Protein quality control in the bacterial periplasm

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein.     

Translational control of exported proteins in Escherichia coli

We recently described the suppression of export of a class of periplasmic proteins of Escherichia coli caused by overproduction of a C-terminal truncated periplasmic enzyme (GlpQ'). This truncated protein was not released into the periplasm but remained attached to the inner membrane and was accessible from the periplasm. The presence of GlpQ' in the membrane strongly reduced the appearance in the periplasm of some periplasmic proteins, including the maltose-binding protein (MBP), but did not affect outer membrane proteins, including the lambda receptor (LamB) (R. Hengge and W. Boos, J. Bacteriol., 162:972-978, 1985). To investigate this phenomenon further we examined the fate of MBP in comparison with the outer membrane protein LamB. We found that not only localization but also synthesis of MBP was impaired, indicating a coupling of translation and export. Synthesis and secretion of LamB were not affected. The possibility that this influence was exerted via the level of cyclic AMP could be excluded. Synthesis of MBP with altered signal sequences was also reduced, demonstrating that export-defective MBP which ultimately remains in the cytoplasm abortively enters the export pathway. When GlpQ' was expressed in a secA51(Ts) strain, the inhibition of MBP synthesis caused by GlpQ' was dominant over the precursor accumulation usually caused by secA51(Ts) at 41 degrees C. Therefore, GlpQ' acts before or at the level of recognition by SecA. For LamB the usual secA51(Ts) phenotype was observed. We propose a mechanism by which GlpQ' blocks an yet unknown membrane protein, the function of which is to couple translation and export of a subclass of periplasmic proteins.     

Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1.

The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1. We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface. The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined. Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids. KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum. A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule. Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells. A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule. However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional. Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane.     

Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm.     

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.     

The secY protein can act post-translationally to promote bacterial protein export

Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Export and localization of N-terminally truncated derivatives of Escherichia coli K-12 outer membrane protein PhoE

To identify export and sorting information in outer membrane protein PhoE of Escherichia coli K-12, a set of deletions was created, resulting in the removal of N-terminal amino acids of the mature protein. Pulse-chase experiments revealed that some mutant proteins were slowly or not at all processed, but there was not correlation between processing rate and the extent of the deletions. The unprocessed precursors were accessible to trypsin in the periplasm showing that processing by leader peptidase rather than translocation is affected by these deletions. The results show that no specific sequences in the N-terminal part of the mature PhoE protein are required for translocation through the inner membrane. The capability of the processed mutant proteins to assemble into the outer membrane was correlated to the exten of the deletions. Thus, mutants which lack up to amino acid residue 14 are normally incorporated into the outer membrane. Larger deletions which removed the first postulated membrane-spanning fragment of the protein affected the efficiency of assembly: in addition to trimers of the protein in the outer membrane, also monomers were detected in the periplasm. If the deletions extended C-terminally to residue 48, only monomeric forms of the proteins were found in the periplasm.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein

The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion. It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane. Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures. In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes. The protein is synthesized as a precursor. Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species. Soluble pIV then integrates into the membrane with a half-time of one to two minutes. Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form. The gene IV protein may be part of the structure through which the assembling phage is extruded.     

On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli

In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present.     

Diverse effects of mutations in the signal sequence on the secretion of β-lactamase in Salmonella typhimurium

Mutations in the β-lactamase structural gene that alter the signal peptide were used to study secretion into the periplasm of Salmonella typhimurium. Processing and cellular location of mutant gene products were followed by pulse-chase and cell-fractionation experiments and by trypsin accessibility in intact and lysed spheroplasts. The precursor proteins examined never appear as a free species in the periplasm. Two of the signal-sequence mutants accumulate a precursor form that is trypsin-accessible in intact spheroplasts; the precursors synthesized by the remaining mutants resemble wild-type in that they remain trypsin-inaccessible. One of the latter mutants does produce mature protein, but at a very reduced rate. It thus appears that signal-sequence mutations can affect more than one step in the secretion process, and that processing of the signal peptide is not required for the protein to be translocated (at least partially) across the inner membrane.     

Use of a β-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM β–lactamase was fused to random positions within the coding region of the penicillin–binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in–frame fusions were determined. All fusion proteins that contained at least the NH₂–terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the β–lactamase moiety had been translocated to the periplasm. Fusion proteins that contained ≤ 63 residues of PBP 1B possessed β–lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the 3–lactamase moiety of these fusion proteins remained in the cytoplasm. The β–lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic trans–membrane segment (residues 64–87), with a short NH₂–terminal domain (residues 1–63), and the remainder of the polypeptide (residues 68–844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.     

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.     

The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export.

The product of the secB gene is required for export of a subset of secreted proteins to the outer membrane and periplasm of Escherichia coli. Precursor maltose-binding protein (MBP) accumulates in the cytoplasm of secB-carrying mutants, but export of alkaline phosphatase is only minimally affected by secB mutations. When export of MBP-alkaline phosphatase hybrid proteins was analyzed in wild-type and secB-carrying mutant strains, the first third of mature MBP was sufficient to render export of the hybrid proteins dependent on SecB. Substitution of a signal sequence from a SecB-independent protein had no effect on SecB-dependent export. These findings show that the first third of mature MBP is capable of conferring export incompetence on an otherwise competent protein.     

DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.

The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.     

The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins

The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.     

A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A is synthesized with a secretion signal peptide typical of proteins whose final destination is the periplasm. However, exotoxin A is released from the cell without a detectable periplasmic pool, suggesting that additional determinants in this protein are important for recognition by a specialized machinery of extracellular secretion. The role of the N terminus of the mature exotoxin A in this recognition was investigated. A series of exotoxin A proteins with amino acid substitutions for the glutamic acid pair at the +2 and +3 positions were constructed by mutagenesis of the exotoxin A gene. These N-terminal acidic residues of the mature exotoxin A protein were found to be important not only for efficient processing of the precursor protein but also for extracellular localization of the toxin. The mutated exotoxin A proteins, in which a glutamic acid at the +2 position was replaced by a lysine or a double substitution of lysine and glutamine for the pair of adjacent glutamic acids, accumulated in precursor forms in the mixed cytoplasmic and membrane fractions, which was not seen with the wild-type exotoxin A. The processing of the precursor form of one exotoxin A mutant, in which the glutamic acid at the +2 position was replaced with a glutamine, was not affected. Moreover, a substantial fraction of the mature forms of all three mutants of exotoxin A accumulated in the periplasm, while wild-type exotoxin A could be detected only extracellularly. The periplasmic pools of these variants of exotoxin A could therefore represent the intermediate state during extracellular secretion. The signal for extracellular localization may be located in a small region near the amino terminus of the mature protein or could consist of several regions that are brought together after the polypeptide has folded. Alternatively, the acidic residues may be important for ensuring a conformation essential for exotoxin A to traverse the outer membrane.     

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.