A genetic selection for supercoiling mutants of Escherichia coli reveals proteins implicated in chromosome structure.
Chromosomes are divided into topologically independent regions, called domains, by the action of uncharacterized barriers. With the goal of identifying domain barrier components, we designed a genetic selection for mutants with reduced negative supercoiling of the Escherichia coli chromosome. We employed a strain that contained two chromosomally located reporter genes under the control of a supercoiling-sensitive promoter and used transposon mutagenesis to generate a wide range of mutants. We subjected the selected mutants to a series of secondary screens and identified five proteins as modulators of chromosomal supercoiling in vivo. Three of these proteins: H-NS, Fis and DksA, have clear ties to chromosome biology. The other two proteins, phosphoglucomutase (Pgm) and transketolase (TktA), are enzymes involved in carbohydrate metabolism and have not previously been shown to affect DNA. Deletion of any of the identified genes specifically affected chromosome topology, without affecting plasmid supercoiling. We suggest that at least H-NS, Fis and perhaps TktA assist directly in the supercoiling of domains by forming topological barriers on the E. coli chromosome.
Hardy
Christine D
CD
Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3204, USA.
Cozzarelli
Nicholas R
NR
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D035021:Factor For Inversion Stimulation Protein; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016254:Mutagenesis, Insertional; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D012641:Selection, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; C031908:H-NS protein, bacteria; D014157:Transcription Factors; C090751:dksA protein, E coli