Identification of N-terminal acetylation of recombinant human prothymosin α in Escherichia coli

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin α is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin α expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin α, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin α produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin α and thymosin α1.     

Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-BΔc). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B_ΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B_ΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B_ΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B_ΔC that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B_ΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

The effect of severing abdominal nerves on egg production in Rhodnius prolixus Stål

Severing the two large abdominal nerves which serve segment VI and the internal genitalia in Rhodnius does not prevent the increase in egg production which follows mating so long as the transport of semen into the spermathecae is unaffected. It is concluded that there is no nervous component in the transmission of the mating stimulus from the genitalia to the central nervous system. Severing the remaining abdominal nerves (i.e. those to segments II–V) on one side reduces the egg production of mated females but does not affect that of virgin females. These results are interpreted in terms of interrupting an inhibitory innervation of the abdominal neurosecretory organs which secrete an antigonadotropin.     

Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SP_MalE:IsPETase and pET22b-SP_LamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.     

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli

Background

Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.

Methods

The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.

Results

Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.

Conclusions

The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.

General significance

We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.     

Noncritical Signaling Role of a Kinase–Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex

In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor–P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5–receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW–receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.     

Is the secondary putative RNA–RNA interaction site relevant to GcvB mediated regulation of oppA mRNA in Escherichia coli?

GcvB is a non-coding RNA that regulates oppA mRNA in different bacterial species by binding a GcvB GU-rich region named R1 to oppA mRNA. A secondary putative interaction site (PS1) was identified in this study that is able to form a second nearly perfect 10 base-pair duplex between these two RNAs in Escherichia coli. In this work, we have studied whether the formation of a second interaction site could help stabilize the previously reported GcvB/oppA complex. Several mutations and the full deletion of PS1 were engineered. None of these modifications affected the ability of GcvB to control OppA expression. Therefore the second, putative, interaction site appears to be unnecessary for the regulatory function of GcvB with regard to its oppA target mRNA.     

Mutation and DNA replication in Escherichia coli treated with low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

N-Methyl-N′-nitro-N-nitrosoguanidine (nitrosoguanidine) causes an unexpectedly high frequency of closely linked double mutants because of its specificity for chromosome regions in replication. Low nitrosoguanidine concentrations (1 μg/ml) in liquid cultures allow replication at the normal rate and are mutagenic. It was expected that mutations would be spread over the chromosome as it replicated, but a high frequency of closely linked double mutants was found.If a thymine auxotroph is grown in the presence of 5-bromodeoxyuridine (BUdR) and nitrosoguanidine, then exposed to 313-nm radiation (which destroys BUdR-substituted DNA), the mutation frequency is much higher among survivors than among non-irradiated cells. It is concluded that nitrosoguanidine inhibits DNA replication in a small fraction of the population and that mutations are induced in that same fraction.Nitrosoguanidine treatment leads to a high frequency of closely linked double mutants under all known conditions.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Factor-free (“Non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes

Systems of poly(U)-directed polyphenylalanino synthesis by Escherichia coli ribosomes in the absence of elongation factors and GTP (factor-free system) or in the presence of one of the elongation factors and GTP (EF-G² and EF-T_u-deperident systems) are described. It is shown that the use of oligouridylates of different length as templates in the factor-free system results in peptides, the degree of polymerization of which does not exceed the number of template codons, i.e. a conjugated translocation of the peptidyl-tRNA and the template takes place. Thus, the function of translocation as well as the specific binding of aminoacyl-tRNA and transpeptidation proved to be intrinsic to the ribosome itself. The study of kinetics of polyphenylalanine synthesis and dependence of the synthesis rate on the Mg²⁺ concentration in the factor-free, EF-T_u-dependent and EF-G-dependent translation systems has demonstrated that the elongation factors with GTP promote ribosomal mechanisms of aminoacyl-tRNA binding and translocation, respectively. It turned out that the factor-free translation system does not display miscoding. It is the promotion of translocation by EF-G with GTP that has been found to be responsible in full measure for miscoding, while EF-T_U with GTP does not contribute to this.     

Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~ 25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80 °C, compared to 66 °C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.     

Immediate stoichiometric appearance of β-galactosidase products in the medium of Escherichia coli cells incubated with lactose

Products of β-galactosidase action on lactose by intact E. coli cells appeared in the medium as soon as lactose was added and the amount of product was equal to the lactose used. No detectable levels of β-galactosidase were found in the medium and lactose was not significantly broken down unless lac permease was present. The appearance did not depend upon the presence of any of the commonly known galactose or glucose permease systems. The Km of product appearance from whole cells was equal to the K_t for lactose transport by lac permease. When the cells were broken the Km became the normal β-galactosidase Km.     

Escherichia coli HflK and HflC can individually inhibit the HflB (FtsH)-mediated proteolysis of λCII in vitro

λCII is the key protein that influences the lysis/lysogeny decision of λ by activating several phage promoters. The effect of CII is modulated by a number of phage and host proteins including Escherichia coli HflK and HflC. These membrane proteins copurify as a tightly bound complex ‘HflKC’ that inhibits the HflB (FtsH)-mediated proteolysis of CII both in vitro and in vivo. Individual purification of HflK and HflC has not been possible so far, since each requires the presence of the other for proper folding. We report the first purification of HflK and HflC separately as active and functional proteins and show that each can interact with HflB on its own and each inhibits the proteolysis of CII. They also inhibit the proteolysis of E. coli σ³² by HflB. We show that at low concentrations each protein is dimeric, based on which we propose a scheme for the mutual interactions of HflB, HflK and HflC in a supramolecular HflBKC protease complex.     

Hybrid dynamic modeling of Escherichia coli central metabolic network combining Michaelis–Menten and approximate kinetic equations

The construction of dynamic metabolic models at reaction network level requires the use of mechanistic enzymatic rate equations that comprise a large number of parameters. The lack of knowledge on these equations and the difficulty in the experimental identification of their associated parameters, represent nowadays the limiting factor in the construction of such models. In this study, we compare four alternative modeling approaches based on Michaelis–Menten kinetics for the bi-molecular reactions and different types of simplified rate equations for the remaining reactions (generalized mass action, convenience kinetics, lin-log and power-law). Using the mechanistic model for Escherichia coli central carbon metabolism as a benchmark, we investigate the alternative modeling approaches through comparative simulations analyses. The good dynamic behavior and the powerful predictive capabilities obtained using the hybrid model composed of Michaelis–Menten and the approximate lin-log kinetics indicate that this is a possible suitable approach to model complex large-scale networks where the exact rate laws are unknown.     

Spermidine triggering effect to the signal transduction through the AtoS–AtoC/Az two-component system in Escherichia coli

Recent analysis revealed that, in Escherichia coli the AtoS–AtoC/Az two-component system (TCS) and its target atoDAEB operon regulate the biosynthesis of short-chain poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles, upon acetoacetate-mediated induction. We report here that spermidine further enhanced this effect, in E. coli that overproduces both components of the AtoS–AtoC/Az TCS, without altering their protein levels. However, bacteria that overproduce either AtoS or AtoC did not display this phenotype. The extrachromosomal introduction of AtoS–AtoC/Az in an E. coli ΔatoSC strain restored cPHB biosynthesis to the level of the atoSC⁺ cells, in the presence of the polyamine. Lack of enhanced cPHB production was observed in cells overproducing the TCS that did not have the atoDAEB operon. Spermidine attained the cPHB enhancement through the AtoC/Az response regulator phosphorylation, since atoC phosphorylation site mutants, which overproduce AtoS, accumulated less amounts of cPHB, compared to their wild-type counterparts. Exogenous addition of N⁸-acetyl-spermidine resulted in elevated amounts of cPHB but at lower levels than those attained upon spermidine addition. Furthermore, AtoS–AtoC/Az altered the intracellular distribution of cPHB according to the inducer recognized by the TCS. Overall, AtoS–AtoC/Az TCS was induced by spermidine to regulate both the biosynthesis and the intracellular distribution of cPHB in E. coli.     

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Paraxial left-sided nodal expression and the start of left–right patterning in the early chick embryo

A common element during early left–right patterning of the vertebrate body is left-sided nodal expression in the early-somite stage lateral plate mesoderm. Leftward cell movements near the node of the gastrulating chick embryo recently offered a plausible mechanism for breaking the presomite-stage molecular symmetry in those vertebrates which lack rotating cilia on the notochord or equivalent tissues. However, the temporal and functional relationships between generation of the known morphological node asymmetry, onset of leftward cell movements and establishment of stable molecular asymmetry in the chick remain unresolved. This study uses high-resolution light microscopy and in situ gene expression analysis to show that intranodal cell rearrangement during the phase of counter-clockwise node torsion at stage 4+ is immediately followed by symmetry loss and rearrangement of shh and fgf8 expression in node epiblast between stages 5− and 5+. Surprisingly, left-sided nodal expression starts at stage 5−, too, but lies in the paraxial mesoderm next to the forming notochordal plate, and can be rendered symmetrical by minimal mechanical disturbance of distant tissue integrity at stage 4. The “premature” paraxial nodal expression together with morphological and molecular asymmetries in, and near, midline compartments occurring at defined substages of early gastrulation help to identify a new narrow time window for early steps in left–right patterning in the chick and support the concept of a causal relationship between a—still enigmatic—chiral (motor) protein, cell movements and incipient left–right asymmetry in the amniote embryo.