RESUMEN
The LysR-type transcriptional regulators (LTTRs) are DNA-binding proteins present in bacteria, archaea, and in algae. Knowledge about their distribution, abundance, evolution, structural organization, transcriptional regulation, fundamental roles in free life, pathogenesis, and bacteria-plant interaction has been generated. This review focuses on these aspects and provides a current picture of LTTR biology.
RESUMEN
LtrR is a LysR-type regulator involved in the positive expression of ompR to promote ompC and ompF expression. This regulatory network is fundamental for the control of bacterial transformation and resistance to the bile salt sodium deoxycholate in Salmonella enterica serovar Typhi. In this work, the transcriptional regulation of ltrR was characterized, revealing that the use of alternative promoters results in two transcripts. The larger one, the ltrR2 mRNA, was repressed at promoter and coding regions by H-NS, whereas Lrp repressed its expression at the coding region. In the case of the second and shorter ltrR1 transcript, it was repressed only at the coding region by H-NS and Lrp. Remarkably, pH 7.5 is a positive signal involved in the transcriptional expression of both ltrR units. Translational fusions and Western blot experiments demonstrated that ltrR2 and ltrR1 mRNAs encode the LtrR2 and LtrR1 proteins. This study adds new data on the complex genetic and regulatory characteristics of one of the most predominant types of transcriptional factors in bacteria, the LysR-type transcriptional regulators.IMPORTANCE The LysR-type transcriptional regulators are present in viruses, archaea, bacteria, and eukaryotic cells. Furthermore, these proteins are the most abundant transcriptional factors in bacteria. Here, we demonstrate that two LysR-type proteins are generated from the ltrR gene. These proteins are genetically induced by pH and repressed at the promoter and coding regions by the global regulators H-NS and Lrp. Thus, novel basic aspects of the complex genetic regulation of the LysR-type transcriptional regulators are described.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Salmonella typhi/metabolismo , Factores de Transcripción/metabolismo , Álcalis/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Concentración de Iones de Hidrógeno , Operón , Salmonella typhi/genética , Factores de Transcripción/genéticaRESUMEN
A characterization of the LtrR regulator, an S. Typhi protein belonging to the LysR family is presented. Proteomics, outer membrane protein profiles and transcriptional analyses demonstrated that LtrR is required for the synthesis of OmpR, OmpC and OmpF. DNA-protein interaction analysis showed that LtrR binds to the regulatory region of ompR and then OmpR interacts with the ompC and ompF promoters inducing porin synthesis. LtrR-dependent and independent ompR promoters were identified, and both promoters are involved in the synthesis of OmpR for OmpC and OmpF production. To define the functional role of the ltrR-ompR-ompC-ompF genetic network, mutants in each gene were obtained. We found that ltrR, ompR, ompC and ompF were involved in the control of bacterial transformation, while the two regulators and ompC are necessary for the optimal growth of S. Typhi in the presence of one of the major bile salts found in the gut, sodium deoxycholate. The data presented establish the pivotal role of LtrR in the regulatory network of porin synthesis and reveal new genetic strategies of survival and cellular adaptation to the environment used by Salmonella.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Ácidos y Sales Biliares/farmacología , Salmonella typhi/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Ácido Desoxicólico/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Salmonella typhi/genética , Transformación Bacteriana/genéticaRESUMEN
The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ΔleuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Oxidorreductasas/metabolismo , Salmonella typhi/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Genoma Bacteriano , Familia de Multigenes , Mutación , Operón , Oxidorreductasas/genética , Regiones Promotoras Genéticas , ARN Bacteriano/genética , Salmonella typhi/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoid-associated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuO-independent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína Reguladora de Respuesta a la Leucina/metabolismo , Operón , Salmonella typhi/genética , Factores de Transcripción/metabolismo , Análisis Mutacional de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU-yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU-yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU-yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU-yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidroxibutirato Deshidrogenasa/metabolismo , Operón , Salmonella typhi/genética , Salmonella typhi/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Represión Catabólica , Humanos , Hidroxibutirato Deshidrogenasa/química , Hidroxibutirato Deshidrogenasa/genética , Mutagénesis Sitio-Dirigida , Salmonella typhi/crecimiento & desarrollo , Fiebre Tifoidea/microbiologíaRESUMEN
The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C(2) compounds by bypassing the CO(2)-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL- and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.
Asunto(s)
Bacterias/enzimología , Bacterias/patogenicidad , Hongos/enzimología , Hongos/patogenicidad , Isocitratoliasa/fisiología , Malato Sintasa/fisiología , Factores de Virulencia/fisiología , Animales , HumanosRESUMEN
OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to -54 and ends at about -197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Salmonella typhimurium/metabolismo , Transactivadores/metabolismo , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Herbicidas/farmacología , Paraquat/farmacología , Regiones Promotoras Genéticas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Análisis de Secuencia de ADN , Activación Transcripcional/efectos de los fármacosRESUMEN
LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of Salmonella. A genomic analysis with Salmonella enterica serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of ompS1, assT, and STY3070 was induced in an hns mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for tpx and STY1978 in an hns background, suggesting that this global regulatory protein has a positive effect. In contrast, ompS2 and ompX expression appeared to be H-NS independent. LeuO specifically bound to the 5' intergenic regions of ompS2, assT, STY3070, ompX, and tpx, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in Salmonella.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Regulación Bacteriana de la Expresión Génica , Salmonella typhi/genética , Transactivadores/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cloranfenicol O-Acetiltransferasa/metabolismo , Biología Computacional , Huella de ADN , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel Bidimensional , Porinas , Regiones Promotoras Genéticas , Regulón , Salmonella typhi/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sitio de Iniciación de la TranscripciónRESUMEN
The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other alpha-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.
Asunto(s)
Isocitratoliasa/genética , Malato Sintasa/genética , Sinorhizobium meliloti/enzimología , Acetatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Isocitratoliasa/metabolismo , Sinorhizobium meliloti/genética , Transcripción GenéticaRESUMEN
We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfully used to clone plasmids of Rhizobium species with sizes ranging from 150 to 270 kb. In addition, a 500-kb fragment of a 600-kb megaplasmid was also cloned. This strategy could be used for cloning indigenous replicons of other gram-negative bacteria into a different host.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Rhizobium/genética , Vectores Genéticos , PlásmidosRESUMEN
Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N(2)-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome. The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB. Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family. Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis. Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation. The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.
Asunto(s)
Plásmidos/genética , Sinorhizobium meliloti/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Metabolismo de los Hidratos de Carbono , Proteínas Portadoras/genética , Chaperoninas/genética , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Enzimas/genética , Regulación Bacteriana de la Expresión Génica , Biblioteca de Genes , Genes Bacterianos , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , ARN de Transferencia de Arginina/genética , Origen de Réplica/genética , Replicón/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Transcripción Genética/genéticaRESUMEN
A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.
Asunto(s)
Proteínas Bacterianas/genética , Transportadores de Ácidos Dicarboxílicos/genética , Ácidos Dicarboxílicos/metabolismo , Mutación , Rhizobium/crecimiento & desarrollo , Ácido Succínico/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico , Transportadores de Ácidos Dicarboxílicos/metabolismo , Fabaceae/microbiología , Datos de Secuencia Molecular , Rhizobium/genética , Análisis de Secuencia de ADN , SimbiosisRESUMEN
The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.
Asunto(s)
Genoma Bacteriano , Análisis de Secuencia de ADN , Sinorhizobium meliloti/genética , Simbiosis/genética , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Cromosomas Bacterianos/genética , Biología Computacional , Elementos Transponibles de ADN , Metabolismo Energético/genética , Evolución Molecular , Duplicación de Gen , Genes Bacterianos , Genes Esenciales , Genes Reguladores , Medicago sativa/microbiología , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Plásmidos , Polisacáridos Bacterianos/genética , Replicón , Rhizobiaceae/genética , Sinorhizobium meliloti/fisiologíaRESUMEN
Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.
Asunto(s)
Fabaceae/microbiología , Fijación del Nitrógeno , Plantas Medicinales , Plásmidos/genética , Rhizobiaceae/fisiología , Rhizobium/genética , Conjugación Genética , Medios de Cultivo , ARN Ribosómico 16S/genética , Rhizobiaceae/genética , Rhizobium/fisiología , Análisis de Secuencia de ADN , SimbiosisRESUMEN
We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.
Asunto(s)
ADN Bacteriano , Genoma Bacteriano , Plásmidos , Origen de Réplica , Sinorhizobium meliloti/genética , Secuencia de Bases , Clonación Molecular , Vectores Genéticos , Datos de Secuencia Molecular , Recombinación GenéticaRESUMEN
The genetic structure of Bradyrhizobium isolates recovered from three Lupinus species (Lupinus campestris, Lupinus montanus, and Lupinus exaltatus) grown in Mexico was examined. Among 41 Bradyrhizobium isolates, 18 electrophoretic types (ETs) were distinguished by multilocus enzyme electrophoresis of five metabolic enzymes. The mean genetic diversity, 0.64, indicated that there was great genetic diversity in the population sampled. Most isolates (63%) fell into two closely related clusters (clusters I and II) and were the types most frequently isolated from the root nodules of L. montanus and L. campestris. ET cluster III isolates were frequent nodule occupants of L. exaltatus. The isolates also were assigned to three main groups by using Curie point pyrolysis mass spectrometry. In general, the multilocus enzyme electrophoretic data and pyrolysis mass spectrometric data agreed. We determined the 16S rRNA sequences of representative Lupinus isolates and of Bradyrhizobium japonicum USDA 6T and found that the lupine isolates were highly related to the B. japonicum type strain, although not all B. japonicum type strains (subcultures maintained in different bacterial collections) had identical small-subunit rRNA.
Asunto(s)
ADN Bacteriano/análisis , Ecosistema , ARN Ribosómico 16S/análisis , Rhizobiaceae/genética , Proteínas Bacterianas/genética , Datos de Secuencia Molecular , Filogenia , Plantas/microbiología , Reacción en Cadena de la Polimerasa , Rhizobiaceae/clasificación , Rickettsiaceae/fisiologíaRESUMEN
Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.
Asunto(s)
Cromosomas Bacterianos/genética , Citrato (si)-Sintasa/genética , Genes Bacterianos , Rhizobium/enzimología , Rhizobium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/enzimología , Escherichia coli/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Plásmidos/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , SimbiosisRESUMEN
We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species. All share the capacity to nodulate Phaseolus vulgaris L., the common bean. Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages. We defined the host ranges of two strains of Rhizobium etli and three strains of R. tropici, comparing them with those of the two most divergently related new strains. Twenty-two of the 43 tested legume species were nodulated by three or more of these strains. All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala.
Asunto(s)
Fabaceae/microbiología , Plantas Medicinales , Rhizobium/genética , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/químicaRESUMEN
We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.