Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology

PprA Contributes to Deinococcus radiodurans Resistance to Nalidixic Acid, Genome Maintenance after DNA Damage and Interacts with Deinococcal Topoisomerases

PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB

Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholera and are stabilized by a double signaling domain receptor DosM

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae’s cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array

Farm Level Economic Implications of Genetic Selection for Improving Milk Fat Composition

The objective of the study was to assess the farm level economic implications of value-adding genetic selection strategies to improve milk fat composition. Selection based on a quantitative trait (ratio of total saturated to total unsaturated fatty acids in milk) or a known genotype (for the DGAT1 gene) was considered. Technical and economic performance of hypothetical herds were computed by a herd optimization and simulation model. It was assumed that the herds are already bred for the specific milk composition, and the transition period was not considered. Correlated effects of the selection scenarios on milk production, female fertility, and functional longevity traits were accounted for. Results showed that increasing the total unsaturated fatty acids in milk by traditional selection leads to lower net revenue, whereas selection based on DGAT1 genotype results in slightly higher net revenue. Our results, therefore, suggest that genetic selection based on DGAT1 genotype is a more profitable strategy for dairy farmers than selection based on phenotypes for SFA/UFA ratio.dairy cattle, genetic selection, milk composition, farm economics, Livestock Production/Industries,

Design of a school-based randomized trial to reduce smoking among 13 to 15-year olds, the X:IT study

BACKGROUND: Adolescent smoking is still highly prevalent in Denmark. One in four 13-year olds indicates that they have tried to smoke, and one in four 15-year olds answer that they smoke regularly. Smoking is more prevalent in socioeconomically disadvantaged populations in Denmark as well as in most Western countries. Previous school-based programs to prevent smoking have shown contrasting results internationally. In Denmark, previous programs have shown limited or no effect. This indicates a need for developing a well-designed, comprehensive, and multi-component intervention aimed at Danish schools with careful implementation and thorough evaluation. This paper describes X:IT, a study including 1) the development of a 3-year school-based multi-component intervention and 2) the randomized trial investigating the effect of the intervention. The study aims at reducing the prevalence of smoking among 13 to 15-year olds by 25%. METHODS/DESIGN: The X:IT study is based on the Theory of Triadic Influences. The theory organizes factors influencing adolescent smoking into three streams: cultural environment, social situation, and personal factors. We added a fourth stream, the community aspects. The X:IT program comprises three main components: 1) smoke-free school premises, 2) parental involvement including smoke-free dialogues and smoke-free contracts between students and parents, and 3) a curricular component. The study encompasses process- and effect-evaluations as well as health economic analyses. Ninety-four schools in 17 municipalities were randomly allocated to the intervention (51 schools) or control (43 schools) group. At baseline in September 2010, 4,468 year 7 students were eligible of which 4,167 answered the baseline questionnaire (response rate = 93.3%). DISCUSSION: The X:IT study is a large, randomized controlled trial evaluating the effect of an intervention, based on components proven to be efficient in other Nordic settings. The X:IT study directs students, their parents, and smoking prevention policies at the schools. These elements have proven to be effective tools in preventing smoking among adolescents. Program implementation is thoroughly evaluated to be able to add to the current knowledge of the importance of implementation. X:IT creates the basis for thorough effect and process evaluation, focusing on various social groups. TRIAL REGISTRATION: Current Controlled Trials ISRCTN77415416

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Development of Mobile Machining Cell

This report covers some initial aspects of development of the mobile InnoMill machining cell. The new machining paradigm where the machine is mounted on the workpiece is compared to the old paradigm where the workpiece is mounted inside the machine, and differences are discussed. Parametric studies of the workpiece case study of the InnoMill project, the Vestas V112-3.0MW wind turbine hub, are performed to supply insight regarding load capacity etc. for the machine designers. The hub finite element model is validated using experimental results from Operational Modal Analysis performed on the hub. Furthermore, the InnoMill concept is described, and work regarding the 6 degree of freedom parallel kinematic manipulator which is present in the concept is performed. A numerical procedure accounting for base deflections due to static loading is proposed and implemented. Additionally, a six degree of freedom spring-mass model vibrational response is compared to vibrational response obtained from experiments on the 6 degree of freedom parallel kinematic manipulator at Aarhus University. The model, which is based on assumptions commonly found in literature, is rejected. Finally, an outlook for the remaining part of the PhD project is presented