Cnu, a novel oriC-binding protein of Escherichia coli

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.     

A mutational study of Cnu reveals attractive forces between Cnu and H-NS

Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the mechanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.     

New roles for key residues in helices H1 and H2 of the Escherichia coli H-NS N-terminal domain: H-NS dimer stabilization and Hha binding

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.     

A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.     

The high-precision solution structure of Yersinia modulating protein YmoA provides insight into interaction with H-NS

The high-resolution solution structure of Yersinia modulating protein YmoA is presented. The protein is all helical with the first three of four helices forming the central core. Structures calculated with only NOE and dihedral restraints exhibit a backbone root-mean-square deviation (rmsd) of 0.77 A. Upon refinement against Halpha-Calpha, HN-N, and Calpha-C' J-modulated residual dipolar couplings, the backbone rmsd improves to 0.22 A. YmoA has a high amino acid sequence identity to and a similar overall fold to Escherichia coli hemolysin expression modulating protein Hha; however, structural differences do occur. YmoA is also found to be structurally similar to the histone-like nucleoid structuring protein H-NS, indicating that YmoA may intercalate into higher-order H-NS suprastructuring by substituting for an H-NS dimer.     

Essential residues in the H-NS binding site of Hha, a co-regulator of horizontally acquired genes in Enterobacteria

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of horizontally acquired genes in Enterobacteria. Systematic mutations of conserved acidic residues in Hha have allowed the identification of D48 as an essential residue for H-NS binding and the involvement of E25. Mutations of these residues resulted in deregulation of sensitive genes in vivo. D48 is only partially solvent accessible, yet it defines the functional binding interface between Hha and H-NS confirming that Hha has to undergo a conformational change to bind H-NS. Exposed acidic residues, such as E25, may electrostatically facilitate and direct the approach of Hha to the positively charged region of H-NS enabling the formation of the final complex when D48 becomes accessible by a conformational change of Hha.     

A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing

The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related.     

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.     

N9L and L9N mutations toggle Hha binding and hemolysin regulation by Escherichia coli and Vibrio cholerae H-NS

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of virulence factors in Enterobacteriaceae. Vibrio cholerae lacks Hha-like proteins and its H-NS (vcH-NS) is unable to bind Hha, in spite of the conservation of a key residue for Hha binding by Escherichia coli H-NS (ecH-NS). Exchange of the residues in position 9 between vcH-NS and ecH-NS strongly reduces Hha binding by ecH-NS and introduces it in vcH-NS. These mutations strongly affect the repression of the hemolysin operon in E. coli and the electrophoretic mobility of complexes formed with a DNA fragment containing its regulatory region.     

YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes. We also demonstrate that YdgT interacts both with H-NS and with StpA. Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell. The StpA protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.     

How can elongation factors EF-G and EF-Tu discriminate the functional state of the ribosome using the same binding site?

Elongation factors EF-G and EF-Tu are structural homologues and share near-identical binding sites on the ribosome, which encompass the GTPase-associated centre (GAC) and the sarcin-ricin loop (SRL). The SRL is fixed structure in the ribosome and contacts elongation factors in the vicinity of their GTP-binding site. In contrast, the GAC is mobile and we hypothesize that it interacts with the alpha helix D of the EF-Tu G-domain in the same way as with the alpha helix A of the G'-domain of EF-G. The mutual locations of these helices and GTP-binding sites in the structures of EF-Tu and EF-G are different. Thus, the orientation of the GAC relative to the SRL determines whether EF-G or EF-Tu will bind to the ribosome.     

FeeM, an N-acyl amino acid synthase from an uncultured soil microbe: structure, mechanism, and acyl carrier protein binding

Attempts to access antibiotics by capturing biosynthetic genes and pathways directly from environmental DNA, which is overwhelmingly derived from uncultured bacteria, have revealed a large and previously unknown family of N-acyl amino acid synthases (NASs). The structure of the NAS FeeM reveals structural similarity to the GCN5-related N-acyl transferases and acylhomoserine lactone synthases. The overall structure has a central beta sheet with alpha helices on both sides. A bound product at a cleft in the beta sheet identifies the active site and the structural basis for catalysis, and sequence conservation in this region indicates a bias for recognition over speed. FeeM interacts with an acyl carrier protein (FeeL), and the structure, mutagenesis, and enzymatic measurements reveal that a small hydrophobic pocket in alpha helix 5 dominates binding of FeeM to FeeL. The structural and mechanistic analyses suggest that the products of FeeM could be bacterial signaling agents.