Export of the outer membrane lipoprotein is defective in secD, secE, and secF mutants of Escherichia coli.

The export of major outer membrane lipoprotein has been found to be affected in secD, secE, and secF mutants of Escherichia coli, which are defective in protein export in general. After a shift to the nonpermissive temperature, the kinetics of accumulation of prolipoprotein and pre-OmpA protein was indistinguishable from that of pre-OmpA protein accumulation in the secD and secF mutants but different in the secE mutant. The prolipoprotein accumulated in the secD, secE, and secF mutants at the nonpermissive temperature was not modified with glyceride. We conclude from these results and those of previous studies that the export of lipoprotein requires all common sec gene products except the SecB protein, i.e., the SecA, SecD, SecE, SecF, and SecY proteins.     

A Mutation Affecting the Regulation of a Seca-Lacz Fusion Defines a New Sec Gene

It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export. Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way. Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene. The properties of the fusion strain provide a new selection for mutants that are defective in protein export. Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY. In addition, mutations in several genes not previously known to affect secA expression were obtained. A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE.     

The secD locus of E.coli codes for two membrane proteins required for protein export.

Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

Genetic and molecular characterization of the Escherichia coli secD operon and its products.

The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.     

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.     

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

In vivo selection and characterization of internal deletions in the lamB::lacZ gene fusion

Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli. As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose. In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac+ mutants of strain Pop3299 on lactose tetrazolium media. Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB::lacZ gene fusion. Thus, it appears that the type of selection employed affects the type of mutations obtained. We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes. In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology.     

Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants.

SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant. An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.     

Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.     

Evidence for specificity at an early step in protein export in Escherichia coli.

We previously described mutations in a gene, secB, which have pleiotropic effects on protein export in Escherichia coli. In this paper, we report the isolation of mutants in which the activity of the secB gene was eliminated. Null mutations in secB affected only a subset of exported proteins. Strains carrying these mutations, although unable to grow on L broth plates, were still viable on minimal media. These secB mutations reversed a block in the translation of an exported protein that was caused by the elimination of another component of the secretion machinery, SecA protein. These results suggest that the secB product acts at an early step in the export process and is involved in the export of only a subset of cell envelope proteins.     

Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants.

Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.     

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.     

Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli.

A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.     

Suppressor mutations that restore export of a protein with a defective signal sequence

A selection procedure is described that should allow the genetic identification of cellular components involved in the process of protein localization in Escherichia coli. This procedure makes use of mutations that alter the signal sequence of the λ receptor protein (product of the lamB gene), and prevent export of this protein to its normal outer membrane location. Several suppressor mutations have been identified that restore export of the mutant λ receptor protein. Mapping experiments show that the suppressor phenotype is the result of mutations in any of at least three different chromosomal loci. One class of suppressor mutations, the class containing the largest number of independent isolates, maps in the major ribosomal gene cluster, suggesting that the suppressor phenotype is the consequence of an altered ribosomal protein. This class of suppressors phenotypically suppresses all known export-defective mutations, internal to the signal sequence region of the lamB gene. These results suggest that ribosomes play an important role in the export of λ receptor to the outer membrane.     

Identification of cutC and cutF (nlpE) genes involved in copper tolerance in Escherichia coli.

It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.     

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope.

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments.     

Genetic analysis of bacteriophage N4 adsorption.

We isolated six mutants of Escherichia coli K-12 that were defective in bacteriophage N4 adsorption. We mapped the mutations to four loci designated nfrA through nfrD (N four resistance). nfrA and nfrB were tightly linked to each other and were mapped to min 12 of the E. coli linkage map. nfrC was mapped to min 85, and nfrD was mapped between min 44 and 58. We isolated a clone carrying both nfrA and nfrB and identified its gene products through maxicell analysis of plasmid subclones. The nfrA gene product was an outer membrane protein of 96,000 apparent molecular weight, whereas nfrB encoded an inner-membrane protein of 69,500 apparent molecular weight. The nfrB1 mutation did not affect the export of the nfrA gene product to the outer membrane and did not affect the alkaline phosphatase activity of an nfrA-phoA fusion. We propose that nfrA encodes the structural receptor for N4 and that the nfrB gene product may be required for irreversible adsorption and injection of the phage genome and virion-encapsulated RNA polymerase through the inner membrane.