Optimization of cell permeabilization in electron flow based mitochondrial function assays.

Permeable cell models have contributed much to the progress in mitochondrial research. Optimization of permeabilization is required to make the cell's plasma membrane permeable to small molecules while keeping the intracellular organelles and their membranes intact and fully functional. Here we report our assessment and optimization of commonly used permeabilizing agents including different saponin preparations, digitonin, and recombinant perfringolysin O employing a new electron flow based mitochondrial assay technology that utilizes a colorimetric redox dye. The results of this study provide guidance in optimizing the conditions for mitochondrial function assays with permeabilized cells using the novel redox dye-based format.

Using phenotype microarrays to determine culture conditions that induce or repress toxin production by Clostridium difficile and other microorganisms.

Toxin production is a central issue in the pathogenesis of Clostridium difficile and many other pathogenic microorganisms. Toxin synthesis is influenced by a variety of known and unknown factors of genetics, physiology, and environment. To facilitate the study of toxin production by C. difficile, we have developed a new, reliable, quantitative, and robust cell-based cytotoxicity assay. Then we combined this new assay with Phenotype MicroArrays (PM) technology which provides high throughput testing of culture conditions. This allowed us to quantitatively measure toxin production by C. difficile type strain ATCC 9689 under 768 culture conditions. The culture conditions include different carbon, nitrogen, phosphorus, and sulfur sources. Among these, 89 conditions produced strong toxin induction and 31 produced strong toxin repression. Strong toxin inducers included adenine, guanosine, arginine dipeptides, γ-D-Glu-Gly, methylamine, and others. Some leucine dipeptides and the triple-leucine tripeptide were among the strongest toxin repressors. While some results are consistent with previous observations, others are new observations that provide insights into toxin regulation and pathogenesis of C. difficile. Additionally, we have demonstrated that this combined assay technology can be applied broadly to a wide range of toxin producing microorganisms. This study is the first demonstration of simultaneous assessment of a large number of culture conditions influencing bacterial toxin production. The new functional cytotoxin quantitation method developed provides a valuable tool for studying toxigenic microorganisms and may also find applications in clinical and epidemiological research.

Assay of the multiple energy-producing pathways of mammalian cells.

BACKGROUND: To elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells. METHODOLOGY AND PRINCIPAL FINDINGS: Here we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation. CONCLUSIONS: This technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype.

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.

Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems.

Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.