Dissimilatory perchlorate reduction: a review.

In the United States anthropogenic activities are mainly responsible for the wide spread perchlorate contamination of drinking water, surface water, groundwater, and soil. Even at microgram levels, perchlorate causes toxicity to flora and fauna and affects growth, metabolism and reproduction in humans and animals. Reports of antithyroid effects of perchlorate and its detection in common food items have raised serious public health concerns, leading to extensive decontamination efforts in recent years. Several physico-chemical removal and biological decontamination processes are being developed. Although promising, ion exchange is a non-selective and incomplete process as it merely transfers perchlorate from water to the resin. The perchlorate-laden spent resins (perchlorate 200-500 mg L(-1)) require regeneration resulting in production of concentrated brine (6-12% NaCl) or caustic waste streams. On the contrary, biological reduction completely degrades perchlorate into O(2) and innocuous Cl(-). High reduction potential of ClO(4)(-)/Cl(-) (E° =∼ 1.28 V) and ClO(3)(-)/Cl(-) pairs (E° =1.03 V) makes these contaminants thermodynamically ideal e(-) acceptors for microbial reduction. In recent years unique dissimilatory perchlorate reducing bacteria have been isolated and detailed studies pertaining to their microbiological, biochemical, genetics and phylogenetic aspects have been undertaken which is the subject of this review article while the various physico-chemical removal and biological reduction processes have been reviewed by others.

Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

Characterization of interactions between and among components of the meiotic silencing by unpaired DNA machinery in Neurospora crassa using bimolecular fluorescence complementation.

Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant.

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.

Cyclosporin A-resistance based gene placement system for Neurospora crassa.

DNA introduced into Neurospora crassa are usually inserted at random ectopic sites of the genome, often in multiple copies. To facilitate the study of gene expression and function, transformation by a single-copy of a gene at a defined locus is desired. Although several targeted gene placement methods are available for N. crassa, they all require a specific genetic background in the recipient. We describe here the development of a new locus for targeted gene placement that does not require any pre-existing marker in the target strain. Our system takes advantage of the fact that disruption of the csr-1 gene, which encodes the cyclosporin A-binding protein, leads to the resistance to cyclosporin A. By cloning a gene of interest into a csr-1 knock-in vector and transforming a fungus with it, one can easily insert any gene, in single-copy, into a defined locus.

Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris.

The study describes expression and purification of recombinant hepatitis B small surface antigen (rHBsAg hereafter) in methylotrophic yeast Pichia pastoris strain GS115. For expression of the rHBsAg, a single copy of 678 bp cDNA was inserted at the unique EcoRI site downstream of the alcohol oxidase (AOX 1) promoter of the 8.2 kb pHIL-D2 vector. The cDNA-pHIL-D2 construct was used to transform the strain GS115, resulting in a Mut(S) (Methanol Utilizing Slow) phenotype in which the 226 amino acids containing active and full-length rHBsAg protein could be expressed intra-cellularly during slow growth and induction with methanol. The recombinant protein from the Mut(S) expressor was harvested by cell disruption, and purified first by adsorption-desorption on aerosil followed by two-step chromatographic separation i.e. anion exchange on DEAE resin followed by gel permeation on Superdex 75. Reversed passive hem-agglutination assay (RPHA) was used to test the antigenicity while SDS-PAGE was performed to check the purity of the 27 kDa rHBsAg and its aggregates. The results showed that disruption at 12 Kpsi (three cycles), or 30 Kpsi (1 cycle), desorption with 10mM carbonate buffer (pH 9-10), and storage at 4 degrees C without detergent did not adversely affect the antigenicity of the rHbsAg. However, the presence of detergents such as TritonX100 and deoxycholate in the disruption and desorption buffers, respectively resulted in reduced antigenicity during storage both at 4 and -20 degrees C in spite of higher initial yields.

Bioremediation potential of a perchlorate-enriched sewage sludge consortium.

The purpose of this work was to explore the reductive bioremediation potential of a perchlorate-enriched facultative anaerobic consortium. Rapid perchlorate reduction and bacterial growth were observed up to 1.84 g l(-1) of perchlorate, but not at 3.82 g l(-1) due to the toxicity. The specific growth rate of the mixed consortium was 0.1 h(-1). The consortium co-reduced perchlorate and nitrate with acetate as e- donor and carbon source. The presence of nitrate slowed down the perchlorate reduction rate. The other e- acceptors utilized include oxygen, chlorate, Cr(VI), and selenate. Over 95% of the 16 mg l(-1) of added Cr(VI) was reduced within 24 h of incubation with a high-density perchlorate-grown consortium. However, the consortium failed to couple growth with reduction of nitrite, sulfate, thiosulfate, and sulfite. During the search for autotrophic perchlorate reduction, many consortia from very diverse natural sources could not use sulfur compounds such as thiosulfate as e- donor.

Role of Citrobacter amalonaticus and Citrobacter farmeri in dissimilatory perchlorate reduction.

The article deals with the novel physiological function of dissimilatory perchlorate reduction by strains JB101 and JB109 isolated from a laboratory-enriched mixed consortium originating from a sewage treatment facility. The biochemical and physiological data of the strains showed good correspondence with members of the family Enterobacteriaceae. The partial 16S rDNA sequence of the strains JB101 and JB109 had similarity of 99.8% to Citrobacter amalonaticus and 98% to Citrobacter farmeri, respectively. The results inferred the possibility of Citrobacter spp. to form an important group of dissimilatory perchlorate reducers among the gamma subclass of Proteobacteria, since the majority of the perchlorate reducers belong to two monophyletic groups, Dechloromonas and Dechlorosoma in beta subclass. The perchlorate-grown Citrobacter strains preferred perchlorate to nitrate as an electron acceptor unlike most of the reported dissimilatory perchlorate reducers.

Interference of thiosulfate during colorimetric analysis of hexavalent chromium using 1,5-diphenylcarbazide method.

Thiosulfate (S(2)O(3)(2-)) contained in the media for autotrophic Cr (VI) reduction was found to interfere with Cr (VI) measurement following the 1,5-diphenylcarbazide (DPC) method. The interference was confirmed at several abiotic and biotic conditions, and was influenced by S(2)O(3)(2-) concentration, pH, and the media components. At neutral to alkaline pH, 500 mg/L of S(2)O(3)(2-) did not cause interference, while 4 mg/L of S(2)O(3)(2-) resulted in the interference at pH 2.0. Atomic absorption spectrophotometry could be an alternative method when the interference by S(2)O(3)(2-) is expected.