Cnu, a novel oriC-binding protein of Escherichia coli

Abstract

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

FIG. 1.

(A) Schematic illustration of the oriC region. The important elements of oriC are marked with ovals and empty boxes in the schematic and with underlines in the nucleotide sequence of the minimal oriC. The 13-mers L, M, and R are the AT-rich 13-mer repeats where the origin initially opens. R1, R2, R3, R4, and R5, and I1, I2, and I3 are DnaA boxes and I sites where the initiator protein DnaA binds. (B) Ori-1, Ori-2, and Ori-3 are the DNA sequences used as operators in plasmids with an artificial rpsL operon. The newly discovered protein-binding site cnb is identified over the sequence. We tried to avoid DNA sequences that are known to bind to proteins (except DnaA) while choosing the operator sequences.

FIG. 2.

(A) SF measurements at various times during growth of HB101/pOri-3. The line graph (growth curve) shows A₆₀₀ values at different time points. The bars represent SF values at the corresponding time points. (B) Western blots of Cnu at various times during growth of HB101. The first lane (Cnu) is the purified Cnu protein (1 μg). The numbers indicate the time points in panel A. Crude extracts (20 μg of protein) from the culture at each time point were loaded. Lane M is the 10-kDa Western size marker (ELPis-Biotech, Taejean, Korea).

FIG. 3.

Description of pHL355 used to construct an expression library of the E. coil genome. pHL355 has been derived from pBluescript and contains the gene for the lac repressor (lacI^q), the ampicillin-resistant determinant (amp), the promoter tac (Ptac), and the transcriptional stop signal (rrnBT1T2). This diagram also shows a piece of genomic DNA cloned in the plasmid that gave a high SF value to the host cells. Serial deletions of the insert DNA (shown in this figure) and the corresponding SF values indicated that the putative b1625 open reading frame is the determinant for the high SF value.

FIG. 4.

Comparison of amino acid sequences of proteins homologous to Cnu. The identical amino acids are shown in boldface.

FIG. 5.

(A and B) SDS-PAGE showing proteins eluted from Ni-NTA agarose after interaction with H-NS alone (A) or a mixture of 6×His-Cnu and H-NS (B). (C) SDS-PAGE of proteins after cross-linking with increasing concentrations of glutaraldehyde (0.00025, 0.005, 0.025, and 0.125%). (D) SDS-PAGE of crude extract of BL21/pETCnu after Cnu overexpression, loading onto a Ni-NTA agarose column, washing of the loaded column, and elution from the column.

FIG. 7.

Flow cytometry of triple mutant MG1655cnuhhahns along with other MG1655 derivatives. Genotypes were indicated in each box. Cells used were exponentially growing in LB medium. The generation times of all six strains tested were in the range of 24 to 28 min.

FIG. 6.

Determination of cell size (A) and chromosome content (B-D) by flow cytometry. Cells used were exponentially growing in different growth media: LB broth (A-B), minimal glucose + CAA (C), and minimal glucose (D). Depending upon the growth medium, the two major peaks of Hoechst fluorescence represent cells with either four and eight (B) or two and four (C-D) chromosomes, but their ratio varied depending upon the genotype of the cells as identified to the right of the figure (see Table 3 for details). The cells used were either MG1655 (WT) or its isogenic derivatives. The dam mutant (BR2786) is from our lab collection and is not derived from MG1655.