The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of Hi-C to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains likely comprised of supercoiled plectonemes arrayed into a bottlebrush-like fiber. These domains are stable throughout the cell cycle and re-established concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly-expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC promote short-range compaction and the cohesion of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo.

Two-component signal transduction systems enable bacteria to sense, respond, and adapt to a wide range of environments, stressors, and growth conditions. In the prototypical two-component system, a sensor histidine kinase catalyzes its autophosphorylation and then subsequently transfers the phosphoryl group to a response regulator, which can then effect changes in cellular physiology, often by regulating gene expression. The utility of these signaling systems is underscored by their prevalence throughout the bacterial kingdom and by the fact that many bacteria contain dozens, or sometimes hundreds, of these signaling proteins. The presence of so many highly related signaling proteins in individual cells creates both an opportunity and a challenge. Do cells take advantage of the similarity between signaling proteins to integrate signals or diversify responses, and thereby enhance their ability to process information? Conversely, how do cells prevent unwanted cross-talk and maintain the insulation of distinct pathways? Here we address both questions by reviewing the cellular and molecular mechanisms that dictate the specificity of two-component signaling pathways.

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.