Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS3

Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.     

Targeted metabolomics profiles are strongly correlated with nutritional patterns in women

Nutrition plays an important role in human metabolism and health. Metabolomics is a promising tool for clinical, genetic and nutritional studies. A key question is to what extent metabolomic profiles reflect nutritional patterns in an epidemiological setting. We assessed the relationship between metabolomic profiles and nutritional intake in women from a large cross-sectional community study. Food frequency questionnaires (FFQs) were applied to 1,003 women from the TwinsUK cohort with targeted metabolomic analyses of serum samples using the Biocrates Absolute-IDQ? Kit p150 (163 metabolites). We analyzed seven nutritional parameters: coffee intake, garlic intake and nutritional scores derived from the FFQs summarizing fruit and vegetable intake, alcohol intake, meat intake, hypo-caloric dieting and a “traditional English” diet. We studied the correlation between metabolite levels and dietary intake patterns in the larger population and identified for each trait between 14 and 20 independent monozygotic twins pairs discordant for nutritional intake and replicated results in this set. Results from both analyses were then meta-analyzed. For the metabolites associated with nutritional patterns, we calculated heritability using structural equation modelling. 42 metabolite nutrient intake associations were statistically significant in the discovery samples (Bonferroni P < 4 × 10^?5) and 11 metabolite nutrient intake associations remained significant after validation. We found the strongest associations for fruit and vegetables intake and a glycerophospholipid (Phosphatidylcholine diacyl C38:6, P = 1.39 × 10^?9) and a sphingolipid (Sphingomyeline C26:1, P = 6.95 × 10^?13). We also found significant associations for coffee (confirming a previous association with C10 reported in an independent study), garlic intake and hypo-caloric dieting. Using the twin study design we find that two thirds the metabolites associated with nutritional patterns have a significant genetic contribution, and the remaining third are solely environmentally determined. Our data confirm the value of metabolomic studies for nutritional epidemiologic research.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

Investigating apoptosis: Characterization and analysis of Trichoplusia ni-caspase-1 through overexpression and RNAi mediated silencing

In both mammals and invertebrates, caspases play a critical role in apoptosis. Although Lepidopteron caspases have been widely studied in Spodoptera frugiperda cells, this is not the case for Trichoplusia ni cells, despite their widespread use for the production of recombinant protein and differences in baculovirus infectivity between the two species. We have cloned, expressed, purified and characterized Tn-caspase-1 in several situations: in its overexpression, in silencing via RNA interference (RNAi), during baculovirus infection, and in interactions with baculovirus protein p35. Overexpression can transiently increase caspase activity in T. ni (High Five?) cells, while silencing results in a greater than 6-fold decrease. The reduction in caspase activity resulted in a reduction in the level of apoptosis, demonstrating the ability to affect apoptosis by modulating Tn-caspase-1. During baculovirus infection, caspase activity remains low until approximately 5 days post infection, at which point it increases dramatically, though not in those cells treated with dsRNA. Our results demonstrate that Tn-caspase-1 is presumably the principal effector caspase present in High Five cells, and that it is inhibited by baculovirus protein p35. Finally, our results indicate differences between RNAi and p35 as effector molecules for modulating caspase activity and apoptosis during cell growth and baculovirus infection.     

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28–82) and its relationship to age, BMI and fasting insulin and glucose serum levels. The overall estimated prevalence of heteroplasmy for both tissues in the control region measured for 37 sites was 17%. The prevalence of heteroplasmy was higher among the older half of the study subjects than in the younger half (23% vs 10% p<0.03), primarily reflecting the increase in the prevalence of a heteroplasmic dinucleotide CA repeat in variable region II (VRII) with age. The VRII 523–524 heteroplasmic site (heteroplasmic in 25 subjects) was also associated with a decrease in BMI. In addition, concordance rates for common heteroplasmy were observed to be near complete for both dizygotic (DZ = 94%) and monozygotic twin pairs (MZ = 100%), consistent with previous reports that suggest variation in heteroplasmy rates between generations are determined by bottlenecks in maternal transmission of mitochondria. Differences in the prevalence of heteroplasmy were observed overall between samples derived from buccal swabs (19%) and blood (15%, p<0.04). These were particularly marked at position 16093 of hypervariable region I (HVI, 7% vs 0%, respectively, p<4×10⁻¹¹). The presence of the C allele at position 16093 in blood was associated with the presence of heteroplasmy in buccal swabs at this position (p = 3.5×10⁻¹⁴) and also at VRII (p = 2×10⁻⁴) suggesting a possible predisposing role for this site in the accumulation of heteroplasmy. Our data indicate that BMI is potentially associated with control region heteroplasmy.     

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.     

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.