YeiR: a metal-binding GTPase from Escherichia coli involved in metal homeostasis

A comparative genomic analysis predicted that many members of the under-characterized COG0523 subfamily of putative P-loop GTPases function in metal metabolism. In this work we focused on the uncharacterized Escherichia coli protein YeiR by studying both the physiology of a yeiR mutant and the in vitro biochemical properties of YeiR expressed as a fusion with the maltose-binding protein (YeiR-MBP). Our results demonstrate that deletion of yeiR increases the sensitivity of E. coli to EDTA or cadmium, and this phenotype is linked to zinc depletion. In vitro, the tagged protein binds several Zn(2+) ions with nanomolar affinity and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group, but GTP hydrolysis is enhanced by Zn(2+) binding. These results support the predicted connection between the COG0523 P-loop GTPases and roles in metal homeostasis.     

The DinG protein from Escherichia coli is a structure-specific helicase

The Escherichia coli DinG protein is a DNA damage-inducible member of the helicase superfamily 2. Using a panel of synthetic substrates, we have systematically investigated structural requirements for DNA unwinding by DinG. We have found that the helicase does not unwind blunt-ended DNAs or substrates with 3'-ss tails. On the other hand, the 5'-ss tails of 11-15 nucleotides are sufficient to initiate DNA duplex unwinding; bifurcated substrates further facilitate helicase activity. DinG is active on 5'-flap structures; however, it is unable to unwind 3'-flaps. Similarly to the homologous Saccharomyces cerevisiae Rad3 helicase, DinG unwinds DNA.RNA duplexes. DinG is active on synthetic D-loops and R-loops. The ability of the enzyme to unwind D-loops formed on superhelical plasmid DNA by the E. coli recombinase RecA suggests that D-loops may be natural substrates for DinG. Although the availability of 5'-ssDNA tails is a strict requirement for duplex unwinding by DinG, the unwinding of D-loops can be initiated on substrates without any ss tails. Since DinG is DNA damage-inducible and is active on D-loops and forked structures, which mimic intermediates of homologous recombination and replication, we conclude that this helicase may be involved in recombinational DNA repair and the resumption of replication after DNA damage.     

Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor

A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.     

EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N-1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.     

lon gene product of Escherichia coli is a heat-shock protein

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

The lipoate synthase from Escherichia coli is an iron-sulfur protein.

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.     

The ammonia channel protein AmtB from Escherichia coli is a polytopic membrane protein with a cleavable signal peptide

The Escherichia coli ammonia channel protein, AmtB, is a homotrimeric polytopic inner membrane protein in which each subunit has 11 transmembrane helices. We have shown that the structural gene amtB encodes a preprotein with a signal peptide that is cleaved off to produce a topology with the N-terminus in the periplasm and the C-terminus in the cytoplasm. Deletion of the signal peptide coding region results in significantly lower levels of AmtB accumulation in the membrane but modification of the signal peptidase cleavage site, leading to aberrant cleavage, does not prevent trimer formation and does not inactivate the protein. The presence of a signal peptide is apparently not a conserved feature of all prokaryotic Amt proteins. Comparison of predicted AmtB sequences suggests that while Amt proteins in Gram-negative organisms utilize a signal peptide, the homologous proteins in Gram-positive organisms do not.     

The Escherichia coli protein YjjG is a house-cleaning nucleotidase in vivo

House-cleaning enzymes protect cells from the adverse effects of noncanonical metabolic chemical compounds. The Escherichia coli nucleotide phosphatase YjjG (B4374, JW4336) functions as a house-cleaning phosphatase in vivo. YjjG protects the cell against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine (5-FdUridine), 5-fluorouridine, 5-fluoroorotic acid (5-FOA), 5-fluorouracil, and 5-aza-2'-deoxycytidine. YjjG prevents the incorporation of potentially mutagenic nucleotides into DNA as shown for 5-bromo-2'-deoxyuridine (BrdU). Its enzymatic activity in vitro towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) is higher than towards canonical thymidine monophosphate (dTMP). The closest homolog in humans, HDHD4, does not show a protective effect against noncanonical nucleotides, excluding an involvement of HDHD4 in resistance against noncanonical nucleotides used for cancer chemotherapy. The substrate spectrum of YjjG suggests that its in vivo substrates are noncanonical pyrimidine derivatives, which might also include oxidized nucleobases such as 5-formyluracil and 5-hydroxyuracil.     

CbiX-homologous protein (CbiXhp), a metal-binding protein, from Streptomyces seoulensis is involved in expression of nickel-containing superoxide dismutase

To find the accessory proteins participating in expression and maturation of nickel-containing superoxide dismutase (NiSOD), a metal-binding protein (CbiXhp) homologous to cobaltochelatase (CbiX) of Bacillus megaterium was isolated by nickel-nitrilotriacetic acid resin from Streptomyces seoulensis. The deduced amino acid sequence of cbiXhp showed 87% and 39% identity to CbiX of Streptomyces coelicolor and that of B. megaterium, respectively. Overexpression of CbiXhp increased the activity and the expression of NiSOD in the presence and absence of nickel, but to a lesser extent in its absence. This result indicates that CbiXhp is involved in the expression of NiSOD.     

Stationary phase protein overproduction is a fundamental capability of Escherichia coli

Although Escherichia coli is well studied and various recombinant E. coli protein expression systems have been developed, people usually consider the rapid growing (log phase) culture of E. coli as optimum for production of proteins. However, here we demonstrate that at stationary phase three E. coli systems, BL21 (DE3)(pET), DH5alpha (pGEX) induced with lactose, and TG1 (pBV220) induced with heat shock could overexpress diversified genes, including three whose products are deleterious to the host cells, more stably and profitably than following the log phase induction protocol. Physical and patch-clamp assays indicated that characteristics of target proteins prepared from cultures of the two different growth phases coincide. These results not only provide a better strategy for recombinant protein preparation in E. coli, but also reveal that rapid rehabilitation from stresses and stationary phase protein overproduction are fundamental characters of E. coli.     

SlyA,a regulatory protein from Salmonella typhimurium,induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

MdfA from Escherichia coli, a model protein for studying secondary multidrug transport

MdfA is a prototypic secondary multidrug transporter from Escherichia coli, which recognizes and exports a broad spectrum of structurally and electrically dissimilar toxic compounds. Here we review recent studies of MdfA, which, on the one hand, provide advanced understanding of certain aspects of secondary multidrug transport, and, on the other, address major mechanistic questions, some of which remain to be elucidated. Using biochemical, genetic, and physiological approaches, we have revealed several surprisingly promiscuous properties of MdfA including its multidrug recognition capacity, proton recognition determinants, aspects of energy utilization, and physiological role.     

Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.     

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.     

Crystal structure of YdaL, a stand-alone small MutS-related protein from Escherichia coli

Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL??-???), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/β/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL??-??? protein were slightly lower than those of full-length YdaL?-???, suggesting that residues 1-38 may be involved in DNA binding.     

Serine acetyltransferase from Escherichia coli is a dimer of trimers

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.