Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems

Abstract

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.

The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic yersiniae

“…Furthermore, given that Y. pestis has essentially lost its intestinal pathogenic potential, as evidenced by numerous examples of pseudogenes in enteric pathogenic factors such as ail and ureA, it can be speculated that rcsD may be important for the enteric survival of Y. pseudotuberculosis and Y. enterocolitica. Additionally, the fact that neither rcsC nor rcsB has accumulated mutations resulting in them becoming pseudogenes in any of the sequenced Y. pestis strains raises the possibility that RcsC and RcsB are still required by Y. pestis and may still signal via an unknown phosphotransfer protein or by cross-talk with other sensory systems (Zhou et al, 2003).…”

Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data

“…The microplates were incubated at 37°C, and the absorbances were measured both at 570 and 750 nm after 24-and 48-h incubations, respectively, resulting in a total of 8 measurements. The detailed experimental conditions were described elsewhere (20). The colorimetric assay was considered as positive when the absorbance corresponding to the reduced dye (indicating substrate utilization) was 1.2 times higher than that of negative control.…”

A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

“…Reduction of this dye due to cell respiration during growth results in formation of a purple color, and it accumulates in the well over the incubation period; the data (see Fig. 5) are presented as a plot of the respiration over time (21,22). Therefore, the more respiratory activity growing cells have, the more purple color should accumulate.…”