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Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

ABSTRACT: 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.

SUBMITTER: Atlung T

PROVIDER: S-EPMC134913 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

Atlung T T Hansen F G FG

Journal of bacteriology 20020401 7

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 no ...[more]

PMID: 11889089

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Project description:UnlabelledThe architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a “slow-growth” type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content.ImportanceIt is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions.

Dataset Information

Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

Publications

Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

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