the water irrigation channels of the NeuquUn River (Argentina). Fifty-six water samples were collected during the spring-summer seasons of 1997-1999 years. Both the heterotrophic plate count bacterial and the number of coliforms oscillated between 110-5050 CFU/ml and 8-1400 CFU/100 ml, respectively, during the period this study was carried out. Fecal coliforms were detected in 91.1 of the water samples investigated. Moreover, the results showed that S. meliloti capable of nodulating alfalfa (Medicago sativa L.) Cuf 101 were present in 68 of the water samples and in effectiveness studies, no isolate out of 25 evaluated could be classified as superior N fixers. That is, they did not produce plants equal in weight to nitrate-grown plants (KNO3 0.05). All the S. meliloti strains were resistant to novobiocin and bacitracin, while 72 of the microsymbionts demonstrated resistance to between seven and ten antibiotics. Results presented in this study showed that irrigation waters of the NeuquUn river could act as dispersal agents of both ineffective S. meliloti strains and thermotolerant coliform bacteria.(AU)
Asunto(s)
RESEARCH SUPPORT, NON-U.S. GOVT , Enterobacteriaceae/aislamiento & purificación , Sinorhizobium meliloti/aislamiento & purificación , Microbiología del Agua , Contaminación del Agua/análisis , Argentina , Bacitracina/farmacología , Farmacorresistencia Microbiana , Resistencia a Múltiples Medicamentos , Calor , Medicago sativa/microbiología , Fijación del Nitrógeno , Novobiocina/farmacología , Estaciones del Año , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/fisiologíaRESUMEN
A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.