Pseudomonas aeruginosa PilY1 Binds Integrin in an RGD- and Calcium-Dependent Manner

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (Tl) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.link_to_subscribed_fulltex

Evaluation of the CSM-CROPGRO-Soybean model for dual-purpose soyabean in Kenya

Limited information is available on the potential performance of introduced dual purpose varieties across different Kenyan soils and agro-ecological environments and consistency across sites and seasons. Crop simulation modeling offers an opportunity to explore the potential of and select introduced cultivars for new areas before establishing costly and time-consuming field trials. Dual purpose soybeans were introduced due to their ability to improve soils and at the same time provide substantial grain yields. The objective of this study was to derive genetic coefficients of recently introduced dual purpose soybean varieties and to explore the reliability of the Cropping System Model (CSM)-CROPGRO-Soybean model in simulating phenology and yield of the dual purpose varieties under different environments. Field trials for seven varieties were conducted across three sites in two seasons and data on phenology and management, soil characteristics and weather was collected and used in the CROPGRO model. A stepwise procedure was used in the calibration of the model to derive the genetic coefficients. Two sets of data from Kakamega and Kitale were used in calibration process while 2006 data for Kakamega and Msabaha, were used for evaluation of the model. The derived genetic coefficients provided simulated values of various development and growth parameters that were in good agreement with their corresponding observed values for most parameters. Model evaluation with independent data sets gave similar results. The differences among the cultivars were also expressed through the differences in the derived genetic coefficients. CROPGRO was able to accurately predict growth, phenology and yield. The model predicted the first flowering dates to within 2¿3 days of the observed values, the first pod dates within 3 days of the observed values and yields within 5¿300 kg ha-1 of the observed yields. The genetic coefficients derived in CROPGRO model can, therefore, be used to predict soybean yield and phenology of the dual purpose soybean varieties across different agro-ecological zones. (Résumé d'auteur