Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.
Flow cytometric analysis showed that the hns205 and hns206 mutants, lacking the abundant nucleoid-associated protein H-NS, have decreased origin concentration, as well as a low number of origins per cell (ploidy). The most striking observation was that the low ploidy was due to a very short replication time, e.g., at 30 degrees C it was halved compared to that of the hns(+) strain. The decreased origin concentration was not caused by a decreased dnaA gene expression, and the hns206 mutant had normal DnaA protein concentrations. The replication phenotypes of the hns206 mutant were independent of RpoS. Cells overproducing H-NS from a LacI-controlled plasmid had a normal origin concentration, indicating that H-NS is not controlling initiation. A wild-type H-NS concentration is, however, required to obtain a wild-type origin concentration, since cells with an intermediate H-NS concentration had an intermediate origin concentration. Two lines of evidence point to an indirect effect of H-NS on initiation. First, H-NS did not show high-affinity binding to any part of oriC, and H-NS had no effect on transcription entering oriC from the mioC promoter. Second, in a shift experiment with the hns206 mutant, when H-NS protein was induced to wild-type levels within 10 min, it took more than one generation before the origin concentration started to increase.
Atlung
T
T
Department of Life Sciences and Chemistry, Roskilde University, DK-4000 Roskilde, Denmark. atlung@ruc.dk
Hansen
F G
FG
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects; D002876:Chromosomes, Bacterial / Q000187:drug effects* / Q000502:physiology; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D015870:Gene Expression / Q000187:drug effects; D007700:Kinetics; D051738:Origin Recognition Complex; D010641:Phenotype; D012808:Sigma Factor / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D005420:Flavoproteins; C031908:H-NS protein, bacteria; C109225:MioC protein, E coli; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D012808:Sigma Factor; D014764:Viral Proteins; C069567:sigma factor KatF protein, Bacteria