Abstract
Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.
Highlights
Species differences pose serious problems for research in drug design, in developing therapeutic treatments for many human diseases, and for advancing gene therapy protocols that are safe for humans [1]
Four significant mysteries include: 1) Different phenotypes for the identical mutation in GyrB; 2) Different phenotypes for mutants in the MukB subunit of the condensin complex; 3) Toxicity of Salmonella GyrB when it is expressed at low levels in E. coli; 4) Significant differences in the supercoil density of plasmid and chromosomal DNA in cells growing on Luria Broth (LB) at all temperatures showed that E. coli is a “high supercoil organism.”
The aim of our work is to explain how supercoil differences are established throughout the chromosome in E. coli and Salmonella and to define the enzymatic mechanism(s) that coordinates pathway flow of transcription, translation, and protein folding for cells of each organism during exponential growth
Summary
Species differences pose serious problems for research in drug design, in developing therapeutic treatments for many human diseases, and for advancing gene therapy protocols that are safe for humans [1]. The basic differences between a man and mouse often involve yet-to-be defined biochemical pathways and responses in the species-specific innate immunological repertoire. With institutional funding’s emphasis on translational medicine and systems biology of all the “omics” (proteomics, transcriptomics, metabalomics and phenomics), little attention is being directed at understanding “core biochemistry” in different species, different cell types and different genetic backgrounds. Species differences in “core biochemistry” can be studied in “simple” bacteria [2]. In 2007, Champion discovered multiple unexpected differences between E. coli and Salmonella [3]. The aim of our work is to explain how supercoil differences are established throughout the chromosome in E. coli and Salmonella and to define the enzymatic mechanism(s) that coordinates pathway flow of transcription, translation, and protein folding for cells of each organism during exponential growth Four significant mysteries include: 1) Different phenotypes for the identical mutation in GyrB; 2) Different phenotypes for mutants in the MukB subunit of the condensin complex; 3) Toxicity of Salmonella GyrB when it is expressed at low levels in E. coli; 4) Significant differences in the supercoil density of plasmid and chromosomal DNA in cells growing on Luria Broth (LB) at all temperatures showed that E. coli is a “high supercoil organism.” The aim of our work is to explain how supercoil differences are established throughout the chromosome in E. coli and Salmonella and to define the enzymatic mechanism(s) that coordinates pathway flow of transcription, translation, and protein folding for cells of each organism during exponential growth
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