The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes.

The measure of the quality of a systems biology model is how well it can reproduce and predict the behaviors of a biological system such as a microbial cell. In recent years, these models have been built up in layers, and each layer has been growing in sophistication and accuracy in parallel with a global data set to challenge and validate the models in predicting the content or activities of genes (genomics), proteins (proteomics), metabolites (metabolomics), and ultimately cell phenotypes (phenomics). This review focuses on the latter, the phenotypes of microbial cells. The development of Phenotype MicroArrays, which attempt to give a global view of cellular phenotypes, is described. In addition to their use in fleshing out and validating systems biology models, there are many other uses of this global phenotyping technology in basic and applied microbiology research, which are also described.

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.

Salmonel typhimurium LT2 induces a set of heatshock proteins analogous to those found previously in Escherichia coli. These are virtually the only proteins synthesized after a temperature shift from 280C to 50TC. Using a two-dimensional thinlayer chromatographic system developed to resolve adenylylated nucleotides, we have found that S. typhimurium and E. coli accumulate P',P4-diadenosine-5'-tetraphosphate (AppppA),

AppppA , ApppGpp , AppppG , ApppG , and ApppA rapidly accumulate to high levels in Salmonella typhimurium following exposure to a variety of oxidizing agents, but not to a variety of other stresses. Among the agents inducing these adenylylated nucleotides are 1-chloro-2,4-dinitrobenzene, diamide, hydrogen peroxide, t-butyl hydroperoxide, N-ethyl maleimide, iodoacetamide, cadmium chloride, and a variety of quinones. Some of these oxidizing agents cause preferential synthesis of specific adenylylated nucleotides, e.g., N-ethyl maleimide induces ApppA and menadione induces ApppGpp . Our data, as well as other evidence in the literature, strongly suggest that oxidation stress is coupled to adenylylated nucleotide synthesis by aminoacyl-tRNA synthetases. Although adenylylated nucleotides are made by tRNA synthetases in vitro, their synthesis in vivo is not a simple consequence of inhibition of synthetase activity. Compounds that inhibit normal charging by aminoacyl-tRNA synthetases do not result in the synthesis of adenylylated nucleotides, nor do mutations in tRNA synthetase structural genes or tRNA structural, modifying, or processing genes. We propose that the family of adenylylated nucleotides are alarmones signaling the onset of oxidation stress, and that particular ones may be alarmones for specific oxidative stresses, e.g., ApppGpp for oxidative damage to amino acid biosynthesis.