RESUMO
The Burkholderia xenovorans LB400 multireplicon genome displays a relatively high proportion of redundant genes, including several genes predicted to be related to arsenic resistance. These comprise an ars gene cluster, composed of the arsR3, acr3, arsC1 and arsH genes, as well as two arsB, arsC2, and seven individual arsR genes. The objective of this work was to elucidate the involvement of the ars gene cluster in arsenic resistance by the LB400 strain. Susceptibility tests showed that B. xenovorans LB400 is highly resistant to arsenate and arsenite. Arsenic resistance was induced by prior exposure of LB400 to arsenate or arsenite. reverse transcription-polymerase chain reaction assays using total RNA from LB400 showed arsenite-induced transcription of the arsR3 gene, suggesting that the ars gene cluster constitutes an arsenite-responsive operon. Transfer of cloned LB400 ars genes to heterologous Escherichia coli or Pseudomonas aeruginosa strains demonstrated that the ArsR3 transcriptional repressor, ArsC1 arsenate reductase, and the Acr3 arsenite efflux pump encoded in the LB400 ars gene cluster, are all associated to the arsenic resistance phenotype of this strain. The ars gene cluster from Burkholderia xenovorans LB400 is responsible for the inducible arsenic-resistance phenotype of the bacterium.
Assuntos
Arsênio/metabolismo , Proteínas de Bactérias/genética , Burkholderia/genética , Farmacorresistência Bacteriana/genética , Família Multigênica/genética , Arseniato Redutases/genética , Arseniatos/metabolismo , Arseniatos/toxicidade , Arsênio/toxicidade , Arsenitos/metabolismo , Arsenitos/toxicidade , Proteínas de Bactérias/metabolismo , Sequência de Bases , Burkholderia/efeitos dos fármacos , Burkholderia/crescimento & desenvolvimento , Clonagem Molecular , DNA Bacteriano/análise , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana , Óperon , Fenótipo , Filogenia , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Transativadores/genéticaRESUMO
The Pseudomonas aeruginosa plasmid pUM505 contains in a pathogenicity island the dsbA2 gene, which encodes a product with similarity to DsbA protein disulfide isomerases, enzymes that catalyze formation and isomerization of disulfide bonds in protein cysteine residues. Using transcriptional fusions, it was found that dsbA2 gene promoter is activated during the stationary phase, suggesting that DsbA2 protein may be required for adaptive changes that occur during this stage of bacterial growth. Transfer of the pUM505 dsbA2 gene to a cadmium-sensitive P. aeruginosa PAO1-derivative affected in the chromosomal dsbA gene, restored cadmium resistance, suggesting a role of DsbA2 in protecting protein disulfide bonds. PAO1 dsbA2 transformants displayed increased sensitivity to intercalating agent mitomycin C, indicating that DsbA2 functions as a thioredoxin enzyme able to modify and activate toxicity of this compound. These results highlight the adaptive role of the pUM505 plasmid in its P. aeruginosa hosts.
Assuntos
Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Isomerases de Dissulfetos de Proteínas/genética , Tiorredoxinas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cádmio/farmacologia , Cádmio/toxicidade , Clonagem Molecular , Farmacorresistência Bacteriana , Ordem dos Genes , Mitomicina/farmacologia , Isomerases de Dissulfetos de Proteínas/química , Pseudomonas aeruginosa/genética , Tiorredoxinas/químicaRESUMO
The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA- mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA- mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA- mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.
Assuntos
Regulação Bacteriana da Expressão Gênica , Plasmídeos , Pseudomonas aeruginosa/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Resposta SOS em Genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Óperon , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.
