Biological effect of phosphate on the dissimilatory arsenate-respiring bacteria-catalyzed reductive mobilization of arsenic from contaminated soils.

Dissimilatory arsenate-respiring prokaryotes (DARPs) are considered to be the major drive of the reductive mobilization of arsenic from solid phases. However, it is not fully understood how phosphate, a structural analog of arsenate, affects the DARPs-mediated arsenic mobilization. This work aimed to address this issue. As-contaminated soils were collected from a Shimen Realgar Mine-affected area. We identified a unique diversity of DARPs from the soils, which possess high As(V)-respiring activities using one of multiple small organic acids as the electron donor. After elimination of the desorption effect of phosphate on the As mobilization, the supplement of additional 10 mM phosphate to the active slurries markedly increased the microbial community-mediated reductive mobilization of arsenic as revealed by microcosm tests; this observation was associated to the fact that phosphate significantly increased the As(V)-respiratory reductase (Arr) gene abundances in the slurries. To confirm this finding, we further obtained a new DARP strain, Priestia sp. F01, from the samples. We found that after elimination of the chemical effect of phosphate, the supplement of 10 mM phosphate to the active slurries resulted in an 82.2% increase of the released As(III) in the solutions, which could be contributed to that excessive phosphate greatly increased the Arr gene abundance, and enhanced the transcriptional level of arrA gene and the bacterial As(V)-respiring activity of F01 cells. Considering that phosphate commonly coexists with As in the environment, and is a frequently-used fertilizer, these findings are helpful for deeply understanding why As concentrations in contaminated groundwater are dynamically fluctuated, and also provided new knowledge on the interactions between the biogeochemical processes of P and As.

Inhibitory Effect of an Acidic Peptide on the Activity of an Antimicrobial Peptide from the Scorpion Mesobuthus martensii Karsch.

Highly acidic peptides with no disulfide bridges are widely present in the scorpion venoms; however, none of them has been functionally characterized so far. Here, we cloned the full-length cDNA of a short-chain highly acidic peptide (referred to as HAP-1) from a cDNA library made from the venom glands of the Chinese scorpion Mesobuthus martensii Karsch. HAP-1 contains 19 amino acid residues with a predicted IP value of 4.25. Acidic amino residues account for 33.3% of the total residues in the molecule of HAP-1. HAP-1 shows 76⁻98% identities to some scorpion venom peptides that have not yet been functionally characterized. Secondary structure prediction showed that HAP-1 contains a beta-sheet region (residues 9⁻17), and two coiled coil regions (residues 1⁻8 and 18⁻19) located at the N-terminal and C-terminal regions of the peptide, respectively. Antimicrobial assay showed that HAP-1 does not have any effect on the growth of the bacterium Staphylococcus aureus AB94004. However, it potently inhibits the antimicrobial activity of a 13-mer peptide from M. martensii Karsch against Staphylococcus aureus AB94004. This finding is the first characterization of the function of such highly acidic peptides from scorpions.

Dissimilatory arsenate-respiring prokaryotes catalyze the dissolution, reduction and release of arsenic from paddy soils into groundwater: implication for the effect of sulfate.

The paddy soils in some areas in Jianghan Plain were severely contaminated by arsenic. However, little is known about the activity and diversity of the dissimilatory arsenate-respiring prokaryotes (DARPs) in the paddy soils, and the effects of sulfate on the microbial mobilization and release of arsenic from soils into solution. To address this issue, we collected arsenic-rich soils from the depths of 1.6 and 4.6 m in a paddy region in the Xiantao city, Hubei Province, China. Microcosm assays indicated that all of the soils have significant arsenate-respiring activities using lactate, pyruvate or acetate as the sole electron donor. Functional gene cloning and analysis suggest that there are diverse DARPs in the indigenous microbial communities of the soils. They efficiently promoted the mobilization, reduction and release of arsenic and iron from soils under anaerobic conditions. Remarkably, when sulfate was amended into the microcosms, the microorganisms-catalyzed reduction and release of arsenic and iron were significantly increased. We further found that sulfate significantly enhanced the arsenate-respiring reductase gene abundances in the soils. Taken together, a diversity of DARPs in the paddy soils significantly catalyzed the dissolution, reduction and release of arsenic and iron from insoluble phase into solution, and the presence of sulfate significantly increased the microbial reactions.

Microbially Mediated Methylation of Arsenic in the Arsenic-Rich Soils and Sediments of Jianghan Plain.

Almost nothing is known about the activities and diversities of microbial communities involved in As methylation in arsenic-rich shallow and deep sediments; the correlations between As biomethylation and environmental parameters also remain to be elucidated. To address these issues, we collected 9 arsenic-rich soil/sediment samples from the depths of 1, 30, 65, 95, 114, 135, 175, 200, and 223 m in Jianghan Plain, China. We used microcosm assays to determine the As-methylating activities of the microbial communities in the samples. To exclude false negative results, we amended the microcosms with 0.2 mM As(III) and 20.0 mM lactate. The results indicated that the microbial communities in all of the samples significantly catalyzed arsenic methylation. The arsM genes were detectable from all the samples with the exception of 175 m, and 90 different arsM genes were identified. All of these genes code for new or new-type ArsM proteins, suggesting that new As-methylating microorganisms are widely distributed in the samples from shallow to deep sediments. To determine whether microbial biomethylation of As occurs in the sediments under natural geochemical conditions, we conducted microcosm assays without exogenous As and carbons. After 80.0 days of incubation, approximately 4.5-15.5 µg/L DMAsV were detected in all of the microcosms with the exception of that from 30 m, and 2.0-9.0 µg/L MMAsV were detected in the microcosms of 65, 114, 135, 175, 200, and 223 m; moreover, approximately 18.7-151.5 µg/L soluble As(V) were detected from the nine sediment samples. This suggests that approximately 5.3, 0, 8.1, 28.9, 18.0, 8.7, 13.8, 10.2, and 14.9% of total dissolved As were methylated by the microbial communities in the sediment samples from 1, 30, 65, 95, 114, 135, 175, 200, and 223 m, respectively. The concentrations of biogenic DMAsV show significant positive correlations with the depths of sediments, and negative correlations with the environmental NH4+ and NaCl concentrations, but show no significant correlations with other environmental parameters, such as NO3-, SO42+, TOC, TON, Fe, Sb, Cu, K, Ca, Mg, Mn, and Al. This work helps to better understand the biogeochemical cycles of arsenic in arsenic-rich shallow and deep sediments.

Draft genome sequence of Arthrobacter sp. strain B6 isolated from the high-arsenic sediments in Datong Basin, China.

Arthrobacter sp. B6 is a Gram-positive, non-motile, facultative aerobic bacterium, isolated from the arsenic-contaminated aquifer sediment in the Datong basin, China. This strain displays high resistance to arsenic, and can dynamically transform arsenic under aerobic condition. Here, we described the high quality draft genome sequence, annotations and the features of Arthrobacter sp. B6. The G + C content of the genome is 64.67%. This strain has a genome size of 4,663,437 bp; the genome is arranged in 8 scaffolds that contain 25 contigs. From the sequences, 3956 protein-coding genes, 264 pseudo genes and 89 tRNA/rRNA-encoding genes were identified. The genome analysis of this strain helps to better understand the mechanism by which the microbe efficiently tolerates arsenic in the arsenic-contaminated environment.

Luteimonas arsenica sp. nov., an arsenic-tolerant bacterium isolated from arsenic-contaminated soil.

A Gram-stain-negative, rod-shaped bacterium that formed yellow and viscous colonies was isolated from arsenic-contaminated soil of the Jianghan plain, Hubei Province, China, and it was designated 26-35T. This strain was capable of resisting arsenate and arsenite with MICs of 40 and 20 mM, respectively. The 16S rRNA gene of the novel isolate displayed 96.7-94.2 % sequence similarities to those of other known species of the genus Luteimonas. The respiratory quinone was ubiquinone-8 (Q-8). The DNA G+C content was 71.4 mol%. The predominant cellular fatty acids were iso-C15 : 0, iso-C16 : 0, iso-C17 : 0, iso-C11 : 0, iso-C11 : 0 3-OH and iso-C17 : 1ω9c. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Phylogenetic and physiological analysis indicated that the isolate represents a novel species of the genus Luteimonas, for which the name Luteimonas arsenica sp. nov. is proposed. The type strain is 26-35T (=KCTC 42824T=CCTCC AB 2014326T).

Unique diversity of the venom peptides from the scorpion Androctonus bicolor revealed by transcriptomic and proteomic analysis.

Androctonus bicolor is one of the most poisonous scorpion species in the world. However, little has been known about the venom composition of the scorpion. To better understand the molecular diversity and medical significance of the venom from the scorpion, we systematically analyzed the venom components by combining transcriptomic and proteomic surveys. Random sequencing of 1000 clones from a cDNA library prepared from the venom glands of the scorpion revealed that 70% of the total transcripts code for venom peptide precursors. Our efforts led to a discovery of 103 novel putative venom peptides. These peptides include NaTx-like, KTx-like and CaTx-like peptides, putative antimicrobial peptides, defensin-like peptides, BPP-like peptides, BmKa2-like peptides, Kunitz-type toxins and some new-type venom peptides without disulfide bridges, as well as many new-type venom peptides that are cross-linked with one, two, three, five or six disulfide bridges, respectively. We also identified three peptides that are identical to known toxins from scorpions. The venom was also analyzed using a proteomic technique. The presence of a total of 16 different venom peptides was confirmed by LC-MS/MS analysis. The discovery of a wide range of new and new-type venom peptides highlights the unique diversity of the venom peptides from A. bicolor. These data also provide a series of novel templates for the development of therapeutic drugs for treating ion channel-associated diseases and infections caused by antibiotic-resistant pathogens, and offer molecular probes for the exploration of structures and functions of various ion channels.

Androcin, a novel type of cysteine-rich venom peptide from Androctonus bicolor, induces akinesia and anxiety-like symptoms in mice.

A novel type of venom peptide with six disulfide bridges, referred to as Androcin, was identified from the scorpion Androctonus bicolor using a cDNA library strategy. The amino acid sequence of Androncin displays little identity to other known peptides from scorpions. We found that the genomic sequence of Androcin consists of three exons interrupted by two introns that are localized in the signal sequence and mature peptide encoding regions, inserted in phase 2 and phase 0, respectively. This genomic organization is unique among those of the cysteine-rich peptides from scorpions described so far. The primary and secondary structures of Androcin are homologous to those of the N-terminal domains of insulin-like growth factor-binding proteins; this suggests that Androcin may block the normal function of IGFs. Toxicological analysis using the recombinant Androcin peptide revealed that Androcin is able to induce severe akinesia and anxiety-like symptoms in mice. Androcin is a novel mammalian toxin with six disulfide bridges that provides the scorpion with another tool to subdue animals.

Genome-wide search and comparative genomic analysis of the trypsin inhibitor-like cysteine-rich domain-containing peptides.

It was shown that peptides containing trypsin inhibitor-like cysteine-rich (TIL) domain are able to inhibit proteinase activities, and thus play important roles in various biological processes, such as immune response and anticoagulation. However, only a limited number of the TIL peptides have been identified and characterized so far; and little has been known about the evolutionary relationships of the genes encoding the TIL peptides. BmKAPi is a TIL domain-containing peptide that was identified from Mesobuthus martensii Karsch. Here, we conducted genome-wide searches for new peptides that are homologous to BmKAPi or possess a cysteine pattern similar to that of BmKAPi. As a result, we identified a total of 80 different TIL peptides from 34 species of arthropods. We found that these peptides can be classified into seven evolutionarily distinct groups. Furthermore, we cloned the genomic sequence of BmKAPi; the genomic sequences of the majority of other TIL peptides were also identified from the GenBank database using bioinformatical approaches. Through phylogenetic and comparative genomic analysis, we found 26 cases of intron gain events occurred in the genes of the TIL peptides; however, no instances of intron loss were observed. Moreover, we found that alternative splicing contributes to the diversification of the TIL peptides. It is interesting to see that four genes of the TIL domain-containing peptides overlap in a DNA region located on the chromosome LG B15 of Bombus terretris. These data suggest that the evolution of the TIL peptide genes are dynamic, which was dominated by intron gain.

Three new antimicrobial peptides from the scorpion Pandinus imperator.

Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.

Molecular and bioinformatical characterization of a novel superfamily of cysteine-rich peptides from arthropods.

The full-length cDNA sequences of two novel cysteine-rich peptides (referred to as HsVx1 and MmKTx1) were obtained from scorpions. The two peptides represent a novel class of cysteine-rich peptides with a unique cysteine pattern. The genomic sequence of HsVx1 is composed of three exons interrupted by two introns that are localized in the mature peptide encoding region and inserted in phase 1 and phase 2, respectively. Such a genomic organization markedly differs from those of other peptides from scorpions described previously. Genome-wide search for the orthologs of HsVx1 identified 59 novel cysteine-rich peptides from arthropods. These peptides share a consistent cysteine pattern with HsVx1. Genomic comparison revealed extensive intron length differences and intronic number and position polymorphisms among the genes of these peptides. Further analysis identified 30 cases of intron sliding, 1 case of intron gain and 22 cases of intron loss occurred with the genes of the HsVx1 and HsVx1-like peptides. It is interesting to see that three HsVx1-like peptides XP_001658928, XP_001658929 and XP_001658930 were derived from a single gene (XP gene): the former two were generated from alternative splicing; the third one was encoded by a DNA region in the reverse complementary strand of the third intron of the XP gene. These findings strongly suggest that the genes of these cysteine-rich peptides were evolved by intron sliding, intron gain/loss, gene recombination and alternative splicing events in response to selective forces without changing their cysteine pattern. The evolution of these genes is dominated by intron sliding and intron loss.