Cnu, a NoveloriC-Binding Protein ofEscherichia coli

Abstract

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoidassociated) 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.The chromosomal DNA replication in Escherichia coli starts from a single locus called oriC that is minimally 258 base pairs (bp) long. This DNA sequence contains DNA-binding sites for many different proteins that participate in DNA replication (Fig. 1). There are eight binding sites (DnaA boxes and I sites) for the initiator protein DnaA (19,35). The IciA and DpiA proteins bind to the AT-rich 13-mer repeats in oriC. IciA inhibits unwinding of the repeats (11), and overexpression of DpiA can cause SOS response (20). Nucleoid proteins such as IHF and Fis bind specifically to oriC and bend oriC upon binding (27). Another nucleoid protein, HU, binds to oriC nonspecifically but modulates the binding of IHF to oriC (5). Binding of these nucleoid proteins was shown in vitro to assist the action of DnaA protein in the unwinding of oriC (12, 28). The SeqA protein known as a negative modulator of replication initiation (17) binds specifically to two sites in oriC and has higher affinity toward hemimethylated rather than fully methylated oriC (33,34). Nonspecific acid phosphatase also preferentially binds to hemimethylated oriC (26). Rob binds to the right region of oriC (31), while phosphorylated ArcA protein binds to the left region of oriC (16). Although deletion of the rob gene has no phenotype, phosphorylated ArcA inhibits chromosomal replication in vitro (16). Finally, CspD, a singlestranded DNA-binding protein, was shown to inhibit DNA replication in vitro (37).The control of chromosomal DNA replication is a complex process in which many proteins are needed to allow initiation at the right time and frequency in accordance with the changing environment. Because the process remains to be satisfactorily understood, we contemplated that there could be more oriC-binding proteins yet to be discovered. In an attempt to find new oriC-binding proteins, we used a genetic strategy that employs transcriptional repression that is caused by DNA binding of a protein to an operator (15). This assay revealed a novel oriC-binding protein, which we have named Cnu (oriCbinding nucle...

Modulation of Rho-Dependent Transcription Termination in Escherichia coli by the H-NS Family of Proteins

“…Binding of H-NS to DNA results in several structural (including supercoiling) alterations that have been variously referred to as bending, bridging, coating, looping, and stiffening of the DNA (16,20,23,32,39,61). YdgT and Hha bear structural resemblance to, and also interact with, the N-terminal oligomerization domains of H-NS and StpA; in this manner, YdgT and Hha are believed to modulate the DNA-binding and nucleoid-organizing properties of H-NS and StpA even though they do not bind DNA by themselves (23,30,33,38,42,55).…”

“…on April 7, 2019 by guest http://jb.asm.org/ suppressors of rho and nusG (27) and, since YdgT is an interacting partner of H-NS (30,33,42), we tested whether it is the presence of YdgT in the plasmid derivatives that was responsible for the suppression phenotype. The minimal ydgT construct, as well as the gene fragment encoding H-NS⌬64 as a positive control, was cloned downstream of the IPTG-inducible trc promoter in the ColE1-plasmid vector pTrc99A.…”

“…YdgT (also called Cnu [30]) and Hha belong to the H-NS family of proteins (33), whose prototypic member H-NS participates in both regulation of transcription initiation and nucleoid structure (reviewed in references 16, 20, 23, and 59). Genetic, biophysical, and structural studies have shown that the 137-amino acid H-NS protein possesses two separate dimerization interfaces, between approximately residues 2 and 47 and residues 58 and 84, respectively, and a third C-terminal DNA-binding domain between residues 92 and 137 (3,5,8,22,54,57,58).…”

A Mutational Study of Cnu Reveals Attractive Forces between Cnu and H-NS

“…These are Cnu and Hha in Escherichia coli (Kim et al, 2005;Nieto et al, 1991), RomA from the plasmid R100-1 (Nieto et al, 1998), and YmoA from Yersinia enterocolitica (Cornelis et al, 1991). The Cnu-H-NS complex binds to cnb (Kim et al, 2005), and the Hha-H-NS complex represses the hly operon that encodes the hemolysin toxin (Nieto et al, 2000).…”

“…Cnu was originally identified as a DNA binding factor for a specific region in the origin of chromosomal DNA replication of E. coli (oriC), cnb (Kim et al, 2005) (Fig. 1B).…”