RESUMO
Acinetobacter calcoaceticus BD413 was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron-scavenging phenotype was associated with the secretion of iron-repressible catechol and the induction of a group of six outer membrane proteins with molecular weights ranging from 34 to 85 kDa. The amount of catechol produced was dependent on medium composition and iron stringency. The relation between iron limitation and lipase production was studied at the level of lipA transcription and extracellular lipase activity. In minimal medium, iron limitation slightly affected lipA expression but decreased exo-lipase activity significantly. However, if iron limitation and rich nitrogen sources were simultaneously present in the culture media, the production of lipase was increased approximately 4 times.
Assuntos
Acinetobacter calcoaceticus/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/biossíntese , Ferro/farmacologia , Sideróforos/biossíntese , Acinetobacter calcoaceticus/crescimento & desenvolvimento , Acinetobacter calcoaceticus/fisiologia , Proteínas da Membrana Bacteriana Externa/análise , Catecóis/metabolismo , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica/efeitos dos fármacos , Lipase/metabolismo , Dados de Sequência Molecular , Sideróforos/análiseRESUMO
Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.
Assuntos
Plasmídeos/genética , Replicon , Vibrio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Primers do DNA/genética , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Origem de Replicação , Salmonidae/microbiologia , Vibrio/metabolismo , Vibrio/patogenicidadeRESUMO
The Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius causes a fatal septicaemic disease, resembling fulminant meningococcal sepsis, in children. When isolate F3031 was grown under iron-limiting conditions, the presence of several iron-regulated proteins of 38-110 kDa was revealed by electrophoretic analysis and a Fur homologue was shown by immunoblotting. Dot-blot assays and immunoblotting indicated that BPF cells bound human transferrin and contained transferrin-binding proteins in the outer membrane. However, the binding activity and the biosynthesis of these proteins were detected even under iron-rich conditions. Immunoblot analysis demonstrated the presence of a periplasmic protein related to the ferric iron-binding protein A (FbpA), the major iron-binding protein described in Neisseria spp. However, the FbpA homologue in strain F3031 was constitutively expressed and was smaller than the periplasmic protein detected in H. influenzae type b strain Eagan. The periplasm of strain F3031 also contained a protein related to the Streptococcus parasanguis FimA protein which recently has been shown to be involved in iron acquisition in Yersinia pestis. Although the Eagan and F3031 FimA homologues had a similar mol. wt, of 31 kDa, the expression of the BPF fimA-like gene was not regulated by the iron concentration of the culture medium.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas de Fímbrias , Infecções por Haemophilus/fisiopatologia , Haemophilus influenzae/patogenicidade , Púrpura/microbiologia , Proteínas Repressoras/fisiologia , Transferrina/fisiologia , Western Blotting , Brasil , Proteínas de Transporte , Criança , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica , Haemophilus influenzae/fisiologia , Hemina/metabolismo , Humanos , Ferro/metabolismo , Peso Molecular , Sepse/fisiopatologiaRESUMO
Haemophilus influenzae biogroup aegyptius (H. aegyptius) is the etiological agent of Brazilian purpuric fever (BPF), a recently described pediatric disease that is often fatal. The vascular destruction that occurs in this disease is a distinctive trait, and little is known about the mechanism(s) of the overwhelming purpura fulminans that causes the high mortality associated with this pediatric infection. Iron is an essential micronutrient for nearly all living cells, and the mechanisms used by bacteria to acquire and internalize iron are often associated with virulence. Therefore, the focus of our studies is the molecular characterization of the iron uptake system used by H. aegyptius. Specifically, we are investigating the high-affinity transferrin binding proteins in the bacterial outer membrane, components of ABC transporter systems, and a possible regulatory mechanism for the genes encoding these proteins. A detailed understanding of the molecular nature of the regulatory genetic components and proteins involved in the acquisition of iron will broaden the knowledge of the pathogenesis of the disease caused by H. aegyptius and will also lead to a better understanding of the nature of other infections that affect the vascular system.
Assuntos
Haemophilus influenzae/genética , Vasculite por IgA/microbiologia , Ferro/metabolismo , Receptores da Transferrina/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Previsões , Haemophilus influenzae/metabolismo , Vasculite por IgA/genética , Vasculite por IgA/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismoRESUMO
The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).
Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , Plasmídeos/biossíntese , Vibrio/genética , Proteínas de Bactérias/biossíntese , Clonagem Molecular , DNA Bacteriano/genética , Mutagênese Insercional , Plasmídeos/genética , Replicon , Mapeamento por Restrição , Deleção de Sequência , Vibrio/metabolismoRESUMO
Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.
Assuntos
Acinetobacter calcoaceticus/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Transporte Biológico , Catecóis/isolamento & purificação , Catecóis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ferro/farmacologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/efeitos dos fármacos , Sideróforos/química , Sideróforos/isolamento & purificação , Transferrina/metabolismoRESUMO
Se analizó el contenido plasmídico de una serie de cepas de Klebsiella pneumoniae resistentes a amikacina y otros antibióticos. Estas cepas se identificaron como causantes de epidemias intrahospitalares en unidades pediátricas situadas en distintas localidades geográficas de Argentina. En todos los casos se encontraron plásmidos que poseían determinantes genéticos para resistencia a amikacina. Mediante análisis realizados con cortes con enzimas de restricción e hibridizaciones se determinó la presencia de elementos transponibles relacionados a Tn 1331 en todos las cepas estudiadas. Estos resultados indican que la transposición de estos elementos ha jugado un papel importante en el proceso de diseminación de la resistencia a la amikacina en el género Klebsiella (y probablemente en otras bacterias gram negativas) en Argentina (AU)
Assuntos
Klebsiella pneumoniae/genética , Amicacina/farmacologia , Fatores R/genética , Resistência Microbiana a Medicamentos/genética , Elementos de DNA Transponíveis/efeitos dos fármacos , Argentina , Fatores R/efeitos dos fármacos , Mapeamento CromossômicoRESUMO
Se analizó el contenido plasmídico de una serie de cepas de Klebsiella pneumoniae resistentes a amikacina y otros antibióticos. Estas cepas se identificaron como causantes de epidemias intrahospitalares en unidades pediátricas situadas en distintas localidades geográficas de Argentina. En todos los casos se encontraron plásmidos que poseían determinantes genéticos para resistencia a amikacina. Mediante análisis realizados con cortes con enzimas de restricción e hibridizaciones se determinó la presencia de elementos transponibles relacionados a Tn 1331 en todos las cepas estudiadas. Estos resultados indican que la transposición de estos elementos ha jugado un papel importante en el proceso de diseminación de la resistencia a la amikacina en el género Klebsiella (y probablemente en otras bacterias gram negativas) en Argentina
Assuntos
Amicacina/farmacologia , Klebsiella pneumoniae/genética , Fatores R/genética , Argentina , Mapeamento Cromossômico , Elementos de DNA Transponíveis/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Fatores R/efeitos dos fármacosRESUMO
Plasmids isolated from Klebsiella pneumoniae strains that caused outbreaks in pediatric units in various geographical regions of Argentina harbored genetic determinants for resistance to amikacin. By using restriction endonuclease and Southern blot hybridization analysis it was determined that all of the strains carried plasmids with Tn1331-related elements indicating that transposition of these elements may have played an important role in the dissemination process of resistance to amikacin.
Assuntos
Amicacina/farmacologia , Klebsiella pneumoniae/genética , Fatores R/genética , Argentina , Mapeamento Cromossômico , Elementos de DNA Transponíveis/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Fatores R/efeitos dos fármacosRESUMO
Plasmids isolated from Klebsiella pneumoniae strains that caused outbreaks in pediatric units in various geographical regions of Argentina harbored genetic determinants for resistance to amikacin. By using restriction endonuclease and Southern blot hybridization analysis it was determined that all of the strains carried plasmids with Tn1331-related elements indicating that transposition of these elements may have played an important role in the dissemination process of resistance to amikacin.