RÉSUMÉ
Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.
Sujet(s)
Paroi cellulaire/métabolisme , Flavanones/pharmacologie , Herbaspirillum/effets des médicaments et des substances chimiques , Herbaspirillum/génétique , Régulation de l'expression des gènes bactériens/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes bactériens/génétique , Racines de plante/microbiologie , RT-PCR , Zea mays/microbiologieRÉSUMÉ
The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Although the diversity of its fauna and flora has been studied fairly well, little is known of its microbial communities. In this work, we analyzed the Atlantic Forest ecosystem to determine its bacterial biodiversity, using 16S rRNA gene sequencing, and correlated changes in deduced taxonomic profiles with the physicochemical characteristics of the soil. DNAs were purified from soil samples, and the 16S rRNA gene was amplified to construct libraries. Comparison of 754 independent 16S rRNA gene sequences from 10 soil samples collected along a transect in an altitude gradient showed the prevalence of Acidobacteria (63%), followed by Proteobacteria (25.2%), Gemmatimonadetes (1.6%), Actinobacteria (1.2%), Bacteroidetes (1%), Chloroflexi (0.66%), Nitrospira (0.4%), Planctomycetes (0.4%), Firmicutes (0.26%), and OP10 (0.13%). Forty-eight sequences (6.5%) represented unidentified bacteria. The Shannon diversity indices of the samples varied from 4.12 to 3.57, indicating that the soils have a high level of diversity. Statistical analysis showed that the bacterial diversity is influenced by factors such as altitude, Ca(2+)/Mg(2+) ratio, and Al(3+) and phosphorus content, which also affected the diversity within the same lineage. In the samples analyzed, pH had no significant impact on diversity.
Sujet(s)
Bactéries/classification , Bactéries/isolement et purification , Biodiversité , Microbiologie du sol , Sol/analyse , Altitude , Bactéries/génétique , Brésil , Calcium/analyse , Analyse de regroupements , ADN bactérien/composition chimique , ADN bactérien/génétique , ADN ribosomique/composition chimique , ADN ribosomique/génétique , Magnésium/analyse , Données de séquences moléculaires , Phosphore/analyse , Phylogenèse , ARN ribosomique 16S/génétique , Analyse de séquence d'ADN , ArbresRÉSUMÉ
AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.
Sujet(s)
Bovins/microbiologie , Escherichia coli/classification , Escherichia coli/isolement et purification , Shiga-toxines/biosynthèse , Animaux , Brésil , Chlorocebus aethiops , Escherichia coli/pathogénicité , Fèces/microbiologie , Génotype , Viande , Phénotype , Réaction de polymérisation en chaîne , Polymorphisme de restriction , Sérotypie , Shiga-toxines/génétique , Cellules Vero , Virulence/génétiqueRÉSUMÉ
The glnZ mutant of Azospirillum brasilense (strain 7611) showed only partial recovery (20 to 40%) after 80 min of ammonia-induced nitrogenase switch-off, whereas the wild type recovered totally within 10 min. In contrast, the two strains showed identical anoxic-induced switch-on/switch-off, indicating no cross talk between the two reactivation mechanisms.
Sujet(s)
Azospirillum brasilense/métabolisme , Protéines bactériennes/physiologie , Nitrogenase/métabolisme , Ammoniac , Anaérobiose , Azospirillum brasilense/enzymologie , Protéines bactériennes/génétique , Mutation , Nitrogenase/antagonistes et inhibiteurs , Facteurs tempsRÉSUMÉ
Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.
Sujet(s)
Betaproteobacteria/génétique , Gènes bactériens , Fixation de l'azote/génétique , Protéines bactériennes/génétique , Protéines de transport/génétique , Régulation de l'expression des gènes bactériens , Ordre des gènes , Protéines de régulation du métabolisme azoté , Facteurs de transcription/génétiqueRÉSUMÉ
The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.
Sujet(s)
Protéines bactériennes , Protéines de liaison à l'ADN/génétique , Glutamate-ammonia ligase/métabolisme , Bactéries à Gram négatif/génétique , Transactivateurs/génétique , Facteurs de transcription , Régulation de l'expression des gènes codant pour des enzymes , Gènes bactériens , Vecteurs génétiques , Glutamate-ammonia ligase/déficit , Glutamate-ammonia ligase/génétique , Bactéries à Gram négatif/enzymologie , Mutation , Fixation de l'azote/génétique , Protéines de régulation du métabolisme azoté , beta-Galactosidase/génétiqueRÉSUMÉ
Azospirillum species are plant-associated diazotrophs of the alpha subclass of Proteobacteria. The genomes of five of the six Azospirillum species were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. The nifHDK operon was identified in the largest replicon.
Sujet(s)
Azospirillum/génétique , ADN bactérien/génétique , Génome bactérien , Chromosomes de bactérie , Électrophorèse en champ pulsé , RépliconRÉSUMÉ
The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 sigma(N)-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5' end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter. Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the sigma(N)-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.
Sujet(s)
Protéines bactériennes/génétique , Betaproteobacteria/génétique , Gènes bactériens , Transactivateurs , Facteurs de transcription/génétique , Protéines bactériennes/métabolisme , Séquence nucléotidique , Betaproteobacteria/métabolisme , Sites de fixation/génétique , Amorces ADN/génétique , ADN bactérien/génétique , Protéines de liaison à l'ADN/métabolisme , Escherichia coli/génétique , Protéines Escherichia coli , Expression des gènes , Opéron lac , Données de séquences moléculaires , Mutation , Protéines de régulation du métabolisme azoté , Plasmides/génétique , Régions promotrices (génétique) , ARN bactérien/génétique , Facteurs de transcription/métabolismeRÉSUMÉ
A 5.1-kb DNA fragment from the nifHDK region of H. seropedicae was isolated and sequenced. Sequence analysis showed the presence of nifENXorf1orf2 but nifTY were not present. No nif or consensus promoter was identified. Furthermore, orf1 expression occurred only under nitrogen-fixing conditions and no promoter activity was detected between nifK and nifE, suggesting that these genes are expressed from the upstream nifH promoter and are parts of a unique nif operon. Mutagenesis studies indicate that nifN was essential for nitrogenase activity whereas nifXorf1orf2 were not. High homology between the C-terminal region of the NifX and NifB proteins from H. seropedicae was observed. Since the NifX and NifY proteins are important for FeMo cofactor (FeMoco) synthesis, we propose that alternative proteins with similar activities exist in H. seropedicae.
Sujet(s)
Betaproteobacteria/génétique , Gènes bactériens , Bactéries à Gram négatif/génétique , Fixation de l'azote/génétique , Opéron , Betaproteobacteria/enzymologie , Conjugaison génétique , Électroporation , Bactéries à Gram négatif/enzymologie , Mutagenèse par insertion , Nitrogenase/génétique , Nitrogenase/métabolisme , Cadres ouverts de lecture , Régions promotrices (génétique) , Analyse de séquence d'ADN , beta-Galactosidase/métabolismeRÉSUMÉ
The structure and activity of the nifA promoter of Azospirillum brasilense was studied using deletion analysis. An essential region for nifA promoter activity was identified between nucleotides -67 and -47 from the identified transcription start site. A sequence resembling a sigma(70) recognition site occurs in this region and may constitute the nifA gene promoter. The regulation of the nifA gene was studied in plasmid and chromosomal nifA::lacZ fusions. Full expression was obtained under low oxygen levels and in the absence of ammonium ions. Repression of nifA expression involves a synergistic effect between oxygen and ammonium.
Sujet(s)
Azospirillum brasilense/génétique , Régulation de l'expression des gènes bactériens/effets des médicaments et des substances chimiques , Oxygène/pharmacologie , Composés d'ammonium quaternaire/pharmacologie , Fusion artificielle de gènes , Protéines bactériennes/génétique , Séquence nucléotidique , Données de séquences moléculaires , Plasmides , Facteurs de transcription/génétiqueRÉSUMÉ
The activity of a truncated form of Herbaspirillum seropedicae NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O2 sensitivity or Fe dependence. The model for nitrogen control involving PII could thus apply to the proteobacteria at large. NifA may have a role in controlling ADP-ribosylation of nitrogenase in Azospirillum brasilense.
Sujet(s)
Ammoniac/pharmacologie , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Régulation de l'expression des gènes bactériens , Gènes bactériens , Bactéries à Gram négatif/génétique , Klebsiella pneumoniae/génétique , Fixation de l'azote/génétique , Nitrogenase/génétique , Oxidoreductases , Oxygène/pharmacologie , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Azospirillum brasilense/enzymologie , Déferoxamine/pharmacologie , Escherichia coli , Régulation de l'expression des gènes bactériens/effets des médicaments et des substances chimiques , Bactéries à Gram négatif/effets des médicaments et des substances chimiques , Bactéries à Gram négatif/métabolisme , Nitrogenase/métabolisme , Régions promotrices (génétique) , Séquences d'acides nucléiques régulatricesRÉSUMÉ
Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.
Sujet(s)
Protéines bactériennes/génétique , Gènes bactériens , Glutamate-ammonia ligase/métabolisme , Bactéries aérobies à Gram négatif/génétique , Chlorure d'ammonium/métabolisme , Conjugaison génétique , Éléments transposables d'ADN , Régulation de l'expression des gènes bactériens , Test de complémentation , Bactéries aérobies à Gram négatif/métabolisme , Klebsiella pneumoniae/génétique , Données de séquences moléculaires , Mutagenèse par insertion , Nitrates/métabolisme , Fixation de l'azote , Protéines de régulation du métabolisme azoté , Phénotype , Composés du potassium/métabolismeRÉSUMÉ
A cosmid able to complement the Nif- and nitrate-dependent growth phenotypes of the Azospirillum brasilense mutant FP9 was isolated from a genomic library of the wild-type strain FP2. A 6-kb DNA region was sequenced and showed two open reading frames (ORFs) identified as the ntrB and ntrC genes. An ORF1 located upstream from the ntrB gene and coding for a 36-kDa polypeptide showed similarity to the nifR3 gene of Rhodobacter capsulatus and the ORF1 of Rhizobium leguminosarum, both located upstream from the ntrB gene in a complex operon. Two other unidentified ORFs (ORF5 and partial ORF4) coding for hydrophobic polypeptides were also observed. delta ORF1-ntrBC, ORF1, ntrB, and ntrC mutants obtained by recombination of suicide plasmids containing an insertion of a promoterless lacZ kanamycin cassette showed decreased nitrogenase activities and were unable to grow on nitrate as the sole N source. These phenotypes were restored by complementation with plasmids containing the ntrC gene. Analysis of lacZ transcriptional fusions suggested that the ORF1-ntrBC operon in Azospirillum brasilense is expressed from a promoter located upstream from the ORF1 and that it is negatively regulated by the ntrC gene product.
Sujet(s)
Azospirillum brasilense/génétique , Gènes bactériens , Séquence d'acides aminés , Séquence nucléotidique , Cartographie chromosomique , Clonage moléculaire , Cosmides , ADN bactérien/génétique , Régulation de l'expression des gènes bactériens , Test de complémentation , Opéron lac , Données de séquences moléculaires , Mutagenèse dirigée , Fixation de l'azote/génétique , Cadres ouverts de lecture , Opéron , Régions promotrices (génétique) , Similitude de séquences d'acides aminésRÉSUMÉ
A recombinant plasmid, pBMR5, carrying a recA-like gene of Herbaspirillum seropedicae, was isolated from a H. seropedicae genomic library by intergeneric complementation of Escherichia coli recA mutant strain HB101. Quantitative survival experiments showed that pBMR5 restored the ultraviolet radiation and methyl methanesulfonate resistances and recombinational proficiency of this strain. Hybridization studies showed that there is DNA sequence homology between the recA gene of E. coli K12 and that of H. seropedicae. Restriction sites for EcoRI, HindIII, BamHI, and Bg/II were found in the DNA insert derived from H. seropedicae in pBMR5. A Tn5 insertional mutant of pBMR5, called pBMR26.2, failed to restore recombination proficiency and methyl methanesulfonate and ultraviolet resistance to recA mutants of E. coli.
Sujet(s)
Gènes bactériens , Bactéries à Gram négatif/génétique , Clonage moléculaire , Éléments transposables d'ADN , ADN bactérien/génétique , Escherichia coli/génétique , Test de complémentation , Mutagenèse par insertion , Fixation de l'azote , Hybridation d'acides nucléiques , Plasmides/génétique , Rec A Recombinases/génétique , Cartographie de restrictionRÉSUMÉ
The deduced amino acid sequence derived from the sequence of a fragment of DNA from the free-living diazotroph Herbaspirillum seropedicae was aligned to the homologous protein sequences encoded by the nifA genes from Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti and Klebsiella pneumoniae. High similarity was found in the central domain and in the C-terminal region. The H. seropedicae putative NifA sequence was also found to contain an interdomain linker similar to that conserved among rhizobial NifA proteins, but not K. pneumoniae or Azotobacter vinelandii. Analysis of the regulatory sequences found 5' from nifA indicated that the expression of this gene in H. seropedicae is likely to be controlled by NifA, NtrC and RpoN, as judged by the presence of specific NifA- and NtrC-binding sites and characteristic -24/-12 promoters. Possible additional regulatory features included an 'anaerobox' and a site for integration host factor. The N-terminus of another open reading frame was found 3' from nifA and tentatively identified as nifB by amino acid sequence comparison. The putative nifB promoter sequence suggests that expression of H. seropedicae nifB may be activated by NifA and dependent on RpoN.
Sujet(s)
Bactéries/génétique , Protéines bactériennes/génétique , Gènes bactériens , Fixation de l'azote/génétique , Facteurs de transcription/génétique , Séquence d'acides aminés , Séquence nucléotidique , Sites de fixation/génétique , Séquence consensus , Klebsiella pneumoniae/génétique , Données de séquences moléculaires , Mutation/génétique , Cadres ouverts de lecture/génétique , Régions promotrices (génétique)/génétique , Séquences d'acides nucléiques régulatrices , Rhizobium/génétique , Similitude de séquences d'acides nucléiquesRÉSUMÉ
A genomic library of Herbaspirillum seropedicae was constructed and screened for the nifA gene by complementation of a nifA mutant of Azospirillum brasilense (FP10). A recombinant plasmid, pEMS1, capable of restoring acetylene reduction activity in the mutant FP10, was isolated and found to hybridize to the nifA gene of Klebsiella pneumoniae. The results suggest that nifA is involved in the regulation of nif genes in H. seropedicae.
Sujet(s)
Bactéries/génétique , Fixation de l'azote/génétique , Azospirillum/génétique , Clonage moléculaire , Électrophorèse sur gel d'agar , Gènes bactériens , Plasmides , Cartographie de restrictionRÉSUMÉ
The pathway for L-arabinose metabolism in Herbaspirillum seropedicae was shown to involve nonphosphorylated intermediates and to produce alpha-ketoglutarate. The activities of the enzymes and the natures of several intermediates were determined. The pathway was inducible by L-arabinose, and two key enzymes, L-arabinose dehydrogenase and 2-keto-glutarate semialdehyde dehydrogenase, were present in all strains of H. seropedicae tested.
Sujet(s)
Arabinose/métabolisme , Bactéries aérobies/métabolisme , Isomérie , Cinétique , Spécificité d'espèceRÉSUMÉ
The growth of Pullularia pullulans on L-rhamnose (6-deoxy-L-mannose) as the sole carbon source induces the synthesis of L-rhamnose dehydrogenase, a nicotinamide adenine dinucleotide-dependent enzyme that catalyzes the oxidation of the deoxy sugar to L-rhamnonolactone. The enzyme induction is inhibited by cycloheximide, suggesting de novo synthesis. The presence of d-glucose (0.2%) or D-galactose (0.2%) simultaneously with the inducer in the induction medium produced 50% repression of dehydrogenase synthesis, but no effect was detected with D-fructose and D-mannose at the same concentration. High levels of D-glucose (2%), under maximal catabolite repression conditions, produced a complete inhibition of enzyme synthesis.
Sujet(s)
Carbohydrate dehydrogenases/biosynthèse , Répression enzymatique , Deuteromycota/enzymologie , Cycloheximide/pharmacologie , Induction enzymatique/effets des médicaments et des substances chimiques , Fructose/pharmacologie , Galactose/pharmacologie , Glucose/pharmacologie , Mannose/pharmacologie , RhamnoseRÉSUMÉ
Growth of Pullularia pullulans on L-rhamnose induces formation of L-rhamnofuranose dehydrogenase, whichreversibly converts L-rhamnofuranose to L-rhamnono-gamma-lactone with the concomitant reduction of NAD, but not of NADP. The dehydrogenase was purified 100-fold by MnCl(2) treatment...