RESUMEN
The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mukB mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Cromosomas Bacterianos/química , Topoisomerasa de ADN IV/química , ADN Bacteriano/química , ADN Superhelicoidal/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Complejos Multiproteicos/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , MutaciónRESUMEN
MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo.
Asunto(s)
Proteínas Cromosómicas no Histona/química , ADN Bacteriano/química , ADN Superhelicoidal/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Multimerización de Proteína , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 µm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 µM.
Asunto(s)
Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Adenosina Trifosfato/farmacología , Proteínas de Escherichia coli/ultraestructura , Magnesio/farmacología , Polimerizacion/efectos de los fármacosRESUMEN
topB, encoding topoisomerase III, was identified as a high copy suppressor of the temperature-sensitive parC1215 allele, encoding one of the subunits of topoisomerase IV. Overexpression of topoisomerase III at the nonpermissive temperature was shown subsequently to restore timely chromosome decatenation and suppress lethality in strains carrying either temperature-sensitive parE or parC alleles. By developing an assay in vitro for precatenane unlinking, we demonstrated directly that both topoisomerase III and topoisomerase IV were efficient at this task, whereas DNA gyrase was very inefficient at precatenane removal. These observations suggest that precatenane unlinking is sufficient to sustain decatenation of replicating daughter chromosomes in the cell.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/enzimología , ADN Bacteriano/metabolismo , Microscopía Fluorescente , Especificidad por SustratoRESUMEN
SetB was identified as a high-copy suppressor of the partition defect of a mutation in parC, encoding one of the subunits of topoisomerase IV. Deletion of this integral inner membrane protein causes a delay in chromosome segregation, whereas its overproduction causes nucleoid disintegration and stretching, leading to a cell division defect. setB deletion mutants also exhibit a synthetic phenotype when combined with mutations that delete the C-terminal motor domain of the septal ring protein FtsK. SetB localizes in the cell as a helix and interacts with MreB, the bacterial actin homologue, which also forms a helix. These observations suggest that there may be a link between chromosome segregation and cellular infrastructure.