Two-stage model for integration of the lysis protein E of ΦX174 into the cell envelope of Escherichia coli

Abstract: As a tool for determining the topology of the small, 91-amino acid ΦX174 lysis protein E within the envelope complex of Escherichia coli , a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.     

The pore-forming domain of colicin A fused to a signal peptide: a tool for studying pore-formation and inhibition

Pore-forming colicins are plasmid-encoded bacteriocins that kill Escherichia coli and closely related bacteria. They bind to receptors in the outer membrane and are translocated across the cell envelope to the inner membrane where they form voltage-dependent ion-channels. Colicins are composed of three domains, with the C-terminal domain responsible for pore-formation. Isolated C-terminal pore-forming domains produced in the cytoplasm of E. coli are inactive due to the polarity of the transmembrane electrochemical potential, which is the opposite of that required. However, the pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of E. coli from the periplasmic side, forming a functional channel. Sp-pfColA is specifically inhibited by the colicin A immunity protein (Cai). This construct has been used to investigate colicin A channel formation in vivo and to characterise the interaction of pfColA with Cai within the inner membrane. These points will be developed further in this review.     

Isolation and characterization of inhibitory factors of DNA polymerase III holoenzyme from Escherichia coli

Abstract Escherichia coli penicillin-binding protein 5 (PBP5) is anchored to the periplasmic face of the inner membrane via a C-terminal amphiphilic α-helix. The results of washing experiments have suggested an electrostatic contribution to the anchoring mechanism which may involve the cationic region of the C-terminal α-helix. Similarities between this anchor domain and some surface active agents, such as melittin, suggest that the cationic region of the PBP5 anchor may require the presence of anionic phospholipids for membrane interaction. Washing experiments performed on membranes of HDL11, an E. coli mutant in which the expression of the major anionic phospholipids is under lac control, found no such requirement. The results are discussed in relation to the hypothesis that the cationic region may interact with other sources of negative charge, possibly arising from a PBP complex.     

Phage lysis: Three steps,three choices,one outcome

The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gramnegative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanin and i-spanin, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the outer membrane is to catalyze fusion of the inner and outer membranes.     

Effect of mutation in phoB and phoM genes for the positive control of alkaline phosphatase biosynthesis on Escherichia coli envelope proteins

The suppression of some envelope proteins, localized in both the periplasm and the outer and inner membranes was shown in phoB and phoM phoR mutants of E. coli. Among these proteins are the proteins of the phosphate regulon and also those not pertaining them. As a result of phoB and phoM phoR mutations, the cytoplasmic membrane was found to be lacking in minor protein of 28,000 Mr, which belongs to the phosphate regulon. Besides, the phoM phoR mutation leads to the loss of protein of 55,000 Mr of the outer membranes, whereas phoB mutation causes loss of protein 37 000 Mr, identified as outer membrane protein OmpT. A damage in the phoB mutant of the protein proteolytic modification, probably determining the suppression of the biosynthesis of E. coli envelope secreted proteins is suggested.     

Evidence that F-plasmid proteins TraV, TraK and TraB assemble into an envelope-spanning structure in Escherichia coli

We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra+ cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain suggested by yeast two-hybrid analysis. We propose that TraV is the outer membrane anchor for a trans-envelope, Tra protein structure required for the assembly of F-pili and possibly for other events of conjugal DNA transfer.     

Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo

Certain small outer envelope membrane proteins of chloroplasts are encoded by the nuclear genome without a cleavable N-terminal transit peptide. We investigated in vivo the targeting mechanism of AtOEP7, an Arabidopsis homolog of the small outer envelope membrane protein. AtOEP7 was expressed as a fusion protein with the green fluorescent protein (GFP) either transiently in protoplasts or stably in transgenic plants. In either case, fluorescence microscopy of transformed cells and protein gel blot analysis of fractionated proteins confirmed that the AtOEP7:GFP fusion protein was targeted to the chloroplast outer envelope membrane. In vivo targeting experiments revealed that two regions, the transmembrane domain (TMD) and its C-terminal neighboring seven-amino acid region, were necessary and sufficient for targeting to the chloroplast outer membrane. Substitution of aspartic acid or lysine residues with glycine residues or scrambling of the amino acid sequence of the seven-amino acid region caused mistargeting to the plasma membrane. Although the amino acid sequence of the TMD is not important for targeting, amino acid residues with large side chains inhibited targeting to the chloroplasts and resulted in the formation of large aggregates in the protoplasts. In addition, introduction of a proline residue within the TMD resulted in inhibition of targeting. Finally, a fusion protein, AtOEP7:NLS:GFP, was targeted efficiently to the chloroplast envelope membranes despite the presence of a nuclear localization signal. On the basis of these results, we conclude that the seven-amino acid region and the TMD are determinants for targeting to the chloroplast outer envelope membrane. The seven-amino acid region plays a critical role in AtOEP7 evading the endomembrane system and entering the chloroplast pathway, and the TMD plays critical roles in migration to the chloroplasts and/or subsequent insertion into the membrane.     

φX174 lysis requires slyD, a host gene which is related to the FKBP family of peptidyl-prolyl cis-trans isomerases

Abstract: Recessive mutations in the slyD (sensitivity to ly sis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage φX174 [1]. The slyD ⁻ mutation, transduced into the normal φX174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues. A segment corresponding to the first 142 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans isomerases, or rotamases. The C-terminal 46 codons of slyD encode a remarkable histidine-rich peptide which is a metal-binding domain [2]. This sequence is dispensable for slyD function in E -mediated lysis. Although there is no obvious phenotype associated with the slyD ⁻ genotype other than the resistance to E -mediated lysis, overexpression of slyD causes cells to filament and to increase significantly in diameter. Mutations in φX174 can restore the plaque-forming ability of the phage on a slyD ⁻ host. These pos ( p lates on s lyD) mutants plate on E. coli C wild-type and slyD ⁻. A model for SlyD involvement in E function and the role of SlyD in the cell is discussed.     

Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS

BACKGROUND: E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS: High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS: The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and subcellular location of native and mutant hTNFα proteins in Escheriahia coli

Abstract Several mutant hTNFα genes were constructed by deletion and stepwise reconstitution of regions coding for C-terminal sequences. The mutant hTNFα proteins behaved differently from native hTNFα when expressed in Escherichia coli . They were either sensitive to proteolytic degradation or formed insoluble aggregates depending on the strains and conditions used for expression. By contrast, native hTNFα was always present in a soluble form and had a tendency to associate with the cytoplasmic membrane. It was even transported to the periplasmic space in E. coli as shown by both cell fractionation and immunoelectron microscopy. The different behaviour of mutant hTNFα proteins probably results from a disturbance of protein folding.     

Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B.

Growing Escherichia coli release envelope material into the medium. Upon infection with T4 phage increased amounts of this material are released and at a greater rate. In order to determine whether both inner and outer membranes are present in this material, and whether the material released by growing cells differs from that released by infected cells, we have examined the protein composition of envelope released by growing and T4-infected E. coli B. Our results show: (a) the protein composition of envelope released from growing or infected cells is similar, (b) the proteins present are representative of the outer membrane, (c) the major outer membrane protein of E. coli B, protein II, is deficient in the released material. We therefore conclude that the envelope material released from growing or infected E. coli represents a special fraction of the outer membrane. This finding is discussed in relation to outer membrane structure and function. In addition, data are presented on the differing outer membrane protein composition of substrains of E. coli B obtained from different laboratories.     

Evidence for membrane-bound oligomerization of bacteriophage phi X174 lysis protein-E

The expression of cloned bacteriophage phi X174 lysis gene E was analyzed in minicells of Escherichia coli using two-dimensional gel electrophoresis. Beside the 10-11-kDa protein-E, at least two additional protein bands were detected, associated with the inner membrane, which showed the same isoelectric point as E. To clarify whether these proteins were E-specific, two different antibodies directed against a beta-galactosidase-E' hybrid protein and a synthetic oligopeptide corresponding to the C-terminal end of protein-E were raised. Immunoadsorption studies with anti-peptide-specific antibodies resulted in the detection of protein-E as well as in the detection of proteins of higher molecular weight. Two of these protein bands were positively recognized by anti beta-galactosidase-E' antibodies. The latter protein bands had the same molecular weight as the putative protein-E bands detected by two-dimensional gel electrophoresis indicating that these bands represent protein-E-specific oligomers. These data support the idea that an E-specific oligomeric structure penetrating the inner and outer membrane of E. coli is formed during the lytic action of protein-E.     

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.     

Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts--a delivery system for the nontypeable Haemophilus influenzae antigen Omp26

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

Changes in host cell phospholipid composition of φX174 gene E product

Abstract Expression of bacteriophage φX174 gene E from plasmid pUH51 induced lysis of Escherichia coli . Before onset of bacterial lysis, cellular phospholipase activity was induced due to the presence of gene E product within the cells. By comparison of the lytic behaviour of phospholipase-negative E. coli strains with the corresponding wild-type strain it was found that neither the action of detergent-resistant phospholipase A nor of detergent-sensitive phospholipase were essential for the lysis-inducing properties of the gene E product. It was concluded that induction of phospholipases after expression of the φX174 gene E was a consequence of membrane perturbation caused by the integration of the gene E product into the cytoplasmic membrane of E. coli .     

Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.     

Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.