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Publisher Summary This chapter describes genome-wide expression profiling of intracellular bacteria. The number of fully sequenced bacterial genomes is large and increasing. The value of genome sequences is, however, limited by the fact that functional information is not available for 30% or more of the open reading frames (orfs) in any given genome. The genome-wide expression profile of a cell is a complex description of its physiological status that includes most of the cell's metabolic activities, and therefore gives access to an unusually holistic approach to the analysis of biological systems. While genome-wide expression profiling contributes to a more complete understanding of cellular physiology, it also demands a careful experimental design. All steps of an experimental protocol have to be investigated with respect to their impact on the expression profile because even routine procedures, such as the concentration of bacteria by centrifugation, can change the expression profile. The chapter focuses on the application of microarrays for the analysis of Mycobacterium tuberculosis inside host cells. It also describes the design of genome-wide expression profiling experiments and discusses the infection of macrophages with M. tuberculosis . The preparation and labeling of RNA from intracellular M. tuberculosis is also described in the chapter.

Publisher Summary This chapter discusses the whole genome DNA Microarry expression analysis of biofilm development by vibrio cholera O1 E1 tor. Asiatic cholera is caused by Vibrio cholerae O1, and results in a purging diarrheal illness that can kill an adult within 24 hours. Laboratory-based studies have demonstrated that V. cholerae O1, biotype E1 Tor, forms a typical three-dimensional biofilm on a variety of abiotic surfaces, and on biotic surfaces composed of chitin. Investigation of this phenotype has showed that the rugose colonial variant forms a thicker and more differentiated biofilm than the smooth colonial variant. This capacity was found to be associated with production of a glucose- and galactose-rich extracellular polysaccharide (EPS) by the rugose form. Transposon mutagenesis studies of the smooth colonial variant by Kolter and colleagues, led to the identification of two other effectors of biofilm formation, a type IV pilus that confers mannose-sensitive hemagglutination and flagella. From this information they proposed a three-step model of biofilm development: first, type IV pili and flagella facilitate the attachment of flee-swimming bacteria to a surface; second, the motility function of flagella causes attached bacteria to spread across the surface; and third, EPS secretion occurs, providing the extracellular matrix of the three-dimensional structure of the mature biofilm. With the completion of the entire V. cholerae O1 genome sequence, it has now become possible to conduct a whole-genome characterization of the biofilm phenotype using microarray-based expression profiling.