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.     

Osmoregulation of the HtrA (DegP) protease of Escherichia coli: an Hha-H-NS complex represses HtrA expression at low osmolarity

The HtrA protein of Escherichia coli is a heat-shock inducible periplasmic protease, essential for bacterial survival at high temperatures. Expression of htrA gene depends on the alternative factor sigmaE and on the two-component regulatory system Cpx. These regulators systems respond, among others factors, to overproduction of misfolded proteins in the periplasm or to high level synthesis of various extracytoplasmic proteins. We describe in this report the osmoregulation of the expression of htrA gene. Low osmolarity conditions result in htrA repression. We report, as well, the role of the nucleoid associated proteins H-NS and Hha in the repression of htrA expression at low osmolarity.     

Functional replacement of the oligomerization domain of H-NS by the Hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.     

Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system.

Summary A fusion gene (ces-hlyA _s) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyA _s) ofEscherichia coli hemolysin (H1yA) to the ces gene for a cholesterol esterase/lipase (CE) from aPseudomonas species. Part (about 30 %) of the expressed fusion protein CE-H1yA_s was secreted inE. coli carryinghlyB andhlyD genes. Following the insertion between the reporter gene andhlyA _s of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-H1yA_s and CE-HlyA_s) were shown to be cleaved by OmpT between the two parts during H1yB/H1yD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increasedompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.     

Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

Mass spectrometric study of the Escherichia coli repressor proteins, IcIR and GcIR, and their complexes with DNA

In Escherichia coli, the IclR protein regulates both the aceBAK operon and its own synthesis. Database homology searches have identified many IclR-like proteins, now known as the IclR family, which can be identified by a conserved C-terminal region. We have cloned and purified one of these proteins, which we have named GclR (glyoxylate carboligase repressor). Although purification is straightforward, both the IclR and GclR proteins are difficult to manipulate, requiring high salt (up to 0.6 M KCl) for solubility. With the advent of nanospray ionization, we could transfer the proteins into much higher concentrations of volatile buffer than had been practical with ordinary electrospray. In 0.5 M ammonium bicarbonate buffer, both proteins were stable as tetramers, with a small amount of dimer. In a separate experiment, we found that IclR protein selected from a random pool a sequence which matched exactly that of the presumed binding region of the GclR protein, although IclR does not regulate the gcl gene. We designed a 29 bp synthetic DNA to which IclR and GclR bind, and with which we were able to form noncovalent DNA-protein complexes for further mass spectrometry analysis. These complexes were far more stable than the proteins alone, and we have evidence of a stoichiometry which has not been described previously with (protein monomer : dsDNA) = (4 : 1).     

Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression.     

Differential protein-protein binding affinities of H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 and IncP-7 plasmid pCAR1

H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 (TurA and TurB) and the IncP-7 plasmid pCAR1 (Pmr) commonly have an N-terminal dimerization/oligomerization domain constituted by a central and a terminal dimerization sites. To clarify the dimerization manner at the central dimerization sites of the three homologs, we performed chemical cross-linking analyses with protein variants inactivated at the terminal dimerization site. Comparison of the hetero-dimer ratios among them suggested stronger affinities between the central dimerization sites of TurA and TurB monomers than between TurA and Pmr or TurB and Pmr. Furthermore, analyses of the interaction between truncated TurB containing only a functional terminal dimerization site and full-length proteins suggested that TurB exhibited higher affinities for oligomer complex formation with TurB itself and TurA but not Pmr. Altogether, we revealed stronger interaction between the N-terminal domains of TurA and TurB than between either of them and Pmr.     

Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli

Abstract Alanine dipeptides normally penetrate into Pseudomonas aeruginosa by the way of two transport systems. In peptidase N-deficient mutants, dialanine is unable to use its low affinity transport system. Uptake competition showed that this system harboured a permease common to the transport of the amino acid alanine. This permease permits the penetration of both alanine and alanyl peptides uniquely in the presence of active peptidase N. The uptake of trialanine is independent of the presence of active peptidase N inside bacteria despite the fact that hydrolysis of this tripeptide absolutely requires this activity.     

Fast microscopical dissection of action scenes played by Escherichia coli RNA polymerase

Using fast-scanning atomic force microscopy, we directly visualized the interaction of Escherichia coli RNA polymerase (RNAP) with DNA at the scan rate of 1-2 frames per second. The analyses showed that the RNAP can locate the promoter region not only by sliding but also by hopping and/or segmental transfer. Upon the addition of 0.05 mM NTPs to the stalled complex, the RNAP molecule pulled the template DNA uni-directionally at the rates of 15 nucleotides/s on average. The present method is potentially applicable to examine a variety of protein-nucleic acid interactions, especially those involved in the process of gene regulation.