Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.     

Study of selective feeding in the marine cladoceran Penilia avirostris by HPLC pigment analysis

Food selection by the marine cladoceran Penilia avirostris was studied in the field by HPLC analysis of phytoplankton marker pigments and in the laboratory by microscopic measurement of cell removal. Comparison between pigment composition in natural phytoplankton and in P. avirostris showed that P. avirostris preferred diatoms, cryptophytes and chlorophytes, and ignored prymnesiophytes and dinoflagellates. Peridinin, the marker pigment for dinoflagellates was found in P. avirostris only when the dinoflagellate populations were dominated by Prorocentrum. Pigment degradation rates ranged from 13.73% for alloxanthin to 36.62% for chlorophyll a. Clearance rates measured in the laboratory provided further evidence of strong preference for diatoms and cryptophytes, and avoidance of dinoflagellates. Microscopic counts suggested that P. avirostris was feeding on prymnesiophytes, although ingestion of prymnesiophytes could not be confirmed by pigment analysis.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Assessment Strategies and the Effects of Fighting Experience on Future Contest Performance in the Green Anole (Anolis carolinensis)

Social experiences can be useful sources of information for animals charged with making fitness‐related decisions. Fighting experience can alter an animal's perception of its fighting ability possibly leading to changes in future contest decisions, which may increase/decrease their probability of winning future contests. Winner and loser effects have been revealed in a wide array of animals, but studies using reptilian models are rare. This study investigated the impact of fighting experience on future contest performance and outcome in the green anole lizard and investigated the assessment strategies used by anoles during contests of different intensities. To determine whether the green anole expresses winner or loser effects, focal animals engaged in a primary contest with a smaller (larger) opponent to gain a winning (losing) experience; opponent size asymmetries were a significant predictor of contest outcome. Focal individuals were isolated for 2 d before being given a secondary contest with a size‐matched, naïve opponent. We found no evidence of winner or loser effects 2 d following a previous contest. Although previous contest outcome did not dictate future contest success, dynamics of the previous contest did. Highly aggressive primary contest losers won a significant proportion of the secondary contests, while less aggressive losers were more apt to lose the secondary contest. Secondary contest success of prior winners was not influenced by earlier contest performance. Further analyses of contest dynamics reveal that individuals may use different assessment strategies depending on the intensity of the contest. Our results demonstrate that future contest success may be driven more by individual performance in a prior contest and less by prior contest outcome.     

Optical colorimetric sensor strip for direct readout glucose measurement

A novel direct readout colorimetric optical glucose sensor strip was constructed based on a three-layer film, including a green-emitted CdTe/CdS quantum dots (QDs) layer as a stable color background, a red-fluorescent platinum-porphyrin oxygen-sensing layer and a glucose oxidase layer. The sensor achieved high resolution (up to 0.2 mmol L⁻¹) glucose determination with a detection range from 0 to 3.0 mmol L⁻¹. A “glucose ruler” which acts as a glucose standard colorimetric card was obtained. Glucose concentration could easily be directly readout using the “glucose ruler”, which made the glucose determination rapid, convenient and easy. The effects of pH, salinity and temperature were systematically investigated. The prepared sensor was finally applied for glucose sample analysis, compared with the “glucose ruler”, accurate results could be directly readout.     

Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli

λ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required.