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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...

bThe gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378:318 -327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3= ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5= ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galEgalT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5= ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3= ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5= portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity. P olycistronic operons in bacteria show a differential expression of the constituent cistrons (1). A Northern blot analysis showed that there are 6 different species of mRNA specific to the galactose operon in wild-type E. coli cells grown exponentially in the presence of galactose (2). Five of the 6 mRNA species, mE1, mE2, mT1, mK1, and mM1, have their 5= ends at the transcription initiation region, and their 3= ends at 5 different locations within the operon, four of which (all but mE2) are at the ends of the galE, galT, galK, and galM cistrons, respectively (Fig. 1A). There is one distinct mRNA species, designated mK2, that has 5= ends not at the promoter region but at the middle of galT. The existence of these mRNA species automatically establishes a gradient of gene expression, higher in the promoter-proximal region and lower in the promoter-distal region, which has been referred to as "natural polarity" (3). Natural polarity is intrinsically different from what has been known as polarity that is caused by a mutation (4), because it can be observed in cells harboring the wild-type operon (2, 5-9). The term "polarity" refers to the phenomenon in which a mutation in one gene of an operon decreases the expression of the subsequent genes of the operon. The cause for polarity is well established. The cessation of translation by a nonsense mutation uncouples transcription from translation, allowing the transcription termination factor, Rho, to bind to the nascent RNA and terminate transcription at the next available termination signal. This Rho-mediated transcription termination leaves the rest of the operon untranscribed, creating polarity ...

Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.

Quantitative analyses of the 5′ end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD600 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD600 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population.

Fig. 1. A schematic presentation of the galactose operon. The -10 regions of the P1 and P2 promoters are shown in boxes. The transcription initiation sites are indicated by arrows and underlined. CRP points to its DNA binding site. OE and OI designate the operators where GalR binds. HU protein binds to hbs. The transcription initiation site of the P1 promoter is assigned as +1, Nucleotide positions of the operon, thus, have been assigned relative to the P1 initiation site.

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