Evaluation of phagocytic activity and nitric oxide generation by molluscan haemocytes as biomarkers of inorganic arsenic exposure

The natural habitats of the freshwater bivalve Lamellidens marginalis face the risk of contamination by the toxic metalloid arsenic. Haemocyte-mediated non-self phagocytosis and generation of nitric oxide (NO) as reactive nitrogen intermediate were examined to establish the reliability of the parameters as biomarkers of sodium arsenite-induced stress on the animal at sublethal concentrations. The studies suggest imposition of a remarkable immune compromise/immune suppression on the animal by the natural contaminant. The animal expressed partial recovery in its phagocytic potential and NO generation over a period of 30 days. Quantitation of phagocytic efficiency and intrahaemocyte NO generation indicates the possibility of the parameters be accepted as cellular biomarkers to estimate and characterize the vulnerability of the freshwater organisms to sodium arsenite-induced stress.     

Sustaining immunity during starvation in bivalve mollusc: A costly affair

Complete or partial depletion of resource in a freshwater habitat is a common phenomenon. As a consequence, aquatic fauna including bivalve molluscs may be exposed to dietary stress on a seasonal basis. Haemocyte based innate immune profile of the freshwater mollusc Lamellidens marginalis (Bivalvia: Eulamellibranchiata) was evaluated under starvation induced stress for a maximum period of 32 days in a controlled laboratory condition. During starvation, the bivalve haemocytes maintained a homeostasis in phagocytic efficacy and nitric oxide generation ability with respect to the control. The mollusc maintained a significantly high protein content in its haemolymph and tissues under the nutritional stress with respect to the control. The dietary stress had no significant impact on the activity of digestive tissue derived α-amylase till sixteenth day but by 32 days the enzyme activity went down significantly. The histopathological profile revealed that the bivalve was adapted to maintain a steady immune profile by incurring degeneration of its own tissue structure. The total haemocyte count surged significantly till 16 days but differed insignificantly with respect to the control at 32 days implying probable haematopoietic exhaustion. The study reflects the instinctive urge of the bivalve to maintain immune physiology at heavy metabolic cost under nutrient limited condition.     

Respiratory arsenate reductase as a bidirectional enzyme

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.     

Engineering the Soil Bacterium Pseudomonas putida for Arsenic Methylation

Accumulation of arsenic has potential health risks through consumption of food. Here, we inserted the arsenite [As(III)] S-adenosylmethionine methyltransferase (ArsM) gene into the chromosome of Pseudomonas putida KT2440. Recombinant bacteria methylate inorganic arsenic into less toxic organoarsenicals. This has the potential for bioremediation of environmental arsenic and reducing arsenic contamination in food.     

Complete genomes of freshwater sulfur oxidizers Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H: Genetic insights into the sulfur oxidation pathway of betaproteobacteria

Despite detailed studies of marine sulfur-oxidizing bacteria, our knowledge concerning their counterparts in freshwater lake ecosystems is limited. Genome sequencing of the freshwater sulfur-oxidizing betaproteobacteria Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H have been completed. Strain skB26 possessed a circular plasmid of 86.6-kbp in addition to its chromosome, and an approximate 18-kbp region of the plasmid was occupied by an arxA-like operon, encoding a new clade of anaerobic arsenite oxidase. Multilocus sequence analysis showed that strain skB26 could not be assigned to any existing order; thus a novel order, Sulfuricellales, is proposed. The genomes of strains skB26 and sk43H were examined, focusing on the composition and the phylogeny of genes involved in the oxidation of inorganic sulfur compounds. Strains skB26 and sk43H shared a common pathway, which consisted of Sqr, SoxEF, SoxXYZAB, Dsr proteins, AprBA, Sat, and SoeABC. Comparative genomics of betaproteobacterial sulfur oxidizers showed that this pathway was also shared by the freshwater sulfur oxidizers Thiobacillus denitrificans and Sideroxydans lithotrophicus. It also revealed the presence of a conserved gene cluster, which was located immediately upstream of the betaproteobacterial dsr operon.     

Electrozymographic evaluation of the attenuation of arsenic induced degradation of hepatic SOD,catalase in an in vitro assay system by pectic polysaccharides of Momordica charantia in combination with curcumin

Momordica charantia (MC) fruit known as bitter gourd, is of potential nutritional and medicinal value. The objectives of the present in vitro study were to evaluate the efficacy of bioactive pectic polysaccharides (CCPS) of MC along with another well-known bioactive compound curcumin in the abrogation of hepatocellular oxidative stress persuaded by sodium arsenite. Electrozymographic method was developed for the assessment of superoxide dismutase (SOD) and catalase activities of liver tissues maintained under an in vitro system. A significant association of CCPS of MC in combination with curcumin was found in the alleviation of oxidative stress induced by sodium arsenite in liver slice. Generated data pointed out that CCPS of MC and curcumin separately or in combination can offer significant protection against alterations in malondialdehyde (MDA), conjugated diene (CD) and antioxidative defense (SOD, CAT) markers. Furthermore, results of hepatic cell DNA degradation strongly supported that both these co-administrations have efficacy in preventing cellular damage. This is the first information of extracted polysaccharides from MC preventing arsenic induced damage in a liver slice of rat.     

Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser

Background  

Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

Toxicity, transformation and accumulation of inorganic arsenic species in a microalga Scenedesmus sp. isolated from soil

Arsenic speciation and cycling in the natural environment are highly impacted via biological processes. Since arsenic is ubiquitous in the environment, microorganisms have developed resistance mechanisms and detoxification pathways to overcome the arsenic toxicity. This study has evaluated the toxicity, transformation and accumulation of arsenic in a soil microalga Scenedesmus sp. The alga showed high tolerance to arsenite. The 72-h 50 % growth inhibitory concentrations (IC₅₀ values) of the alga exposed to arsenite and arsenate in low-phosphate growth medium were 196.5 and 20.6 mg? L^?1, respectively. When treated with up to 7.5 mg? L^?1 arsenite, Scenedesmus sp. oxidised all arsenite to arsenate in solution. However, only 50 % of the total arsenic remained in the solution while the rest was accumulated in the cells. Thus, this alga has accumulated arsenic as much as 606 and 761 μg? g^?1 dry weight when exposed to 750 μg? L^?1 arsenite and arsenate, respectively, for 8 days. To our knowledge, this is the first report of biotransformation of arsenic by a soil alga. The ability of this alga to oxidise arsenite and accumulate arsenic could be used in bioremediation of arsenic from contaminated water and soil.     

Toxicological properties of the thiolated inorganic arsenic and arsenosugar metabolite thio-dimethylarsinic acid in human bladder cells

Thio-dimethylarsinic acid (thio-DMA^V) has recently been identified as human metabolite after exposure toward both the human carcinogen inorganic arsenic and arsenosugars, which are the major arsenical constituents of marine algae. This study aims to get further insight in the toxic modes of action of thio-DMA^V in cultured human urothelial cells. Among others effects of thio-DMA^V on eight cell death related endpoints, cell cycle distribution, genotoxicity, cellular bioavailability as well as for the first time its impact on DNA damage induced poly(ADP-ribosyl)ation were investigated and compared to effects induced by arsenite. The data indicate that thio-DMA^V exerts its cellular toxicity in a similar or even lower concentration range, however most likely via different mechanisms, than arsenite. Most interestingly, thio-DMA^V decreased damage-induced cellular poly(ADP-ribosyl)ation by 35,000-fold lower concentrations than arsenite. The inhibition of this essential DNA-damage induced and DNA-repair related signaling reaction might contribute to inorganic arsenic induced toxicity, at least in the bladder. Therefore, and also because thio-DMA^V is to date by far the most toxic human metabolite identified after arsenosugar intake, thio-DMA^V should contemporary be fully (also in vivo) toxicologically characterized, to assess risks to human health related to inorganic arsenic but especially arsenosugar dietary intake.     

Environmental Stress and Antibiotic Resistance in Food-Related Pathogens

This study investigated the possibility that sublethal food preservation stresses (high or low temperature and osmotic and pH stress) can lead to changes in the nature and scale of antibiotic resistance (ABR) expressed by three food-related pathogens (Escherichia coli, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus). The study found that some sublethal stresses significantly altered antibiotic resistance. Incubation at sublethal high temperature (45°C) decreased ABR. Incubation under increased salt (>4.5%) or reduced pH (<5.0) conditions increased ABR. Some of the pathogens continued to express higher levels of ABR after removal of stress, suggesting that in some cases the applied sublethal stress had induced stable increases in ABR. These results indicate that increased use of bacteriostatic (sublethal), rather than bactericidal (lethal), food preservation systems may be contributing to the development and dissemination of ABR among important food-borne pathogens.     

Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea

Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics.     

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.     

Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO₄:AsO₄, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.     

Effects of phosphate on arsenate and arsenite sensitivity in two rice (Oryza sativa L.) cultivars of different sensitivity

Arsenate and arsenite sensitivity and arsenate influx tests were conducted for two rice cultivars of different arsenic sensitivity, Azucena and Bala. These were to establish if the mechanism of reduced arsenic sensitivity is achieved through an altered phosphate uptake system, as shown for Holcus lanatus. High phosphate treatments (≥50 μM) provided protection against both arsenate and arsenite. Unlike the H. lanatus tolerance mechanism, in the less sensitive cultivar Bala, arsenate influx did not decrease with phosphate treatment and phosphate transporters appeared to be constitutively upregulated; V_max for arsenate influx remain similar when Bala was grown in the presence or absence of phosphate (V_max - 0.90 and 0.63 nmol g⁻¹ f.wt min⁻¹ respectively). Although mean K_m appear different, Bala did not show lower affinity to arsenate than Azucena in the absence of phosphate (K_m - Azucena, 0.30 mM and Bala, 0.18), while in phosphate treatment, Bala arsenate affinity was half that observed for Azucena (K_m - Azucena, 0.14 and Bala, 0.36 mM). These were low compared to a 4 and 6 fold decrease seen for similar studies on H. lanatus in the absence and presence of phosphate. Phosphate-induced arsenic protection was observed but the mechanism does not resemble that of H. lanatus. Alternative mechanisms were discussed.     

A new aerobic chemolithoautotrophic arsenic oxidizing microorganism isolated from a high Andean watershed

Biological arsenic oxidation has been suggested as a key biogeochemical process that controls the mobilization and fate of this metalloid in aqueous environments. To the best of our knowledge, only four aerobic chemolithoautotrophic arsenite-oxidizing (CAO) bacteria have been shown to grow via direct arsenic oxidation and to have the essential genes for chemolithoautotrophic arsenite oxidation. In this study, a new CAO bacterium was isolated from a high Andean watershed evidencing natural dissolved arsenic attenuation. The bacterial isolate, designated TS-1, is closely related to the Ancylobacter genus, in the Alphaproteobacteria class. Results showed that TS-1 has genes for arsenite oxidation and carbon fixation. The dependence of bacterial growth from arsenite oxidation was demonstrated. In addition, a mathematical model was suggested and the kinetic parameters were obtained by simultaneously fitting the biomass growth, arsenite depletion curves, and arsenate production. This research increases the knowledge of chemolithoautotrophic arsenic oxidizing microorganisms and its potential role as a driver for natural arsenic attenuation.