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O M Aguilar

Publications and source records attributed to O M Aguilar.

At least 19 recordsLinked to original sources
PubMed2004

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level.

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.

Animals↗
PubMed2001

The diversity of rhizobia nodulating beans in Northwest Argentina as a source of more efficient inoculant strains.

The common bean (Phaseolus vulgaris L.) is cultivated widely in Central and South America and particularly in the Northwest of Argentina. In order to describe the diversity of the common bean nodulating rhizobial population from the bean producing area in Northwest Argentina (NWA), a collection of about 400 isolates of common beans recovered from nodules and soil samples from NWA were characterized by using nifH-PCR, analysis of genes coding for 16S rRNA and nodC, and REP-fingerprinting, respectively. It was found that species Rhizobium etli is predominant in common bean nodules although a high degree of diversity was found within the species. Other bean nodulating genotypes recovered from soils by using Leucaena sp. as the trapping host was found to have the 16S rDNA alleles of species such as Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium loti, and Rhizobium tropici. Some of the bean genotypes that were found to be more efficient in green house experiments were selected and assayed in two successive bean-cropping seasons in the field environment in NWA, and an increase in yields with inoculation was found. The performance of strains isolated from the region indicates potential for exploiting the diversity.

Argentina↗
PubMed2001

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa.

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

Alcohol Oxidoreductases↗
PubMed2001

Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates.

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.

Amino Acid Sequence↗
PubMed2000

A guaB mutant strain of Rhizobium tropici CIAT899 pleiotropically defective in thermal tolerance and symbiosis.

Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using TnS-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.

Allopurinol↗
PubMed2000

Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance.

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.

DNA Transposable Elements↗
PubMed2000

Low molecular weight heparins.

Since its discovery, nearly 90 years, heparin has been used successfully for the treatment of thromboembolic processes. However, therapy with heparin has several important limitations. Most importantly, the poor predictability of its anticoagulant effects has led to the development of the low molecular weight heparins (LMWHs), which are derived from unfractionated heparin and appear to have pharmacologic advantages, require no laboratory monitoring and are more predictable than their parent compounds. LMWHs have been used for several years in the treatment of venous thromboembolic disorders. More recently, the LMWHs have been used to treat patients with acute coronary interventions. As the results of new studies are revealed, we will learn whether the use LMWH can be extended to all disorders where unfractionated heparin is currently the standard therapy.

Animals↗
PubMed1999

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina.

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS-PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.

Animals↗
PubMed1998

Characterization of isolates of Paenibacillus larvae subsp. larvae from diverse geographical origin by the polymerase chain reaction and BOX primers.

Ninety-nine strains of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease (AFB) of honeybees, were isolated from different regions of Argentina and other countries. The isolates were characterized on the basis of DNA fingerprints by a polymerase chain reaction technique (PCR) with BOX sequence-specific primers. Isolates from Argentina generated three groups of patterns (designated A, B, and C), while P. larvae subsp. larvae strains obtained from other countries yielded two distinguishable patterns (coincident with A and B). Strains from U.S. A. and Germany were identical and related to Group A, while all Czech and English isolates belonged to Group B. Strains from France, Poland, Italy, Sweden, and New Zealand showed two different patterns (A and B). Comparisons of the biochemical type and genotype of isolates rendered no obvious linkage between both features. These results suggest that AFB in Argentina resulted from multiple sources of contaminated material.

Animals↗
PubMed1998

Prevalence of the Rhizobium etli-like allele in genes coding for 16S rRNA among the indigenous rhizobial populations found associated with wild beans from the Southern Andes in Argentina.

A collection of rhizobial isolates from nodules of wild beans, Phaseolus vulgaris var. aborigineus, found growing in virgin lands in 17 geographically separate sites in northwest Argentina was characterized on the basis of host range, growth, hybridization to a nifH probe, analysis of genes coding for 16S rRNA (16S rDNA), DNA fingerprinting, and plasmid profiles. Nodules in field-collected wild bean plants were largely dominated by rhizobia carrying the 16S rDNA allele of Rhizobium etli. A similar prevalence of the R. etli allele was observed among rhizobia trapped from nearby soil. Intragroup diversity of wild bean isolates with either R. etli-like or Rhizobium leguminosarum bv. phaseoli-like alleles was generally found across northwest Argentina. The predominance of the R. etli allele suggests that in this center of origin of P. vulgaris the coevolution of Rhizobium spp. and primitive beans has resulted in this preferential symbiotic association.

Alleles↗
PubMed1992

Induction of C4-dicarboxylate transport genes by external stimuli in Rhizobium meliloti.

We have characterized two mutants of Rhizobium meliloti L5-30 obtained by random mutagenesis using Mu-lacZ that were defective in transport of C4-dicarboxylic acids. These mutants induced ineffective nodules on alfalfa. Mutations in the two strains appeared to be located in a dctA gene. Levels of dctA gene expression were determined under different environmental conditions using dctA-lacZ fusions, and beta-galactosidase activities increased in response to osmotic stress and also when the cells were incubated in medium low in calcium. The transcriptional induction of the dctA gene by environmental signals was decreased by DNA gyrase inhibitors such as novobiocin and coumermycin.

Aminocoumarins↗
PubMed1991

The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa.

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.

Alcohol Oxidoreductases↗
PubMed1990

The nifEN genes participating in FeMo cofactor biosynthesis and genes encoding dinitrogenase are part of the same operon in Bradyrhizobium species.

The nucleotide sequences of genes homologous to the Klebsiella pneumoniae nifEN genes have been determined in Bradyrhizobium japonicum 110. The coding regions for the nifE and nifN consist, respectively, of 1641 and 1407 nucleotides. The nifD gene (coding for the beta-subunit of dinitrogenase) and nifE are linked, and separated by 95 nucleotides. In the region of 12 nucleotides that separates nifE from nifN the stop codon for nifE overlaps the putative ribosome binding site for nifN. In contrast to Klebsiella and Azotobacter vinelandii, the B. japonicum nifEN genes are linked to the nifDK genes in the same operon. Comparison of dinitrogenase polypeptides (nifDK products) and the polypeptides of the nifE and nifN genes reveals considerable homology between nifD and nifE, and between nifK and nifN. Several protein domains, containing highly conserved cysteine residues, are conserved among the gene products of nifD, nifK, nifE and nifN. This result allows us to propose a probable evolutionary pathway for the common origin of these genes.

Amino Acid Sequence↗
PubMed1987

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.

Amino Acid Sequence↗
PubMed1985

Characterization of a Rhizobium meliloti fixation gene (fixF) located near the common nodulation region.

Rhizobium meliloti 2011 DNA from pRmSL26, a plasmid which is known to carry genes involved in the early stages of nodulation, was used to construct Tn5 mutations by site-directed Tn5 mutagenesis. Tn5 mutations located within an 8.7 kilobase EcoRI fragment defined two adjacent loci encoding functions for nodulation (nod) and symbiotic N2 fixation (fix). We investigated the organization and regulation of the fix locus and the characteristics of alfalfa nodules induced by these Fix- mutants. By monitoring expression in Escherichia coli minicells, we determined that the fix locus encoded a 36-kilodalton polypeptide. The gene corresponding to this locus was designated fixF. Morphological and ultrastructural studies of the ineffective nodules formed by R. meliloti fixF mutants showed infected host cells similar to those of the wild type. The ineffective nodules were able to accumulate leghemoglobin, but at lower levels than those found in the wild-type nodules. Expression of the nifHDK operon was unaffected by Tn5 insertions in the fixF gene. Expression of the fixF gene was monitored in E. coli by using translational lacZ fusions. It was shown that transcription of the fixF gene in E. coli could be activated by Klebsiella pneumoniae nifA and the R. meliloti nifA-like regulatory gene products. Expression of the fixF gene was also studied in free-living and symbiotic R. meliloti cells. It was found that the fixF gene was transcribed in the symbiotic state.

Chromosome Mapping↗