A Critical Review of the Arsenic Uptake Mechanisms and Phytoremediation Potential of Pteris vittata

The discovery of the arsenic hyperaccumulator, Pteris vittata (Chinese brake fern), has contributed to the promotion of its application as a means of phytoremediation for arsenic removal from contaminated soils and water. Understanding the mechanisms involved in arsenic tolerance and accumulation of this plant provides valuable tools to improve the phytoremediation efficiency. In this review, the current knowledge about the physiological and molecular mechanisms of arsenic tolerance and accumulation in P. vittata is summarized, and an attempt has been made to clarify some of the unresolved questions related to these mechanisms. In addition, the capacity of P. vittata for remediation of arsenic-contaminated soils is evaluated under field conditions for the first time, and possible solutions to improve the remediation capacity of Pteris vittata are also discussed.     

Diarrhea caused by enterotoxigenic Bacteroides fragilis in children less than 5 years of age in Hanoi, Vietnam

Enterotoxigenic Bacteroides fragilis (ETBF) are considered as an emerging enteropathogen causing diarrhea in children. Eight hundred and thirty-six (836) children less than 5 years of age including 587 children with diarrhea and 249 age-matched controls were involved in the study. Within the group of children with diarrhea, 7.3% (43/587) ETBF was detected by immunoseparation in combination with polymerase chain reaction. The corresponding figure for the controls was 2.4% (6/249) (P<0.01). Within the diarrhea group, the prevalence was significantly higher in children older than 1 year of age. Three subtypes of ETBF isolates have been identified with the prevalence of 67.4%, 18.6%, and 16% for bft-1, bft-2, and a new bft, respectively. In the controls, two of the subtypes were identified, 5 bft-1 and 1 bft-2. More than half (55.8%) of the samples harboring ETBF also had other identified pathogens. The clinical symptoms of the single ETBF infection were not different from those of co-infections. This is the first study of the role of ETBF in children's diarrhea in Vietnam and it is concluded that this pathogen is an important causative agent of diarrhea in children in Hanoi, Vietnam.     

A novel electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas hydrophila,isolated from a microbial fuel cell

A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA-DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila.     

Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.     

Inhibition of cytotoxicity by the Nhe cytotoxin of Bacillus cereus through the interaction of dodecyl maltoside with the NheB component

Nhe ('nonhaemolytic enterotoxin') is a three-component cytotoxin implicated in the pathogenesis of diarrhoea by Bacillus cereus. Nhe forms pores in pure lipid bilayers, but the function of the individual components (NheA, NheB and NheC) remains unclear. NheB and NheC are structural homologues of ClyA, a pore-forming cytotoxin of Escherichia?coli. The non-ionic detergent dodecyl maltoside (DDM) has been shown to inhibit haemolysis of ClyA. We used DDM as a probe to examine the response of the Nhe proteins to DDM micelles. At its critical micellar concentration (0.2?mM), DDM inhibited propidium uptake by the native Nhe complex in Vero and HT29 cell suspensions. Pre-incubation of NheC with DDM did not inhibit cytotoxicity. NheB exhibited marked changes in 1-anilinonaphthalene-8-sulphonic acid (ANS) fluorescence after pre-exposure to DDM. Pre-incubation of NheB with DDM resulted in large molecular weight complexes as detected by size exclusion chromatography and diffusion through sized dialysis membranes and prevented binding of NheB to Vero cell monolayers. These data support a model in which conformational changes and oligomerization of NheB are prerequisite events in the process of pore formation.     

Genetic engineering of Treponema pallidum subsp. pallidum,the Syphilis Spirochete

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kan^R) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan^R gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kan^R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan^R cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl₂-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan^R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan^R message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprA^ko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.