Exploring the low-pressure growth limit: evolution of Bacillus subtilis in the laboratory to enhanced growth at 5 kilopascals

Growth of Bacillus subtilis cells, normally adapted at Earth-normal atmospheric pressure (～101.3 kPa), was progressively inhibited by lowering of pressure in liquid LB medium until growth essentially ceased at 2.5 kPa. Growth inhibition was immediately reversible upon return to 101.3 kPa, albeit at a slower rate. A population of B. subtilis cells was cultivated at the near-inhibitory pressure of 5 kPa for 1,000 generations, where a stepwise increase in growth was observed, as measured by the turbidity of 24-h cultures. An isolate from the 1,000-generation population was obtained that showed an increase in fitness at 5 kPa when compared to the ancestral strain or a strain obtained from a parallel population that evolved for 1,000 generations at 101.3 kPa. The results from this preliminary study have implications for understanding the ability of terrestrial microbes to grow in low-pressure environments such as Mars.     

Cutting Edge: Multiple autoimmune pathways in kd/kd mice

The kidney disease (kd) mutation was transferred to a C57BL/6 (B6) background by selection for closely linked microsatellite markers. The resulting congenic strain, B6.kd, was mated with partners homozygous for targeted mutations of CD4, CD8, CD28, IL-2, recombinase-activating gene-1 (Rag-1), ICAM-1, or beta(2)-microglobulin. In most of the resulting double mutants, kidney disease occurred as readily and as severely as in the B6.kd controls, although disease occurred somewhat less frequently in age-matched CD28(-/-) kd/kd mice. Immunohistology demonstrated a predominance of macrophages in the lesions of B6.kd and most of the double mutants, with the remaining cells consisting of T cells and variable numbers of NK cells. In Rag-1(-/-) kd/kd, approximately 50% of infiltrating cells were macrophages, and approximately 50% were NK cells. These results suggest that the initial lesion caused by the mutant gene is intrinsic to the kidney and that the immune response that subsequently occurs can involve any one of several different cellular compositions.     

PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE,DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES1

Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis.     

Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.     

Chromium picolinate and conjugated linoleic acid do not synergistically influence diet- and exercise-induced changes in body composition and health indexes in overweight women

This study assessed the effects of combined chromium picolinate (CP) and conjugated linoleic acid (CLA) supplementation on energy restriction and exercise-induced changes in body composition, glucose metabolism, lipid lipoprotein profile and blood pressure in overweight, premenopausal women. For 12 weeks, 35 women [age 36+/-1 years (mean+/-S.E.M.); BMI 28.0+/-0.5 kg/m2] were counseled to consume a 2092 kJ/day (500 kcal/day) energy deficit diet and performed 30 min of moderate-intensity walking or jogging 5 days/week. The women were randomly assigned to ingest either CP-CLA [400 mug chromium (Cr), 1.8 g CLA in 2.4 g tonalin oil, n=19] or placebo (<0.1 microg Cr, 2.4 g canola oil, n=16). Compared to baseline, urinary Cr excretion increased 22-fold, plasma CLA isomer 18:2 (c9,t11) content increased 79% and plasma CLA isomer 18:2 (t10,c12) became detectable in CP-CLA and were unchanged in Placebo. Over time, body weight decreased 3.5+/-0.5% (CP-CLA -2.6+/-0.5; placebo -2.5+/-0.5 kg) and fat mass decreased 8.9+/-1.3% (CP-CLA -2.7+/-0.5, placebo -2.4+/-0.5 kg), with no differences in responses between groups. Fasting blood hemoglobin A1c, plasma glucose and insulin, a homeostatic assessment of insulin resistance, serum total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol, triacylglycerol (TG), CHOL/HDL ratio, TG/HDL ratio and sitting systolic and diastolic blood pressures were not changed over time or influenced by CP-CLA. The use of a combined CP and CLA supplement for 3 months does not affect diet- and exercise-induced changes in weight and body composition or improve indexes of metabolic and cardiovascular health in young overweight women.     

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Selenium Supplementation Alters Gene Expression Profiles Associated with Innate Immunity in Whole-Blood Neutrophils of Sheep

Footrot (FR) is a common, contagious bacterial disease of sheep that results in lameness and significant economic losses for producers. We previously reported that sheep affected with FR have lower whole-blood (WB) selenium (Se) concentrations and that Se supplementation in conjunction with routine control practices accelerates recovery from FR. To determine whether oral Se-yeast administered at supranutritional levels (>4.9 mg Se/week) alters the ability of sheep to resist or recover from FR infection, 60 ewes with and 60 ewes without FR were drenched once weekly for 62.5 weeks with 0, 4.9, 14.7, or 24.5 mg organic Se-yeast (30 ewes per treatment group). Footrot prevalence and severity were measured at 0, 20, 28, 40, and 60 weeks of Se supplementation. Genomic expression of eight WB-neutrophil genes for selenoproteins and seven WB-neutrophil genes for proteins involved in innate immunity was determined at the end of the treatment period using SYBR Green and quantitative polymerase chain reaction methodology. Supranutritional Se-yeast supplementation successfully increased Se status in sheep but did not prevent FR. Supranutritional Se-yeast supplementation increased WB-neutrophil expression of genes involved in innate immunity: l-selectin, interleukin-8 receptor, and toll-like receptor 4, which were or tended to be lower in ewes affected with FR. Furthermore, supranutritional Se-yeast supplementation altered the expression of selenoprotein genes involved in innate immunity, increasing selenoprotein S and glutathione peroxidase 4 and decreasing iodothyronine deiodinases 2 and 3. In conclusion, supranutritional Se-yeast supplementation does not prevent FR, but does alter WB-neutrophil gene expression profiles associated with innate immunity, including reversing those impacted by FR.