Genetic tools for constructing gene disruptions and reporter fusions play an important role in gene function and regulation studies for all organisms, no matter the subject is a eubacterium, such as E. coli, or a higher eukaryotic organism, such as a mouse. These studies have contributed significantly to our current knowledge in life sciences, such as bacterial virulence, cell division, cell development, and human diseases, etc. To facilitate functional genomic studies in E. coli and other bacteria, a new genetic tool for high-throughput (HT) studies, a conjugative att shuttle (CAS) CRIM system, has been developed in this work, and has been shown to work in diverse Gram-negative bacteria, including Pseudomonas aeruginosa, Shewanella oneidensis, and Vibrio cholerae. Signal transduction by protein phosphorylation plays a central role in regulation of numerous cellular processes. Two component systems (TCSs) are the most prevalent signal transduction systems in bacteria. With a goal towards understanding the roles of all E. coli TCSs and regulatory interactions among them, we have constructed defined deletion mutants for each TCS systematically and then analyzed these mutants with a new technology called Phenotype MicroArrays that permits assaying nearly 2000 growth phenotypes simultaneously. New roles for some TCSs were uncovered and potential regulatory interactions among different TCSs are discussed. In particular, the PhoR(HK)/PhoB(RR) TCS, which controls genes for phosphorus acquisition, has been investigated in several aspects. Eleven putative PhoR/PhoB regulated promoters were tested for their expression dependency on the PhoR/PhoB TCS under phosphate (Pi) starvation. Roles of the PboR/PhoB TCS in transcription of several polyphosphate kinase (PPK) orthologs under Pi starvation were also examined by using the CAS CRIM system. Results showed that there are two groups of PPK orthologs, one is PhoR/PhoB dependent and the other is PhoR/PhoB independent. Five additional noncognate HKs besides CreC were shown to activate PhoB by earlier bulk enzyme assays. Single cell studies of cross activation of PhoB by these six HKs were carried out in this work and stochastic characteristics of cross talk among TCSs were uncovered and are discussed.