Chip calorimetry and biomagnetic separation: Fast detection of bacterial contamination at low cell titers

We present a new chip calorimeter for fast and quantitative measurement of metabolic heat rates of microorganisms attached to magnetic beads. In biomagnetic separation (BMS) experiments, Escherichia coli K12 immobilized on nonspecifically functionalized beads has a specific heat rate of around 1 pW per cell at 37°C. Therefore, at least 2 × 10⁴ bacteria are required to exceed the calorimetric signal resolution of 20 nW. If the samples to be analyzed have the original volume of 4 mL, bacteria at less than 10⁴ cells mL^?1 should be detectable. In practice, we achieved the detection of approximately 2 × 10⁴ cells mL^?1. The method presented here might also find some applications in the investigation of biofilms and study of biomolecular interactions.     

Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K_m value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the β-anomeric form. Its apparent K_m for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K_m (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K_m fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of β-glucose 1,6-bisphosphate from β-glucose 1-phosphate and ATP is described.     

Climate change and the recent emergence of bluetongue in Europe

Bluetongue, a devastating disease of ruminants, has historically made only brief, sporadic incursions into the fringes of Europe. However, since 1998, six strains of bluetongue virus have spread across 12 countries and 800 km further north in Europe than has previously been reported. We suggest that this spread has been driven by recent changes in European climate that have allowed increased virus persistence during winter, the northward expansion of Culicoides imicola, the main bluetongue virus vector, and, beyond this vector's range, transmission by indigenous European Culicoides species - thereby expanding the risk of transmission over larger geographical regions. Understanding this sequence of events may help us predict the emergence of other vector-borne pathogens.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Obesity and Persistent Organic Pollutants: Possible Obesogenic Effect of Organochlorine Pesticides and Polychlorinated Biphenyls

Persistent organic pollutants (POPs) are endocrine‐disrupting chemicals associated with the development of the metabolic syndrome and type 2 diabetes. In humans, little is known about their role in the potential origin of obesity. This study aims to assess the associations between serum levels of POPs and the prevalence of obesity in a cohort of obese and lean adult men and women. POP serum samples were investigated cross‐sectionally in 98 obese and 47 lean participants, aged ≥18 years. Serum samples were analyzed for the presence of polychlorinated biphenyl (PCB) congeners 153, 138, 180, and 170 and for the organochlorine pesticides, dichloro‐diphenyl‐dichloroethylene (pp‐DDE), and β‐hexachlorocyclohexane (βHCH). We established a significant negative correlation between BMI, waist, fat mass percentage, total and subcutaneous abdominal adipose tissue, and serum levels of PCB 153, 180, 170, and the sumPCBs. For βHCH, we demonstrated a positive correlation with BMI, waist, fat mass percentage, and total and subcutaneous abdominal adipose tissue. PCBs 180, 170, and the sum of PCBs correlated significantly negative with homeostasis model assessment for insulin resistance (HOMA_IR). βHCH correlated significantly positively with HOMA_IR. A strong correlation was established between all POP serum levels and age. We established a positive relationship between high serum levels of βHCH and BMI and HOMA_IR, whereas serum PCB levels were inversely correlated with BMI and HOMA_IR. Combined, these results suggest that the diabetogenic effect of low‐dose exposure to POPs might be more complicated than a simple obesogenic effect.     

G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor

The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.     

Post-pandemic seroprevalence of pandemic influenza A (H1N1) 2009 infection (swine flu) among children <18 years in Germany

Background

We determined antibodies to the pandemic influenza A (H1N1) 2009 virus in children to assess: the incidence of (H1N1) 2009 infections in the 2009/2010 season in Germany, the proportion of subclinical infections and to compare titers in vaccinated and infected children.

Methodology/Principal Findings

Eight pediatric hospitals distributed over Germany prospectively provided sera from in- or outpatients aged 1 to 17 years from April 1^st to July 31^st 2010. Vaccination history, recall of infections and sociodemographic factors were ascertained. Antibody titers were measured with a sensitive and specific in-house hemagglutination inhibition test (HIT) and compared to age-matched sera collected during 6 months before the onset of the pandemic in Germany. We analyzed 1420 post-pandemic and 300 pre-pandemic sera. Among unvaccinated children aged 1–4 and 5–17 years the prevalence of HI titers (≥1∶10) was 27.1% (95% CI: 23.5–31.3) and 53.5% (95% CI: 50.9–56.2) compared to 1.7% and 5.5%, respectively, for pre-pandemic sera, accounting for a serologically determined incidence of influenza A (H1N1) 2009 during the season 2009/2010 of 25,4% (95% CI : 19.3–30.5) in children aged 1–4 years and 48.0% (95% CI: 42.6–52.0) in 5–17 year old children. Of children with HI titers ≥1∶10, 25.5% (95% CI: 22.5–28.8) reported no history of any infectious disease since June 2009. Among vaccinated children, 92% (95%-CI: 87.0–96.6) of the 5–17 year old but only 47.8% (95%-CI: 33.5–66.5) of the 1–4 year old children exhibited HI titers against influenza A virus (H1N1) 2009.

Conclusion

Serologically determined incidence of influenza A (H1N1) 2009 infections in children indicates high infection rates with older children (5–17 years) infected twice as often as younger children. In about a quarter of the children with HI titers after the season 2009/2010 subclinical infections must be assumed. Low HI titers in young children after vaccination with the AS03_B-adjuvanted split virion vaccine need further scrutiny.     

Industrial microbiology of solar salt production

Solar salterns can be modeled as giant outdoor chemostats, much like a series of dams on a slow-moving river. Microorganisms and their products play an essential, but sometimes uncharacterized, role in salt production in these ponds, from seawater salinity up through NaCl saturation. They may physically affect the evaporation process and their by-products may chemically modify or bind with dissolved ions. Many solar salt facilities engage microbiologists to establish monitoring programs for analyses of nutrients, standing crop and associated biological variables in the ponds. Other solar salt companies engage microbiologists only when there are “crises” in the ponds that interfere with salt production. Journal of Industrial Microbiology & Biotechnology (2002) 28, 42–47 DOI: 10.1038/sj/jim/7000173 Received 20 May 2001/ Accepted in revised form 13 June 2001