RESUMEN
Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.
Asunto(s)
Antibacterianos/metabolismo , Bacillus subtilis/metabolismo , Cromatos/metabolismo , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatos/farmacología , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Orden Génico , Iones/metabolismo , Iones/farmacología , Proteínas de Transporte de Membrana/genética , Operón , Transcripción GenéticaRESUMEN
Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cromatos/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleotidiltransferasas/metabolismo , Porinas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Cromatos/farmacología , Proteínas de Unión al ADN/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Insercional , Nucleotidiltransferasas/genética , Sistemas de Lectura Abierta , Porinas/genética , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.
Asunto(s)
Compuestos de Cromo/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Animales , Transporte Biológico , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Compuestos de Cromo/química , Compuestos de Cromo/metabolismo , Humanos , Oxidación-Reducción , Estrés OxidativoRESUMEN
Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted within the PAO1 genome showed that mutant strains GGP-64 and AJ-22 were affected in open reading frames PA0967 and PA5345, which correspond to the ruvB and recG genes, respectively. These genes encode helicases RuvB and RecG involved in DNA replication, recombination and repair. The chromate resistance phenotype in mutants GGP-64 and AJ-22 was restored by cosmids bearing wild type ruvB or recG genes, respectively. Also, both mutant strains showed an increased susceptibility to the toxic oxyanions tellurite and selenite as well as to mitomycin C, but not to arsenite, paraquat and hydrogen peroxide. It was concluded that P. aeruginosa RuvB and RecG helicases are involved in repairing DNA damage caused by chromate or its derivatives.