Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative λ_max at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S₈ configuration. No labeled elemental sulfur was detected when the cells were grown on [³⁵S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.     

Cysteine Is Not the Sulfur Source for Iron-Sulfur Cluster and Methionine Biosynthesis in the Methanogenic Archaeon Methanococcus maripaludis

Three multiprotein systems are known for iron-sulfur (Fe-S) cluster biogenesis in prokaryotes and eukaryotes as follows: the NIF (nitrogen fixation), the ISC (iron-sulfur cluster), and the SUF (mobilization of sulfur) systems. In all three, cysteine is the physiological sulfur source, and the sulfur is transferred from cysteine desulfurase through a persulfidic intermediate to a scaffold protein. However, the biochemical nature of the sulfur source for Fe-S cluster assembly in archaea is unknown, and many archaea lack homologs of cysteine desulfurases. Methanococcus maripaludis is a methanogenic archaeon that contains a high amount of protein-bound Fe-S clusters (45 nmol/mg protein). Cysteine in this archaeon is synthesized primarily via the tRNA-dependent SepRS/SepCysS pathway. When a ΔsepS mutant (a cysteine auxotroph) was grown with ³⁴S-labeled sulfide and unlabeled cysteine, <8% of the cysteine, >92% of the methionine, and >87% of the sulfur in the Fe-S clusters in proteins were labeled, suggesting that the sulfur in methionine and Fe-S clusters was derived predominantly from exogenous sulfide instead of cysteine. Therefore, this investigation challenges the concept that cysteine is always the sulfur source for Fe-S cluster biosynthesis in vivo and suggests that Fe-S clusters are derived from sulfide in those organisms, which live in sulfide-rich habitats.     

Origin and Evolution of Sulfadoxine Resistant Plasmodium falciparum

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.     

Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport

Polysulphide was formed according to reaction (1), when tetrathionate was (1) $${\text{S}}_4 {\text{O}}_6^{2 - } + {\text{HS}}^ - \to 2{\text{S}}_2 {\text{O}}_3^{2 - } + {\text{S(O)}} + {\text{H}}^ + $$ added to an anaerobic buffer (pH 8.5) containing excess sulphide. S(O) denotes the zero oxidation state sulphur in the polysulphide mixture S _infn ^sup2- . The addition of formate to the polysulphide solution in the presence of Wolinella succinogenes caused the reduction of polysulphide according to reaction (2). The bacteria grew in a medium containing formate and sulphide, (2) $${\text{HCO}}_2^ - + {\text{S(O)}} + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + {\text{HS}}^ - + {\text{H}}^ + $$ when tetrathionate was continuously added. The cell density increased proportional to reaction (3) which represents the sum of reactions (1) and (3) $${\text{HCO}}_2^ - + {\text{S}}_{\text{4}} {\text{O}}_6^{2 - } + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + 2{\text{S}}_{\text{2}} {\text{O}}_3^{2 - } + 2{\text{H}}^ + $$ (2). The cell yield per mol formate was nearly the same as during growth on formate and elemental sulphur, while the velocity of growth was greater. The specific activities of polysulphide reduction by formate measured with bacteria grown with tetrathionate or with elemental sulphur were consistent with the growth parameters. The results suggest that W. succinogenes grow at the expense of formate oxidation by polysulphide and that polysulphide is an intermediate during growth on formate and elemental sulphur.     

Inhibition of Sulfur Use by Sulfite Ion in Thiobacillus ferrooxidans

In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans AP19-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mm and 1.5 mm NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mm NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mm, indicating that sulfite ions didn’t inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe^{2 +} - or S0-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans AP19-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.     

Sulfur Species Investigation in Extra- and Intracellular Sulfur Globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus

The sulfur chemical speciation in extracellular and intracellular sulfur globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus were investigated with an integrated approach including scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy and sulfur K-edge X-ray absorption near edge structure spectroscopy (XANES). The results indicated that both strains can accumulate extracellular sulfur globules when grown on thiosulfate, and the major sulfur chemical speciation of which were S₈ for A. ferrooxidans and mixture of ring sulfur and polythionate for A. caldus, respectively. In contrast, A. ferrooxidans can accumulate both linear sulfur and S₈ internally when grown with sulfur powder and thiosulfate, whereas A. caldus did not accumulate intracellular sulfur globules. In addition, the fitted results of sulfur K-edge XANES spectra indicated that the reduced glutathione (containing thiols groups) were involved in sulfur bio-oxidation of both strains and the tetrathionate were the intermediate products during thiosulfate metabolism by two strains.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation

Background

The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules.

Methodology/Principal Findings

Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0–6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs'' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE.

Conclusions

Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates.     

Fine Mapping of a QTL Associated with Kernel Row Number on Chromosome 1 of Maize

The genetic factors underlying changes in ear morphology, and particularly the inheritance of kernel row number (KRN), have been broadly investigated in diverse mapping populations in maize (Zea mays L.). In this study, we mapped a region on the long arm of chromosome 1 containing a QTL for KRN. This work was performed using a set of recombinant chromosome nearly isogenic lines (RCNILs) derived from a BC₂S₃ population produced using the inbred maize line W22 and teosinte (Zea mays ssp. parviglumis) as the parents. A set of 48 RCNILs was evaluated in the field during the summer of 2013 in order to perform the mapping. A QTL for KRN was found that explained approximately 51% of the phenotypic variance and had a 1.5-LOD confidence interval of 203 kb. Seven genes are described in this interval. One of these candidate genes may have been the target of domestication processes in maize and contributed to the shift from two kernel row ears in teosinte to a highly polystichous ear in maize.     

Nickel Inhibition of the Growth of a Sulfur-oxidizing Bacterium Isolated from Corroded Concrete

A sulfur-oxidizing bacterium strain NB1-3 isolated from corroded concrete was a Gram negative, non-spore-forming, and rod-shaped bacterium (0.5–1.0x 1.5–2.0μm) with a polar flagellum. Strain NB1-3 had its optimum temperature and pH for growth at 30°C and 3.0–4.0, respectively. Strain NB1-3 had enzyme activities that oxidized elemental sulfur, thiosulfate, tetrathionate, and sulfide and the activity to incorporate ¹⁴CO₂ into the cells. The mean G+C content of the DNA was 52.9 mol%. These results indicate that strain NB1-3 is Thiobacillus thiooxidans. Since nickel has been known to protect concrete from corrosion, the effect of Ni on the growth of strain NB1-3 was studied. The cell growth on tiosulfate-, elemental sulfur-, or tetrathionate-medium was completely inhibited by 0.1% metal nickel or 5mM NiSO₄. Both cellular activities of elemental sulfur oxidation and CO₂ incorporation were strongly inhibited by 5mM NiSO₄. The amounts of Ni in cells with or without nickel treatment were 1.7 and 160.0 nmol/mg protein, respectively. These results indicate that nickel binds to strain NB1-3 cells and inhibits enzymes involved in sulfur oxidation of this bacterium, and as a result, inhibits cell growth.     

Right Bundle Branch Block: A Predictor of Mortality in Early Systemic Sclerosis

Objective

To evaluate the prognostic significance of baseline electrocardiogram (ECG) abnormalities in a multiethnic cohort of patients with early systemic sclerosis (SSc) and to determine the serological, clinical, and echocardiogram correlates of ECG findings.

Methods

SSc patients with disease duration of≤5 years were enrolled in the GENISOS (Genetics versus Environment in Scleroderma Outcome Study) cohort. At the first visit, a standard 12 lead ECG was obtained along with demographic information, clinical data, and autoantibodies. The results of echocardiograms were also recorded. All ECGs were interpreted by a cardiologist unaware of the patients'' clinical data.

Results

Of 265 SSc patients with average disease duration at enrollment of 2.5 years, 140 (52.8%) had abnormal ECG findings. These findings were not associated with SSc disease type or autoantibody profile but were associated with more severe heart and lung involvement. A total of 75 patients (28.3%) died over a follow up time of 9.9 years. Complete right bundle branch block (± left anterior hemiblock) on ECG, present in 7 (2.6%) patients, predicted a higher risk of mortality (HR: 5.3; 95% CI: 2.1 to 13.4; p<0.001). The predictive significance of right bundle branch block was independent of age at enrollment, gender, ethnicity and risk factors for coronary artery disease.

Conclusion

ECG abnormalities are common in patients with early SSc and are associated with the severity of lung and heart involvement. Right bundle branch block is an independent predictor of mortality, and should be considered a marker of disease severity in SSc.     

MMS21/HPY2 and SIZ1, Two Arabidopsis SUMO E3 Ligases,Have Distinct Functions in Development

The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.     

Structural and Functional Characterization of the Enantiomers of the Antischistosomal Drug Oxamniquine

Background

For over two decades, a racemic mixture of oxamniquine (OXA) was administered to patients infected by Schistosoma mansoni, but whether one or both enantiomers exert antischistosomal activity was unknown. Recently, a ~30 kDa S. m ansoni sulfotransferase (SmSULT) was identified as the target of OXA action.

Methodology/Principal Findings

Here, we separate the OXA enantiomers using chromatographic methods and assign their optical activities as dextrorotary [(+)-OXA] or levorotary [(-)-OXA]. Crystal structures of the parasite enzyme in complex with optically pure (+)-OXA and (-)-OXA) reveal their absolute configurations as S- and R-, respectively. When tested in vitro, S-OXA demonstrated the bulk of schistosomicidal activity, while R-OXA had antischistosomal effects when present at relatively high concentrations. Crystal structures R-OXA•SmSULT and S-OXA•SmSULT complexes reveal similarities in the modes of OXA binding, but only the S-OXA enantiomer is observed in the structure of the enzyme exposed to racemic OXA.

Conclusions/Significance

Together the data suggest the higher schistosomicidal activity of S-OXA is correlated with its ability to outcompete R-OXA binding the sulfotransferase active site. These findings have important implications for the design, syntheses, and dosing of new OXA-based antischistosomal compounds.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

Draft Genome Sequence of the Extremely Acidophilic Bacterium Acidithiobacillus caldus ATCC 51756 Reveals Metabolic Versatility in the Genus Acidithiobacillus

Acidithiobacillus caldus is an extremely acidophilic, moderately thermophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and reduced inorganic sulfur compounds. Here we present the draft genome sequence of Acidithiobacillus caldus ATCC 51756 (the type strain of the species), which has permitted the prediction of genes for survival in extremely acidic environments, including genes for sulfur oxidation and nutrient assimilation.Acidothiobacillus caldus is one of the three recognized species of the genus Acidithiobacillus, which also circumscribes A. thiooxidans and A. ferrooxidans. These bacteria live in extremely acidic environments (pH 1 to 3) typically associated with copper mining operations (bioleaching) (15, 17) and natural acid drainage systems (7). All of these bacteria have the capacity to gain energy by the oxidation of sulfur and reduced inorganic sulfur compounds and to thrive in extremely high concentrations of heavy metals (16). Of the three species, A. ferrooxidans is unique in also being able to obtain energy through the oxidation of ferrous iron, as well as being a facultative anaerobe capable of using ferric iron as an alternative electron acceptor. Acidithiobacilli have been shown to be able to fix atmospheric carbon via the Calvin-Benson-Bassham cycle (1, 11, 21) and to synthesize extracellular polymeric substances that are postulated to promote adhesion to mineral surfaces (3).As opposed to A. ferrooxidans, for which substantial bioinformatic and experimental evidence exists for these and other properties (4, 14, 19, 20), A. caldus is poorly characterized, although it is known that it cannot carry out iron oxidation or nitrogen fixation (13). In contrast to the other two species of the genus, A. caldus thrives at temperatures up to 45 to 50°C. In order to unravel strategies for energy and nutrient assimilation used by A. caldus to survive and proliferate in extremely acidic environments, we have generated and annotated a draft genome sequence of A. caldus and performed a genome-based metabolic reconstruction to address these questions.The draft genome sequence of the type strain of A. caldus, ATCC 51756, was determined by a whole-genome shotgun strategy. Genomic libraries of 4 kb and 40 kb were constructed and sequenced, assembled using the Phred/Phrap programs (5), leading to the generation of a draft assembly based on 41,813 high-quality reads. Using Consed (8), assemblies that contained only contig segments with at least twofold coverage were edited and curated. Gene modeling was performed using CRITICA (2) and Glimmer (6). Predicted coding sequences were annotated based on comparisons with public databases (COG, KEGG, Pfam, TIGRFAMs, Unipprot, and NR-NCBI). Automatic metabolic reconstruction was carried out using the PRIAM and Pathways tools for prediction and curation.The A. caldus ATCC 51756 draft genome sequence has a total of 2,946,159 bp distributed in 139 contigs with an average GC content of 61.4%. Two 5S-16S-23S operons and 47 tRNAs on the draft assembly were identified, as were complete sets of genes for the synthesis of amino acids, nucleotides, and prosthetic groups. As in the case of A. ferrooxidans ATCC 23270 and other chemolithoautotrophic representatives, an incomplete tricarboxylic acid (TCA) cycle was detected, in which genes for the 2-oxogluatarate dehydrogenase enzyme complex were absent. The incomplete TCA cycle has been hypothesized to be an ancient biosynthetic pathway, instead of an energy generation cycle characteristic of the complete TCA cycle (22).A detailed inspection of the genome sequence revealed the presence of a complete set of genes encoding flagellum formation and chemotaxis. All of the genes of the classical Calvin-Benson-Bassham pathway were predicted, including genes for two RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) enzymes (type I and type II) and carboxysome formation genes. In addition, genes for sulfur and reduced inorganic sulfur compound oxidation were identified, including two gene clusters harboring the genes encoding the sulfur oxidation complex SOX (soxYZB-hyp-resB-soxAX-resC and soxYZA-hyp-SoxB), previously characterized in neutrophilic, chemoautotrophic bacteria (9); genes for the sulfur quinone oxidoreductase enzyme (sqr); a sulfur oxygenase:reductase gene (sor); and genes for a tetrathionate hydrolase and a thiosulfate quinone oxidoreductase (doxD) previously characterized in this strain (18). Several terminal oxidases were identified, including two copies of the genes encoding a bo₃-type quinol oxidase (cyoBACD), six copies of the genes encoding a bd-type quinol oxidase (cydAB), and one putative aa₃-type quinol oxidase gene cluster termed “quoxBACD.” No gene was detected that could encode rusticyanin, which has been shown to be involved in electron flow during iron oxidation in A. ferrooxidans ATCC 23270 (10).Genes for ammonia uptake were predicted, while those for atmospheric nitrogen fixation were not found in the draft genome as expected. In addition, an alternative nitrogen assimilatory pathway in A. caldus can be proposed based on the presence of a gene cluster encoding a membrane-associated nitrate reductase (narGHJI). A similar complex has been shown to carry out nitrogen assimilation in Mycobacterium tuberculosis (12).The information provided in the draft genome sequence of A. caldus ATCC 51756 reported here will facilitate additional bioinformatic and experimental investigations to elucidate the role of this microorganism in bioleaching and in natural and man-made acidic environments. This information also provides a first overview of the comparative metabolic diversity of the genus Acidithiobacillus and generates a more comprehensive picture of the metabolic diversity and adaptability of organisms that dwell in extreme acidic environments.     

Schistosoma mansoni Stomatin Like Protein-2 Is Located in the Tegument and Induces Partial Protection against Challenge Infection

Background

Schistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease.

Methodology/Principal Findings

Herein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%–32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-γ and TNF-α, while no IL-4 production was detected, suggesting a Th1-predominant immune response.

Conclusions/Significance

Data presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.     

Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species

Background

The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO₂ fixation via the Calvin-Benson-Bassham cycle.

Methodology/Principal Findings

To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition.

Conclusions/Significance

These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.     

The Putative Thiosulfate Sulfurtransferases PspE and GlpE Contribute to Virulence of Salmonella Typhimurium in the Mouse Model of Systemic Disease

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H₂O₂. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.     

SDA,a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells,the First and Pivotal Step in Transendothelial Migration during Metastasis Formation

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K _d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.