To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. P. aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the IPTG-regulated expression of their B. pseudomallei orthologs on a broad-host-range plasmid. P. aeruginosa genes which are upregulated in response to depletion of each target gene product were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-SpR to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologs in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA), to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06% including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products.
bioluminescence; reporter; screen
SecA is a central component of the general secretion system that is essential for bacterial growth and thus an ideal target for the development of antimicrobial agents. A series of fluorescein analogs were first screened against the ATPase activity using the truncated unregulated SecA catalytic domain. Rose Bengal (RB) and Erythrosin B (EB) were found to be potent inhibitors with IC50 values of 0.5 µM and 2 µM, respectively. RB and EB inhibit the catalytic SecA ATPase more than the F1F0-proton ATPase. We used three assays to test the effect of these compounds on full length SecA ATPase: in solution (intrinsic ATPase), in membrane preparation, and translocation ATPase. RB and EB show the following trend in terms of IC50 values: translocation ATPase < membrane ATPase < intrinsic ATPase. Very importantly, the potency of these fluorescein analogs in inhibiting the truncated SecA ATPase correlates with their ability to inhibit the biologically relevant protein translocation activity of SecA. The in vitro translocation of proOmpA precursors into membrane vesicles is strongly inhibited by RB with IC50 of about 0.25 µM, making RB the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation thus far. The ability of these compounds to inhibit SecA directly translates into antibacterial effects as well. Our findings show the value of fluorescein analogs as probes for mechanistic studies of SecA functions, and for the potential development of new antimicrobial agents with SecA as the target.
SecA; ATPase inhibitor; protein translocation; antimicrobial; membrane protein
The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localizes to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localization of DivIVA in these mutants. Surprisingly, DivIVA was delocalized, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localization depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localization is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.
SecA ATPase; DivIVA; cell division; membrane binding; protein localization
Previous studies showed that E. coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.
SecA-liposomes; SecYEG complex; pPhoA translocation; Protein-conducting channel; Channel activity
The twin-arginine translocase (TAT) in some bacterial pathogens, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis, contributes to pathogenesis by translocating extracellular virulence determinants across the inner membrane into the periplasm, thereby allowing access to the Xcp (type II) secretory system for further export in Gram-negative organisms, or directly to the outside surface of the cell, as in M. tuberculosis. TAT-mediated secretion appreciably contributes to virulence in both animal and plant models of bacterial infection. Consequently, TAT function is an attractive target for small-molecular-weight compounds that alone or in conjunction with extant antimicrobial agents could become novel therapeutics. The TAT-transported hemolytic phospholipase C (PlcH) of P. aeruginosa and its multiple orthologs produced by the above pathogens can be detected by an accurate and reproducible colorimetric assay using a synthetic substrate that detects phospholipase C activity. Such an assay could be an effective indicator of TAT function. Using carefully constructed recombinant strains to precisely control the expression of PlcH, we developed a high-throughput screening (HTS) assay to evaluate, in duplicate, >80,000 small-molecular-weight compounds as possible TAT inhibitors. Based on additional TAT-related functional assays, purified PlcH protein inhibition experiments, and repeat experiments of the initial screening assay, 39 compounds were selected from the 122 initial hits. Finally, to evaluate candidate inhibitors for TAT specificity, we developed a TAT titration assay that determines whether inhibition of TAT-mediated secretion can be overcome by increasing the levels of TAT expression. The compounds N-phenyl maleimide and Bay 11-7082 appear to directly affect TAT function based on this approach.
At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.
Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems.
An amber mutation in the secA gene of Escherichia coli causes a pleiotropic decrease in the synthesis of secreted proteins, including maltose-binding protein (MBP) and alkaline phosphatase. Reversal of the inhibition of MBP synthesis in secA(Am) strains by signal sequence mutations in the malE gene has been reported. These results suggest a coupling between secretion and translation which involves an interaction between the signal sequence of nascent polypeptides and a cellular secretion machinery. Further analysis reported here indicated that signal sequence mutations of MBP or alkaline phosphatase did not selectively overcome the inhibition of MBP or alkaline phosphatase synthesis in secA(Am) strains. Rather, at a given time in parallel experiments there was substantial variability among closely isogenic secA(Am) strains in the magnitude of the synthesis block; this variability could account for the earlier results. Further experiments suggested that the inhibition of MBP synthesis in secA(Am) strains was caused by depletion of cyclic AMP, leading to decreased transcription of the malE gene. However, the secretion defects in secA(Am) strains were not affected by cyclic AMP levels. Therefore, we conclude that the reduction in MBP synthesis was a secondary consequence of the primary export defect in the secA(Am) strains.
Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.
The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive posttranslational secretion of pre-proteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, ‘closed’ state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more ‘open’, has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1), and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.
The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.
The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have demonstrated that this secretion system is also linked to the onset of long-term CD8+ T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8+ T cells that are required for protection against secondary challenge with wild-type (wt) L. monocytogenes. Since the SecA2 ATPase is well conserved among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3+ memory CD8+ T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3+ memory CD8+ T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.
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.
The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.
SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors of bacterial SecA and will be very useful for mechanistic studies.
The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ΔsecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ΔsecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.
Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.
The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential “housekeeping” SecA, and SecA2, the accessory secretion factor. Here, we report that a ΔsecA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ΔsecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The ΔsecA2 mutant was also attenuated for growth in macrophages from phox−/− mice and from NOS2−/− mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the ΔsecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.
SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.
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.
Bacterial protein secretion is catalyzed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantitation, mutagenesis and functional assays. Our data reveal that: a. SecA binds with low affinity on several, peripheral, exposed SecYEG sites. This largely electrostatic association is modulated by temperature and nucleotides. b. Binding sites cluster in five major binding “regions”: three that are exclusively cytoplasmic and two that reach the periplasm. c. Both the aminoterminal and carboxyterminal regions of SecA participate in binding interactions and share some sites. d. Several of these sites are essential for translocase catalysis. Our data provide residue-level dissection of the SecYEG-SecA interaction. Two models of assembly of SecA on dimeric SecYEG are discussed.
secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.
Anthocyanins have been studied as potential antimicrobial agents against Helicobacter pylori. We investigated whether the biosynthesis and secretion of cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) could be suppressed by anthocyanin treatment in vitro. H. pylori reference strain 60190 (CagA+/VacA+) was used in this study to investigate the inhibitory effects of anthocyanins; cyanidin 3-O-glucoside (C3G), peonidin 3-O-glucoside (Peo3G), pelargonidin 3-O-glucoside (Pel3G), and malvidin 3-O-glucoside (M3G) on expression and secretion of H. pylori toxins. Anthocyanins were added to bacterial cultures and Western blotting was used to determine secretion of CagA and VacA. Among them, we found that C3G inhibited secretion of CagA and VacA resulting in intracellular accumulation of CagA and VacA. C3G had no effect on cagA and vacA expression but suppressed secA transcription. As SecA is involved in translocation of bacterial proteins, the down-regulation of secA expression by C3G offers a mechanistic explanation for the inhibition of toxin secretion. To our knowledge, this is the first report suggesting that C3G inhibits secretion of the H. pylori toxins CagA and VacA via suppression of secA transcription.
Helicobacter pylori; CagA; VacA; SecA; anthocyanin; cyanidin 3-O-glucoside.
SecA is found in Escherichia coli both tightly associated with the cytoplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R. J. Cabelli, K. M. Dolan, L. Qian, and D. B. Oliver, J. Biol. Chem. 266:24420-24427, 1991; D. B. Oliver and J. Beckwith, Cell 30:311-319, 1982; W. Wickner, A. J. M. Driessen, and F.-U. Hartl, Annu. Rev. Biochem. 60:101-124, 1991). Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of maltose-binding protein, an exported, periplasmic protein. In addition, under conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the distribution of precursor maltose-binding protein changes in parallel. These results support the idea that cytosolic SecA plays a role in export. With the aim of determining the roles of the multiple binding sites for ATP on SecA, we compared the export defect in a culture of E. coli expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide. The results indicate that the mutational change and treatment with sodium azide inhibit export by affecting different steps in the cycle of ATP binding and hydrolysis by SecA.
There are a number of genetic tools available for studying Francisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for F. tularensis based on the Tet repressor, TetR. For the inducible system, a tet operator sequence was cloned into a modified F. tularensis groESL promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon, luxCDABE, was cloned under the hybrid Francisella tetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with tetO. Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of FTRp regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the lux reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring F. tularensis protein function.