The aerobactin iron uptake system in enteropathogenic Escherichia coli: evidence for an extinct transposon

Abstract It has previously been demonstrated that the precursor form of the phosphate-binding protein (pre-PhoS) is accumulated in both the cytoplasmic membrane and the cytoplasm under conditions of phosphate-binding protein (PhoS) hyperproduction in Escherichia coli [11]. In this study, the trypsin accessibility of these 2 pre-PhoS pools has been investigated in spheroplasts. The results demonstrate that the membrane-associated pre-PhoS is not accessible to trypsin, and thus has not been translocated. The sensitivity to trypsin of mature PhoS, membrane-associated pre-PhoS and cytoplasmic pre-PhoS was compared. The results suggest a difference in conformation between membrane-associated and cytoplasmic pre-PhoS since the former is more trypsin-sensitive than the latter. Mature PhoS is resistant to trypsin. The significance of these results with regard to the export mechanism for periplasmic proteins is discussed.     

Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.     

Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli.

phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.     

Lack of effect of leader peptidase overproduction on the processing in vivo of exported proteins in Escherichia coli

The kinetics of maturation of certain exported proteins were analysed in Escherichia coli strains that also concomitantly overproduce either a periplasmic protein or the leader peptidase. The results led to three conclusions. Overproduction of leader peptidase has no effect on the rate of maturation of at least two exported proteins, one periplasmic (TEM beta-lactamase), one outer membrane (PhoE); therefore, the quantity of leader peptidase is not rate-limiting for normal export. Overproduction of PhoS reduces the rate of maturation of two other periplasmic proteins (beta-lactamase and PhoA) and itself, presumably by competing for the rate-limiting component of the export apparatus. Overproduction of leader peptidase in a strain overproducing PhoS has no effect on the retarded maturation of PhoS. Therefore even in these conditions, leader peptidase is not rate limiting.     

Conditions leading to secretion of a normally periplasmic protein in Escherichia coli.

The phosphate-binding protein (PhoS) is a periplasmic protein which is part of the high-affinity phosphate transport system of Escherichia coli. Hyperproduction of PhoS in strains carrying a multicopy plasmid containing phoS led to partial secretion of the protein. By 6 h after transfer to phosphate-limiting medium, about 13% of the total newly synthesized PhoS was secreted to the medium. Kinetic studies demonstrated that this secretion consists of newly synthesized PhoS. This secretion occurs in PhoS-hyperproducer strains but not in a PhoS-overproducer strain. Another type of secretion concerning periplasmic PhoS was observed in both PhoS-hyperproducer and PhoS-overproducer strains. This mode of secretion depended upon the addition of phosphate to cells previously grown in phosphate-limiting medium.     

Expression vector promoting the synthesis and export of the human growth-hormone-releasing factor in Escherichia coli

We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein. A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF). The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space. Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.     

Requirement of the SecB chaperone for export of a non-secretory polypeptide in Escherichia coli

Summary The SecB protein of Escherichia coli is a cytosolic component of the export machinery which can prevent some precursors from prematurely folding into export-incompatible conformations by binding to the newly synthesised polypeptide. The feature(s) of target proteins recognised by SecB, however, are unclear and have been a matter of controversy. Also, it has not been asked if binding of SecB is specific for secretory proteins. We demonstrate here that a non-secretory polypeptide, a fragment of a tail fiber protein of phage T4, fused to the signal peptide of the outer membrane protein OmpA has a very strong SecB requirement for export and that the signal peptide itself cannot, at least not alone, be responsible for this action of SecB. The data reported, together with those of the literature, suggest that SecB recognizes the polypeptide backbone of the target protein.     

Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli

The export of beta-lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN-III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.     

Flipping the switch: bringing order to flagellar assembly

The bacterial flagellum is a complex self-assembling nanomachine that contains its own type III protein export apparatus. Upon completion of early flagellar structure, this apparatus switches substrate specificity to export late structural subunits, thereby coupling sequential flagellar gene expression with flagellar assembly. The switch is achieved by a conformational change of the export apparatus component FlhB driven by the flagellar hook-length control protein FliK. Two basic models of FliK- and FlhB-based switching are currently being pursued, together with the investigation of another factor, Flk, which prevents premature export of late substrates. Here, we review in detail each of these three export switch components and present the current understanding of how they work in concert in the making of a flagellum.     

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.     

Engineering of Escherichia coli to improve the purification of periplasmic Fab' fragments: changing the pI of the chromosomally encoded PhoS/PstS protein

Escherichia coli is a widely used host for the heterologous expression of proteins of therapeutic and commercial interest. The scale and speed at which it can be cultured can result in the rapid generation of large quantities of product. However, to achieve low costs of production a simple and robust purification process is also required. The general factors that impact on the cost of a purification process are the scale at which a process can be performed, the cost of the purification matrix, and the number and complexity of the chromatographic steps employed. Purification of Fab' fragments of antibodies from the periplasm of E. coli using ion exchange chromatography can result in the co-purification of E. coli host proteins having similar functional pI: such as the periplasmic phosphate binding protein, PhoS/PstS. In such circumstances, an additional chromatographic step is required to separate Fab' from PhoS. Here, we change the functional pI of the chromosomally encoded PhoS/PstS to effect its non-purification with Fab' fragments, enabling the removal of an entire chromatographic step. This exemplifies the strategy of the modification of host proteins with the aim of simplifying the production of heterologous proteins.     

A microsomal GTPase is required for glycopeptide export from the mammalian endoplasmic reticulum

Bidirectional transport of proteins via the Sec61p translocon across the endoplasmic reticulum (ER) membrane is a recognized component of the ER quality control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported from the ER lumen back to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eukaryotic phenomenon. Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established system to study glycopeptide export and conventional liquid chromatography, we isolated a single polypeptide species of 23 kDa from rat liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by mass spectrometric sequence analysis as guanylate kinase (GK), a housekeeping enzyme critical in the regulation of cellular GTP levels. We confirmed the ability of GK to substitute for complete cytosol by reconstitution of glycopeptide export from rat liver microsomes using highly purified recombinant GK from Saccharomyces cerevisiae. Most significantly, we found that the GK (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a requirement for GTP hydrolysis. Membrane integrity was fully maintained under assay conditions, as no ER luminal proteins were released. Competence for glycopeptide export was abolished by very mild protease treatment of microsomes, indicating the presence of an essential protein on the cytosolic face of the ER membrane. These data demonstrate that export of glycopeptide export is controlled by a microsomal GTPase and is independent of cytosolic protein factors.     

Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli

A recombinant gene comprising phoS (the gene for the phosphate-binding protein PhoS) fused to a synthetic gene for a modified human growth-hormone-releasing factor (mhGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS-mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre-PhoE and pre-OmpA. The pre-PhoS-mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/l culture.     

Effect of 5-bromodeoxyuridine on vaccinia virus-induced polypeptide synthesis: selective inhibition of the synthesis of some post-replicative polypeptides.

The effect of 5-bromodeoxyuridine (BrdU) on vaccinia virus-induced polypeptide synthesis in BSC-1 cells has been investigated. Most virus-induced pre- and post-replicative polypeptides were synthesized in concentrations of 5-bromodeoxyuridine that inhibited virus growth. The synthesis of a few post-replicative polypeptides, however, was severely inhibited under these conditions; included in this group was the precursor of a major core component, polypeptide P4b. A delay in the switch-off of pre-replicative polypeptide synthesis and in the onset of post-replicative polypeptide synthesis was also observed. The significance of these observations is discussed.     

The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

In vivo assembly of active maltose binding protein from independently exported protein fragments.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is synthesized in the cytoplasm with an N-terminal signal peptide that is cleaved upon export. We examined whether active MBP could assemble into an active protein in bacteria, from N- and COOH-terminal complementary protein fragments encoded by distinct, engineered segments of its structural gene. We found export and functional periplasmic assembly of MBP fragments, despite the complex polypeptide chain topology of this protein, if two conditions were satisfied. First, each of the two fragments must carry a signal peptide. Second, the boundaries between the two fragments must correspond to a permissive site within the protein. Functional assembly of active MBP occurred in five cases where these conditions were met: sites after residues 133, 161, 206, 285 and 303; but not in three other cases where the break junction corresponded to a non-permissive site: after residues 31, 120 and 339. Thus, permissive sites which were initially characterized because they could accept extensive genetic insertion/deletion modifications without loss of most biological properties provide a means of defining complementing protein fragments. This observation opens a way to study genetically the relationships between protein export and folding into the periplasm.     

Monocistronic messenger RNA in yeast

We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods.These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae, mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.