YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

Changes in streptonigrin lethality during adaptation of Escherichia coli to picolinic acid correlation with intracellular picolinate and iron uptake

Uptake studies with [¹⁴C]picolinate and ⁵⁵Fe³⁺ have provided an explanation for the change in streptonigrin killing on adaptation of Escherichia coli to picolinate, in terms of the available iron within the cell. When picolinic acid is added to a growing culture of E. coli an interval of bacteriostasis ensues; this adaptation period is followed by resumption of exponential growth. Addition of picolinate (4 mM) to a log phase culture of strain W3110 gave protection from the lethal action of streptonigrin (30 μM) when the two agents were added simultaneously. In contrast streptonigrin killed cells that had adapted to picolinate; however, a preincubation of adapted W3110 with phenethyl alcohol protected the cells from streptonigrin lethality. [¹⁴C]Picolinate uptake studies showed that initially picolinate entered the cells, but that it was excluded from adapted cells; addition of phenethyl alcohol permitted the entry of picolinate into adapted W3110. The changes in streptonigrin killing parallel the changes in concentration of intracellular picolinate, which can chelate the iron required by streptonigrin for its bactericidal action. ⁵⁵Fe³⁺ uptake studies showed that initially picolinate prevented iron accumulation by strain W3110, whereas adapted cells did take up iron in the presence of picolinate. Addition of phenethyl alcohol prevented any observed uptake of iron by adapted W3110. This modulation of iron transport by picolinate also affects streptonigrin lethality. Experiments with iron transport mutants showed that picolinate acted on both the enterochelin and citrate routes of uptake. Therefore picolinate affects the concentration of available iron within the cell both by (a) its intracellular presence resulting in chelation of iron and (b) its action on iron uptake; these effects explain the change in streptonigrin killing on adaptation of E. coli to picolinate.     

The Asymmetric Flagellar Distribution and Motility of Escherichia coli

Rod-shaped bacteria such as Escherichia coli divide by binary fission. They inherit an old pole from the parent cell. The new pole is recently derived from the septum. Because the chemoreceptor accumulates linearly with time on the cell pole, the old pole carries more receptors than does the new pole. Here, further evidence is provided that the old pole appears more frequently at the rear when bacteria swim. This phenomenon had been observed, yet not extensively explored in the literature. The biased swimming orientation is the consequence of the asymmetric distribution of flagella over the cell surface. On about 75% of cells, there are more flagella on the old-pole half of the cell than on the new-pole half, regardless of growth conditions. Most flagella are lateral, and few were found on the cell pole per se. The asymmetric flagellar distribution makes cells more efficient in chemotaxis. Both swimming orientation and receptor localization are components of chemotaxis, by which bacteria follow environmental stimuli. If unipolarly flagellated cells, such as the swarmer cells of Caulobacter crescentus, are regarded as 100% polar with respect to chemotaxis, E. coli is about 75%. The difference is quantitative. The peritrichous flagellation might enhance the motility and chemotaxis in the viscous environment of enteric bacteria.     

Genome-scale genetic engineering in Escherichia coli

Genome engineering has been developed to create useful strains for biological studies and industrial uses. However, a continuous challenge remained in the field: technical limitations in high-throughput screening and precise manipulation of strains. Today, technical improvements have made genome engineering more rapid and efficient. This review introduces recent advances in genome engineering technologies applied to Escherichia coli as well as multiplex automated genome engineering (MAGE), a recent technique proposed as a powerful toolkit due to its straightforward process, rapid experimental procedures, and highly efficient properties.     

The transport and mediation mechanisms of the common sugars in Escherichia coli

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.     

Escherichia coli ghosts promote innate immune responses in human keratinocytes

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.     

Lateral phase separations in Escherichia coli membranes

Membranes from unsaturated fatty acid auxotrophs of Escherichia coli were studied by spin labeling and freeze-fracturing. From measurements of the partition of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) between the aqueous phase and fluid lipids in isolated membranes, temperatures, corresponding to the onset and completion of a lateral phase separation of the membrane phospholipids were determined. By freeze-fracture electron microscopy a change in the distribution of particle in the membrane was observed around the temperature of the onset of the lateral phase separation. When cells were frozen from above that temperature a netlike distribution of particles in the plasma membrane was observed for unfixed preparations. When frozen after fixing with glutaraldehyde the particle distribution was random. In membranes of cells frozen with or without fixing from a temperature below the onset of the phase separation, the particles were aggregated and large areas void of particles were present. This behavior can be understood in terms of the freezing rate with the aid of phase diagrams.     

Genetic activity of bleomycin in Escherichia coli

The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated λ-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies. (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes. (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain. (3) The plating efficiency of UV-irradiated λ-phage increased by Blm treatment of the host cell. This increase was not observed in the umuC mutant. The plating efficiency of UV-irradiated λ-phage was drastically reduced in the lexA and recA strains treated with Blm. (4) No significant increase of the reversion of nonsense mutation (his-4 to His⁺) in AB1157 by the treatment of Blm was observed. Possible implications of these results are discussed.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

RecA-mediated SOS response provides a geraniol tolerance in Escherichia coli

Geraniol is an important industrial material and a potential candidate of advanced biofuels. One challenge of microbial geraniol production is the toxicity to hosts. However, the poor understanding on geraniol tolerance mechanism is an obstacle for developing geraniol tolerant host. This study genome-widely screened a shot-gun DNA library of Escherichia coli and found that recA is able to confer geraniol tolerance in E. coli. The recA knockout mutant was found extremely sensitive to geraniol. Based on our data, it was deciphered that recA provided tolerance through SOS response network responding to DNA damage caused by geraniol. RecA-mediated SOS response activates the homologous recombinational repair by RecB and RecN for corrective DNA maintenance. This protection mechanism suggests an effective strategy to combat geraniol toxicity in E. coli.     

A second Escherichia coli protein with CL synthase activity

The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned. Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL. A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity. Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Inducible gluconate permease in a gluconate kinase-deficient mutant of Escherichia coli

Gluconate-resistant mutants were isolated from Escherichia coli strain DF 1070 deficient in phosphogluconate dehydrogenase (EC 1.1.1.44) and in phosphogluconate dehydrogenase (EC 4.2.1.12) which is inhibited by gluconate. Among the resistant mutants, AR 13 has been identified as a gluconate kinase (EC 2.7.1.12)-deficient strain.This mutant exhibits an inducible gluconate transport system capable of concentrating gluconate in the cytoplasm against a concentration gradient. The accumulated gluconate is subject to permanent turnover, and is not chemically modified.The kinetics of induction and deinduction indicate a single inducible component, rate limiting for the transport function, and the distribution of transport capacity among non-induced progeny of induced parents indicates that the inducible protein is membrane bound.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.     

Inhibition of NAD biosynthesis by paraquat in Escherichia coli

Niacin significantly reduced the bacteristatic effect of 1 mM paraquat for Escherichia coli. Without niacin (an intermediate in the salvage pathway for pyridine nucleotide coenzyme biosynthesis), the NAD concentration was decreased rapidly and significantly in E. coli during paraquat poisoning. Niacin prevented the decline in NAD in paraquat-poisoned cells; quinolinate (an intermediate in de novo NAD biosynthesis prior to the entry point of niacin) did not. These data suggest that paraquat poisons the de novo pathway of pyridine nucleotide coenzyme biosynthesis. Similar consequences have been reported to result from hyperbaric oxygen poisoning of E. coli; thus, there is growing evidence for a common mechanism of toxicity for hyperoxia and paraquat.     

Assembly and overexpression of membrane proteins in Escherichia coli

The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class. Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins. In addition, we will briefly review and discuss how E. coli has been used as a vehicle for the overexpression of membrane proteins.     

Evidence that both ligand binding and covalent adaptation drive a two-state equilibrium in the aspartate receptor signaling complex.

The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations ("on" and "off") yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor-kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated.     

Induction Kinetics of a Conditional pH Stress Response System in Escherichia coli

The analysis of stress response systems in microorganisms can reveal molecular strategies for regulatory control and adaptation. In this study, we focused on the Cad module, a subsystem of Escherichia coli’s response to acidic stress that is conditionally activated at low pH only when lysine is available. When expressed, the Cad system counteracts the elevated H⁺ concentration by converting lysine to cadaverine under the consumption of H⁺ and exporting cadaverine in exchange for external lysine. Surprisingly, the cad operon displays a transient response, even when the conditions for its induction persist. To quantitatively characterize the regulation of the Cad module, we experimentally recorded and theoretically modeled the dynamics of important system variables. We established a quantitative model that adequately describes and predicts the transient expression behavior for various initial conditions. Our quantitative analysis of the Cad system supports negative feedback by external cadaverine as the origin of the transient response. Furthermore, the analysis puts causal constraints on the precise mechanism of signal transduction via the regulatory protein CadC.