Regulation of the Escherichia coli secA Gene Is Mediated by Two Distinct RNA Structural Conformations

Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X. SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation. Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution. The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus. The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence. The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus. The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence. As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene. The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain. Received: 30 July 1998 / Accepted: 18 September 1998     

The use of extragenic suppressors to define genes involved in protein export in Escherichia coli

Summary The secA gene codes for a membrane component involved in protein export in E. coli. In order to define other genes whose products play such a role, we have characterized extragenic suppressors of a secA(Ts) mutation. These suppressors fall into at least three genetic loci. One such locus is the prlA gene, previously identified by mutations which suppress signal sequence mutants. Thus, this approach may allow the identification of new genes involved in the export process.     

Cloning and expression of the secA gene of a marine bacterium,Vibrio alginolyticus,and analysis of its function in Escherichia coli

We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation. Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V. alginolyticus. It was shown to encode a 103.3-kDa protein. The deduced amino acid sequence of V. alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E. coli (EcSecA). The secA gene of E. coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V. alginolyticus secA. Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide. Expression of VaSecA in E. coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature. VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms. VaSecA was overproduced in V. alginolyticus and purified to homogeneity for N-terminal sequencing. The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA.     

Critical regions of secM that control its translation and secretion and promote secretion-specific secA regulation

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.     

Thesec andprl genes ofEscherichia coli

Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec,secretion). The other identifies dominant suppressors of signal sequence mutations (prl,proteinlocalization). Subsequent characterization reveals that in at least three cases,prlA/secY,prlD/secA, andprlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.     

Mutations alter secretion of fukutin-related protein

Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker–Warburg syndrome, muscle–eye–brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases.     

secG and Temperature Modulate Expression of Azide-Resistant and Signal Sequence Suppressor Phenotypes of Escherichia coli secA Mutants

SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the Escherichia coli inner membrane. To understand more about this process, azide-resistant (azi) and signal sequence suppressor (prlD) alleles of secA were studied. We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable. secG function is required for expression of azide-resistant and signal sequence suppressor activities of azi and prlD alleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a secG null mutant. These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

Inducible Secretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis

A host-vector system for inducible secretion during the logarithmic growth phase in Bacillus subtilis has been developed. The B. subtilis levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of Clostridium thermocellum was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of B. subtilis showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing B. subtilis sacU(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.     

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.     

SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center

As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNA^Pro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmicβ-galactosidase (β-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that allβ-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus α-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct α-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of α-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.     

Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.     

The effect of combining signal sequences with the N28 fragment on GFP production in Aspergillus oryzae

Effective secretion of green fluorescent protein (GFP) was investigated by the screening signal sequences for GFP secretion in Aspergillus oryzae. GFP production in A. oryzae was evaluated using fusions with signal sequences from Taka-amylase A (TAA), glucoamylase A, glucoamylase B, and triacylglycerol lipase. The TAA signal sequence promoted the highest protein secretion of GFP. Fusing this signal sequence with an N-terminal 28-amino acid region (N28 fragment) from the Rhizopus oryzae lipase signal sequence increased protein secretion. In addition, using multiple copies of this signal sequence, instead of the N28 fragment, also induced protein secretion. These results show that using multiple signal sequences or combining a signal sequence with the N28 fragment can be used to improve heterogeneous protein secretion in A. oryzae.     

Live imaging of Yersinia translocon formation and immune recognition in host cells

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.     

Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA₂₁₈) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 10⁴ E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA₂₁₈ fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA₂₁₈ was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA₂₁₈ fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate Pe1B signal sequence

To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels     

Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.