Phenotype MicroArrays are ideally suited to the study of transporters. Loss of function mutations in amino acid or peptide transporters result in negative tests for substrates the mutant fails to import. Knock-out of multidrug resistance transporters exhibit hypersensitivity to chemicals that may be substrates of the transporter. The papers below show some of the ways that transporters have been studied with Biolog’s Phenotype MicorArray technology.
A Novel Fungal Family of Oligopeptide Transporters Identified by Functional Metatranscriptomics of Soil Eukaryotes
Marmeisse and colleagues converted polyadenylated soil RNA to cDNA for expression in a yeast mutant unable to transport peptides. They recovered transformants containing environmentally sourced peptide transport genes that functionally complemented the yeast mutant. Phenotype MicroArrays were used to functionally characterize the transporters and the genes were also mapped phylogenomically. Using a Xenopus oocyte model, one of the transporter genes was shown to induce peptide-dependent ion currents.
High-throughput Phenotype Characterization of Pseudomonas Aeruginosa Membrane Transport Genes
Paulsen and colleagues used PM metabolic assay to investigate the accuracy of bioinformatic predictions for carbon and nitrogen membrane transport genes. Twenty-seven of the 78 knockouts gave clear transporter phenotypes, and of these only 12 (44%) precisely matched the predicted annotation. In 10, a more precise annotation was obtained, and in 5 (18%), a significant re-annotation was enabled. New transporters were found for hydroxy-L-proline, N-acetyl-L-glutamate and histamine. The latter is the first histamine transporter to be identified.
The Phagosomal Transporter A Couples Threonine Acquisition to Differentiation and Replication of Legionella Pneumophila in Macrophages
Swanson and colleagues used Phenotype MicroArrays to study the mutant of a phagosomal transporter that is conserved in two other vacuolar pathogens. PM analysis indicates that the protein transports threonine. They propsosed a link between this transporter, vacuolar nutrient supply, and the cell cycle.
Harnessing Natural Diversity to Probe Metabolic Pathways
Following up on clues from PM analysis of peptide metabolism in diverse natural isolates of yeast, Oliver Homann and colleagues in Susan Lindquist’s laboratory at MIT have identified previously unknown cellular activities that contribute to peptide utilization. Their observations also fit beautifully with the N-end Rules of proteolysis. The peptide containing PMs (PM6-PM8) are proving very useful in cellular analysis.
Phenotype MicroArray Technology
Biolog’s Phentoype MicroArray technology enables researchers to evaluate nearly 2000 phenotypes of a microbial cell in a single experiment. This integrated system of cellular assays, instrumentation and bioinformatics software provides cellular knowledge that complements molecular information, helping you interpret and find the relevant aspects in massive amounts of gene expression or proteomics data. Through comprehensive and precise quantitation of phenotypes, researchers are able to obtain an unbiased perspective of the effect on cells of genetic differences, environmental change, exposure to chemicals or drugs, and more.