The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.Resistance to arsenite (As 3ϩ ), antimonite (Sb 3ϩ ), and arsenate (As 5ϩ ) is found in both gram-negative and gram-positive bacteria (17). High-level resistance has been associated with plasmid-encoded arsenical resistance (ars) operons (12,25,26,40). These plasmid-encoded metalloid resistances were widespread in bacterial species even before the emergence of resistances to most antibiotics (14). The ars operon carried on the Escherichia coli R factor R773 (10, 12) encodes a transport system that extrudes arsenate, arsenite, and antimonite; the lowering of the intracellular concentration of toxic oxyanion produces resistance (24,30,36). The ars operon of the E. coli conjugal plasmid R773 has five genes, arsRDABC (4, 34, 45). The arsR and arsD genes encode regulatory proteins (43-45). The arsA and arsB genes encode the subunits of an ATP-driven arsenite pump (7). The ArsA protein is the catalytic subunit of the pump (13), while the ArsB protein is the membrane sector (8, 42). In the absence of the arsA gene, the arsB gene product alone provides partial arsenite resistance, most likely by functioning as a secondary uniporter (7a, 9). Arsenate resistance is conferred by reduction to arsenite by the arsC gene product; the resulting arsenite is extruded by the transport system (11,27).The staphylococcal plasmids pI258 and pSX267 also carry ars operons (15, 31). Those operons have only three genes, arsRBC, lacking arsD and arsA genes. Resistance also results from active extrusion of arsenite, and the gram-positive homologs of the ArsB protein have also been proposed to function as secondary porters (3). Although the R773 and pI258 ArsB proteins are only 58% similar in sequence, secondary structural predictions suggest that the two proteins are topologically much more similar, and functional chimeras of the two have been constructed, suggesting that the two ArsB proteins function similarly at the biochemical level (9). Similarly, the ArsC protein of plasmid pI258 is also an arsenate reductase (16), even though there is little sequence similarity between the ArsC proteins of plasmids R773 and pI258. The ArsR proteins of the plasmids of the gram-positive and gram-negative organisms exhibit only 30% sequence similarity, but both are arsenite-responsive repressor proteins (31,43).Recently Sofia and coworkers (39) determined the sequence of a 225.4-kb segment of the E. coli genome corresponding to min 76.0 to 81.5 on the genetic map. Three open reading frames, termed arsE, arsF, and ars...
The ars operon of the Escherichia coli plasmid R773 that confers arsenical and antimonial resistance is negatively regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates. However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coordinate. A site-directed mutagenic approach was employed in a search for a third arsenic ligand. ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-ars DNA complexes could not be dissociated by inducers. Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the operon. While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand. Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations. Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo. In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex. Thus, two ArsR-arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide affinity resin. ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin. Alteration of any two of the three resulted in loss of binding. Arsenic X-ray absorption spectroscopy of ArsR treated stoichiometrically with arsenite confirmed the average arsenic coordination as AsS3 These results suggest that all three cysteine thiolates are arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction.
Aluminum (Al) toxicity is a serious limitation to worldwide crop production. Rice is one of the most Al-tolerant crops and also serves as an important monocot model plant. This study aims to identify Al-responsive proteins in rice, based on evidence that Al resistance is an inducible process. Two Al treatment systems were applied in the study: Al 31 -containing simple Ca solution culture and Al 31-containing complete nutrient solution culture. Proteins prepared from rice roots were separated by 2-DE. The 2-DE patterns were compared and the differentially expressed proteins were identified by MS. A total of 17 Al-responsive proteins were identified, with 12 of those being up-regulated and 5 down-regulated. Among the up-regulated proteins are copper/ zinc superoxide dismutase (Cu-Zn SOD), GST, and S-adenosylmethionine synthetase 2, which are the consistently known Al-induced enzymes previously detected at the transcriptional level in other plants. More importantly, a number of other identified proteins including cysteine synthase (CS), 1-aminocyclopropane-1-carboxylate oxidase, G protein b subunit-like protein, abscisic acid-and stress-induced protein, putative Avr9/Cf-9 rapidly elicited protein 141, and a 33 kDa secretory protein are novel Al-induced proteins. Most of these proteins are functionally associated with signaling transduction, antioxidation, and detoxification. CS, as consistently detected in both Al stress systems, was further validated by Western blot and CS activity assays. Moreover, the metabolic products of CS catalysis, i.e. both the total glutathione pool and reduced glutathione, were also significantly increased in response to Al stress. Taken together, our results suggest that antioxidation and detoxification ultimately related to sulfur metabolism, particularly to CS, may play a functional role in Al adaptation for rice.
BackgroundClimate oscillation may have a profound effect on species distributions, gene flow patterns and population demography. In response to environmental change, those species restricted to montane habitats experienced expansions and contractions along elevation gradients, which can drive differentiation among sky islands.ResultsThe Shangcheng stout salamander (Pachyhynobius shangchengensis) is a cool stream amphibian restricted to high-elevation areas in the Dabie Mountains, East China. In the present study, we used mtDNA genes (Cyt b and ND2) of 193 individuals and 12 nuclear microsatellite loci genotyped on 370 individuals, representing 6 populations (JTX, KHJ, MW, TTZ, BYM and KJY) across the taxon’s distribution area, to investigate their genetic variation and evolutionary history of P. shangchengensis. Most populations showed unusually high levels of genetic diversity. Phylogenetic analyses revealed five monophyletic clades with divergence times ranging from 3.96 to 1.4 Mya. Accordingly, significant genetic differentiation was present between these populations. Bayesian skyline plot analyses provided that all populations underwent long-term population expansions since the last inter-glacial (0.13 Mya ~ 0.12 Mya). Msvar analyses found recent signals of population decline for two northern populations (JTX and KHJ) reflecting a strong bottleneck (approximately 15-fold decrease) during the mid-Holocene (about 6000 years ago). Ecological niche modelling has shown a discontinuity in suitable habitats for P. shangchengensis under different historical climatic conditions.ConclusionsOur results suggest that the niche conservatism of P. shangchengensis and sky island effects may have led to long-term isolation between populations. In sky island refuges, the mid-latitude Dabie Mountains have provided a long-term stable environment for P. shangchengensis, which has led to the accumulation of genetic diversity and has promoted genetic divergence.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1333-8) contains supplementary material, which is available to authorized users.
A bacterial sensing system that responds selectively to antimonite and arsenite has been investigated. The bacteria used in these studies have been genetically engineered to produce the enzyme beta-galactosidase in response to these ions. This is accomplished by using a plasmid that incorporates the gene for beta-galactosidase (reporter gene) under the control of the promoter of the ars operon. This plasmid also encodes for the ArsR protein, a regulatory protein of the ars operon, which, in the absence of antimonite or arsenite, restricts the expression of beta-galactosidase. In the presence of antimonite or arsenite the ArsR protein is released from the operator/ promoter region of the ars operon and beta-galactosidase is expressed. The activity of this enzyme was monitored electrochemically using p-aminophenyl beta-D-galactopyranoside as the substrate. The bacterial sensing system responds selectively to arsenite and antimonite (and to a lesser extent arsenate) and shows no significant response to phosphate, sulfate, nitrate, and carbonate.
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