Characterization of disulfide exchange between DsbA and HtrA proteins from Escherichia coli

DsbA is the major oxidase responsible for generation of disulfide bonds in proteins of E. coli envelope. In the present work we provided the first detailed characterization of disulfide exchange between DsbA and its natural substrate, HtrA protease. We demonstrated that HtrA oxidation relies on DsbA, both in vivo and in vitro. We followed the disulfide exchange between these proteins spectrofluorimetrically and found that DsbA oxidizes HtrA with a 1:1 stoichiometry. The calculated second-order apparent rate constant (kapp) of this reaction was 3.3x10(4)+/-0.6x10(4) M-1s-1. This value was significantly higher than the values obtained for nonfunctional disulfide exchanges between DsbA and DsbC or DsbD and it was comparable to the kapp values calculated for in vitro oxidation of certain non-natural DsbA substrates of eukaryotic origin.     

Purification and Characterization of Escherichia coli MreB Protein

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm.     

Role of the Escherichia coli SurA Protein in Stationary-Phase Survival

SurA is a periplasmic peptidyl-prolyl isomerase required for the efficient folding of extracytoplasmic proteins. Although the surA gene had been identified in a screen for mutants that failed to survive in stationary phase, the role played by SurA in stationary-phase survival remained unknown. The results presented here demonstrate that the survival defect of surA mutants is due to their inability to grow at elevated pH in the absence of ς^S. When cultures of Escherichia coli were grown in peptide-rich Luria-Bertani medium, the majority of the cells lost viability during the first two to three days of incubation in stationary phase as the pH rose to pH 9. At this time the surviving cells resumed growth. In cultures of surA rpoS double mutants the survivors lysed as they attempted to resume growth at the elevated pH. Cells lacking penicillin binding protein 3 and ς^S had a survival defect similar to that of surA rpoS double mutants, suggesting that SurA foldase activity is important for the proper assembly of the cell wall-synthesizing apparatus.     

Analysis and Expansion of the Role of the Escherichia coli Protein ProQ

The decrease in proline transport by the proline porter ProP in a ΔproQ strain has been well documented; however, the reason for this phenotype remains undefined. Previous studies have speculated that ProQ facilitates translation of proP mRNA. Here, we demonstrate that ProQ is enriched in the polysome fractions of sucrose gradient separations of E. coli lysates and the 30S fractions of lysates separated under conditions causing ribosomal subunit dissociation. Thus, ProQ is a bona fide ribosome associated protein. Analysis of proQ constructs lacking predicted structural domains implicates the N-terminal domain in ribosome association. Association with the ribosome appears to be mediated by an interaction with the mRNA being translated, as limited treatment of lysates with Micrococcal Nuclease maintains ribosome integrity but disrupts ProQ localization with polysomes. ProQ also fails to robustly bind to mRNA-free 70S ribosomes in vitro. Interestingly, deletion of proP does not disrupt the localization of ProQ with translating ribosomes, and deletion of proP in combination with the proU operon has no effect on ProQ localization. We also demonstrate that ProQ is necessary for robust biofilm formation, and this phenotype is independent of ProP. Binding studies were carried out using tryptophan fluorescence and in vitro transcribed proP mRNAs. proP is transcribed from two differentially regulated promoters, and ProQ interacts with proP mRNA transcribed from both promoters, as well as a control mRNA with similar affinities. In total, these data suggest that ProQ is positioned to function as a novel translational regulator, and its cellular role extends beyond its effects on proline uptake by ProP.     

Isolation and Characterization of Mutations in the Escherichia coli Regulatory Protein XapR

In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in Escherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.     

The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.     

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.     

Analysis of the Link between the Redox State and Enzymatic Activity of the HtrA (DegP) Protein from Escherichia coli

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.     

Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock

The protective action of chaperone-like activity of HtrA protease against protein aggregation was studied. High levels of proteolytically inactive HtrAS210A (active center serine replaced by alanine) suppressed the temperature-sensitive phenotype of the htrA mutants. The ability of HtrAS210A to alleviate the lethality of htrA bacteria at high temperatures correlated well with the observed decrease of cellular level of large protein aggregates in cells overproducing HtrAS210A. The in vitro experiments proved that HtrA was very efficient in inhibiting the unfolded substrate (lysozyme) aggregation over a wide range of temperatures (30-45 °C). HtrA was able to bind to the denatured polypeptides and as a consequence limited their ability to form large aggregates. Our results suggest that HtrA may protect the bacterial cells from deleterious effects of heat shock not only by degrading the damaged proteins but by combination of the proteolytic and chaperoning activities.     

Mutant of Escherichia coli with Thermosensitive Protein in the Process of Cellular Division

A new thermosensitive mutant of Escherichia coli deficient in cell division was isolated by means of membrane filtration after nitrosoguanidine mutagenesis. The mutant cells grow normally at 30 C but stop dividing immediately after shift to 42 C, resulting in multinucleated filaments lacking septa. The number of colony-forming units does not decrease for at least 6 hr at 42 C. The maximum length of the filaments is 10 to 16 times that of normal cells. Addition of a high concentration of NaCl fails to stimulate cell division at 42 C. The filaments formed at 42 C divide abruptly 30 min after shift to 30 C, and synchronous increase of cell number is shown for 3 hr. The macromolecular synthesis of protein and nucleic acids at 42 C is normal on the whole. The cell division shown after the shift from 42 to 30 C is observed in the absence of thymine, but not in the presence of chloramphenicol or in a medium deficient in amino acids. However, the filament can divide to some extent in the presence of chloramphenicol if some protein synthesis is allowed to proceed at 30 C before the addition of the antibiotic. The elongated cells divide at 42 C provided that they are exposed to 30 C before being shifted to high temperature.     

Role of the Two Structural Domains from the Periplasmic Escherichia coli Histidine-binding Protein HisJ

Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.     

Thiamine-Binding Protein of Escherichia coli

The ability to transport thiamine in Escherichia coli was reduced by osmotic shock treatment with a concomitant release of a thiamine-binding protein; its formation was repressed by thiamine added to the growth medium.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Manganese-Resistant Mutants of Escherichia coli: Physiological and Genetic Studies

Manganese is growth inhibitory for Escherichia coli. The manganese concentration required for inhibition is dependent upon the magnesium concentration of the medium. Mutants have been isolated which are partially resistant to manganese inhibition in both liquid and solid media. From conjugation experiments, the genetic locus for manganese-resistance, mng, appears to be between 34 and 37 min on the E. coli genetic map. Experiments with radioactive (28)Mg lead to the tentative conclusion that the mng mutants are altered in the inhibition constant for manganese as a competitive inhibitor for the mangnesium accumulation system. Once high manganese enters the cells, it displaces internal magnesium and leads to a net cellular loss and hence growth inhibition. The mng mutants are somewhat less subject to manganese-induced magnesium loss under comparable conditions than are manganese-sensitive wild-type cells.