RESUMO
It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (8699%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.
Assuntos
Azospirillum brasilense , DNA Bacteriano , Genes Bacterianos/fisiologia , Genoma Bacteriano/fisiologia , Plasmídeos , Polissacarídeos Bacterianos , Sorogrupo , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genéticaRESUMO
In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR_p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR_p60120 gene in the A. brasilense Sp245 LPSI- Cal- KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Genes Bacterianos/fisiologia , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Sequência de Bases , Benzenossulfonatos/química , Dados de Sequência MolecularRESUMO
We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Leptospira/imunologia , Leptospirose/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Clonagem Molecular , Proteína de Ligação ao Complemento C4b , Ensaio de Imunoadsorção Enzimática , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Antígenos de Histocompatibilidade , Humanos , Immunoblotting , Leptospira/genética , Leptospira/fisiologia , Leptospira interrogans/imunologia , Microscopia Imunoeletrônica , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , Proteínas RecombinantesRESUMO
Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator: the beta-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.
Assuntos
Biodegradação Ambiental , Cupriavidus necator/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Redes Reguladoras de Genes/fisiologia , Hidrocarbonetos Aromáticos/metabolismo , Biologia de Sistemas/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/crescimento & desenvolvimento , Retroalimentação Fisiológica , Genes Bacterianos/fisiologia , Biossíntese de Proteínas/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.
Assuntos
Bacteriocinas/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Ferro/farmacologia , Xylella/genética , 2,2'-Dipiridil/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Quelantes/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulon/genética , Regulon/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xylella/efeitos dos fármacos , Xylella/crescimento & desenvolvimento , Xylella/metabolismoRESUMO
Brucella is an intracellular facultative bacterium able to survive and multiply in professional and non-professional phagocytes. However, its adhesion and invasion mechanisms have not been elucidated yet. In this work, we assess the interruption of a BMEI0216 gene of Brucella melitensis, by using HeLa epithelial cells and murine macrophages for invasion and replication assays. The mutation did not affect survival or multiplication within macrophages. Likewise, invasion assays with HeLa cells revealed no differences at 30 and 45 min, whereas, at 1 and 2h, the infection ability of the mutant was drastically reduced. These results suggest that the BMEI0216 gene is required for B. melitensis internalization.
Assuntos
Proteínas de Bactérias/fisiologia , Brucella melitensis/genética , Brucella melitensis/patogenicidade , Genes Bacterianos/fisiologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Ordem dos Genes , Genes Bacterianos/genética , Teste de Complementação Genética , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Mutação , Fatores de TempoRESUMO
Previously, we demonstrated that Escherichia coli tolC mutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 microg/ml to resistance at 40 microg/ml. In this study, we found that the addition of an sbmA mutation to a tolC::Tn10 mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 microg/ml. Inactivation of sbmA alone partially inhibited high-level tetracycline resistance, from resistance at 200 microg/ml to resistance at 120 microg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10 tetracycline resistance.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Elementos de DNA Transponíveis/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Resistência a Tetraciclina/genética , Antibacterianos/farmacologia , Antiporters/genética , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Testes de Sensibilidade Microbiana , Tetraciclina/farmacologia , Transposases/genéticaRESUMO
Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Herbaspirillum/genética , Mutagênese Insercional/métodos , Compostos de Amônio Quaternário/farmacologia , Genes Bacterianos/fisiologia , Herbaspirillum/química , Herbaspirillum/efeitos dos fármacos , Herbaspirillum/metabolismo , Nitrogênio/fisiologiaRESUMO
Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1(-) mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m(-2 )no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1(-) strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.
Assuntos
Proteínas de Bactérias/genética , Complexos de Proteínas Captadores de Luz/genética , Mutação/genética , Rhodovulum/genética , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Teste de Complementação Genética/métodos , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Genéticos , Mutagênese/genética , Peptídeos/genética , Peptídeos/metabolismo , Rhodovulum/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
The protein GlnB-Hs (GlnB of Herbaspirillum seropedicae) in diazotroph micro-organisms signalizes levels of nitrogen, carbon, and energy for a series of proteins involved in the regulation of expression and control of the activity of nitrogenase complex that converts atmospheric nitrogen in ammonia, resulting in biological nitrogen fixation. Its structure has already been determined by X-ray diffraction, revealing a trimer of (36 kDa) with lateral cavities having hydrophilic boundaries. The interactions of GlnB-Hs with the well-known Si(111) surface were investigated for different incubation times, protein concentrations in initial solution, deposition conditions, and substrate initial state. The protein solution was deposited on Si(111) and dried under controlled conditions. An atomic force microscope operating in dynamic mode shows images of circular, linear, and more complex donut-shaped protein arrangement, and also filament types of organization, which vary from a few nanometers to micrometers. Apparently, the filament formation was favored because of protein surface polarity when in contact with the silicon surface, following some specific orientation. The spin-coating technique was successfully used to obtain more uniform surface covering.