INTRODUCTION

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In Escherichia coli, the cell envelope, including the inner membrane, the periplasm, and the outer membrane, is the center of vital physiological processes. Protein trafficking in the envelope is therefore particularly sensitive to any perturbations. Accordingly, the production of abnormal proteins in the envelope has proven useful to studying essential features of envelope physiology. This importance of envelope integrity for bacterial life raised the question of the existence of pathways to detect and respond to extracytoplasmic perturbations. Recently, toxic hybrid proteins have been used to characterize two envelope stress response pathways in E. coli, the ^E and Cpx pathways (reviewed in reference 51). These pathways detect misfolding and perturbations of envelope structure and trigger cellular response, but with their own specificity. The ^E stress response appears to be associated with outer membrane integrity. First, it can be activated by a modification of the outer membrane protein content (42). Second, this activation leads to an elevated level of cytoplasmic transcription factor ^E, which results in the expression of genes encoding proteins involved in outer membrane protein folding and degradation (19, 24, 50, 53). The Cpx pathway is a typical two-component signal transduction system, composed of a histidine kinase sensor (CpxA) and a cytoplasmic response regulator (CpxR) (23, 65). It responds to various extracytoplasmic stresses, such as misfolded protein production and elevated pH (18, 35, 44). Its activation leads to an increased level of cytoplasmic phosphorylated CpxR, which in turn induces the expression of genes encoding proteins involved in envelope protein physiology (48) and motility and chemotaxis (22).

Although they have their own response specificity, the two pathways' responses partially overlap. The only known target common to the E^E RNA polymerase and to the phosphorylated CpxR regulator is the degP gene, which encodes a periplasmic protease (20, 38, 50, 53, 61). degP mutations were originally described as preventing the degradation of aberrant proteins in the envelope (60). Since then, the involvement of DegP in essential house cleaning functions in the envelope has been highlighted. Indeed, several envelope toxicities, like those due to the production of -galactosidase (LacZ) in the periplasm, outer membrane protein overproduction, and misfolded outer membrane porin LamB production, are suppressed or reduced by increased expression of DegP (14, 16, 58). However, although these studies demonstrated that DegP is necessary for toxicity suppression, they also suggested that other factors may be involved.

Our laboratory previously constructed and characterized periplasmic hybrid proteins whose production is toxic to the cell (5, 31). The hybrid proteins were generated by random TnphoA fusions to a plasmid-borne dsbA gene, and they consist of alkaline phosphatase (PhoA) fused to an amino-terminal part of variable length of disulfide oxidase (DsbA) (2). Although both constituents of these hybrid proteins are of periplasmic origin, their production in the envelope is conditionally lethal to the cell. Toxicity depends on (i) the expression level, (ii) the ability of the DsbA' part to fold or not into a proteolysis-resistant form, and (iii) the presence of the DegP protease. Hence, hybrid proteins carrying amino acids 1 to 160 of DsbA or more are tolerated, while those carrying amino acids 1 to 136 or less are toxic. This toxicity is more pronounced in a strain deficient for DegP, whose proteolytic activity protects bacteria by reducing the amount of toxic hybrid protein. The folding-proficient hybrid proteins are efficiently exported to the periplasm. In contrast, the export of the folding-deficient ones is blocked at a late stage of the process. A fraction of the hybrid protein produced remains bound to the cytoplasmic membrane. Such hybrid proteins not only stick to the membrane, inhibiting the export of newly made molecules, but also inhibit the export of unrelated periplasmic proteins such as maltose-binding protein.

In the present approach, we have taken advantage of the enhanced sensitivity of DegP-deficient strains to screen for putative genes whose overexpression protects bacteria against the lethal effects of unfoldable DsbA'-PhoA hybrid proteins. An untranslated small gene, uptR, producing a 92-nucleotide RNA, was isolated. UptR RNA restores viability by a new way of suppressing toxicity in E. coli, and this is discussed.

	MATERIALS AND METHODS

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Media and chemicals. Bacteria were grown aerobically in M9 liquid minimal medium supplemented with glucose or glycerol (0.5%), thiamine (0.01%), and required amino acids (each at 50 µg/ml) or in Luria-Bertani (LB) medium (43). Antibiotics were added to the following concentrations: chloramphenicol (CHL), 25 µg/ml; tetracycline (TCY), 20 µg/ml; streptomycin (STR), as indicated; spectinomycin (SPC), as indicated; ampicillin (AMP), from 10 to 500 µg/ml according to the experiment; and rifampin (RIF), 100 µg/ml. 5-Bromo-4-chloro-3-indolylphosphate (XP; 40 µg/ml) was used in LB plates as a chromogenic substrate for alkaline phosphatase.

Strains and bacteriophages. The E. coli strains used and constructed in this study are listed in Table 1. A library of DNA fragments originating from SBS2613 was obtained in vivo, using the MudII4042 phagemid [Cm^r, Mucts62 A⁺ B⁺ rep_P15A lac('ZYA)931] (29). Transduction experiments with P1vir were done as previously described (43).

Plasmids and DNA manipulations. The plasmids used and constructed in this study are listed in Table 1. Standard DNA manipulations were used in plasmid construction and manipulation (55). The nucleotide sequences were determined by the dideoxy chain termination method on double-stranded DNA, using an ABI Prism 310 genetic analyzer according to the manufacturer's instructions (56). PCR experiments were performed in a Perkin-Elmer Cetus 480 system using the Vent DNA polymerase according to the manufacturer's instructions.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Subcloning of the protective DNA region from the protective MudII recombinant clone pAG2646. (A) Different DNA fragments of pAG2646 were subcloned into vector pHSG575 (dashed and dotted line), and the protective effect of the recombinant plasmids obtained was determined. DNA fragments from the MudII cloning vector are represented by the thin line, and the cloned DNA fragments are shown by the thick line. (B) Different DNA fragments obtained after PCR amplification from pAG3268 were subcloned into pHSG575, and the protective effect of the recombinant plasmids was determined. B, BamHI; Bg, BglII; H, HindIII; Nc, NcoI; Sp, SphI; Ss, SspI.

Construction of a yjiE null mutant. Plasmid pJP3611 (yjiE⁺) was constructed by cloning a 2.6-kb MluI (blunt ended)-StyI DNA fragment of pJDG100 between the EcoRI (blunt ended) and StyI sites of pBR322. A 2-kb EcoRI-EcoRI  Str^r/Spc^r cassette from pHP45 was inserted between the MfeI sites of pJP3611, interrupting yjiE after nucleotide 388 (49). The resulting plasmid, pJP3619, carries the mutation yjiE1:: and was introduced into strain SBS2171 [polA(T_s) strain] by transformation. This mutation was integrated into the chromosome by homologous recombination as previously described (4, 27). The chromosomal disruption of yjiE was verified by PCR amplification.

Isolation of multicopy suppressors. Bacteria of strain SBS2614 (K10 degP Mucts) were infected with a lysate of MudII4042 obtained on SBS2613 (K10 Mucts), spread on LB plates containing CHL, and incubated at 30°C. The Cm^r colonies representing a random K10 DNA library cloned on MudII were pooled and made competent. They were transformed with the toxic fusion plasmid pAG2694 and plated on LB plates containing CHL, AMP (50 µg/ml), and XP. The Cm^r Ap^r PhoA⁺ transformants carrying both pAG2694 and a mini Mu-derived plasmid encoding and expressing a putative protective factor were isolated on LB plates containing AMP (50 µg/ml), CHL, and XP. Moreover, in order to eliminate the possibility of suppression of the toxicity by spontaneous chromosomal mutations, mixed plasmid DNA was extracted from each of several Cm^r Ap^r PhoA⁺ transformants isolated and again used to transform cells of strain SBS2614. Identical aliquots of the transformation mixture were plated on LB with XP containing (i) AMP (50 µg/ml), (ii) CHL, or (iii) AMP (50 µg/ml) plus CHL, and the number of transformants on each medium was compared. Plasmid DNA preparations giving comparable numbers of colonies in i and iii and much lower numbers in ii were analyzed further. In this case, the colonies growing on LB with AMP (50 µg/ml) were all found to be Cm^r, suggesting that the Cm^r recombinant phagemids selected very likely protected the degP bacteria against the toxicity of pAG2694.

Determination of the origin of transcription. The solutions used for mRNA extraction were prepared with RNase-free reagents. Water was treated with the alkylating reagent diethylpyrocarbonate before use, and glassware was dry-autoclaved at 180°C for 5 h. Bulk mRNA was extracted from cells of strain SBS2146 carrying either pAG3284 or the void vector pHSG575 according to a previously described procedure with some modifications (1). The cultures (25 ml) were harvested at an A₆₀₀ of 0.85 and centrifuged, and the pellet was resuspended in 400 ml of 20 mM sodium acetate-1 mM EDTA (pH 5.5) containing 0.5% sodium dodecyl sulfate (SDS). Then 400 µl of hot phenol (pH 4.5) was added to the suspension. After vortexing, the mixture was incubated for 5 min at 60°C and centrifuged. The aqueous layer was collected, and the phenol extraction was repeated three times. RNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate and 3 volumes of ethanol at 70°C and centrifuged and resuspended in distilled water. These preparations were incubated at 25°C for 1 h in the presence of RNase-free DNase. One volume of phenol-chloroform (1:1) was then added. After shaking and centrifuging, the aqueous phase was collected and extracted with chloroform. The RNA was precipitated as above and dissolved in water. The RNA concentration was determined by measuring absorbance at 260 nm, and its purity was checked by means of the A₂₆₀/A₂₈₀ ratio. Primer extension analysis was made with the deoxyoligonucleotide 5'-CGTAGTGGGGGTGTTGAAACTTC-3' labeled at its 5' end with [-³²P]ATP and polynucleotide kinase (55). Hybridization and primer extension with avian myeloblastosis virus reverse transcriptase (Pharmacia) were carried out at 37°C according to Georgellis et al. (28). The incubation time for primer extension was 2 h. The reaction products were analyzed by electrophoresis in an 8% urea-polyacrylamide gel followed by autoradiography.

Bacterial fractionation. Periplasmic proteins were released from bacteria either by the osmotic shock procedure or by spheroplast formation using the classical lysozyme-EDTA procedure (9, 45). The membrane fraction was separated from the cytoplasm by subjecting spheroplasts to both low osmotic pressure and mechanical disruption. The spheroplast pellet corresponding to a 10-ml culture was resuspended in 1 ml of 1 mM Tris-HCl (pH 8.0) containing 1 mM MgCl₂ and DNase (100 µg/ml). It was vortexed at 4°C for 5 min in the presence of a glass bead (3 mm diameter). The membrane fraction was collected by centrifugation for 1 h at 30,000 × g, washed with Tris-HCl (pH 8.0), and repelleted.

Enzymatic activities. Alkaline phosphatase was assayed as previously described (4). Activities are expressed as nanomoles of p-nitrophenol formed at 37°C per minute and per milligram of bacterial protein. -Lactamase was measured in periplasmic extracts (shock fluids) using nitrocefin as the substrate (Unipath, Dardilly, France), (63). The protein concentration was determined on sonicated bacteria (11).

Pulse-labeling and immunoprecipitation. Bacteria were grown in minimal medium supplemented with all amino acids except methionine and cysteine. Pulse-chase experiments were performed as described previously (31). Samples were immunoprecipitated with an anti-MalE/DegP serum and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) as described by Ito et al. (33).

Disk sensitivity assays. Disk sensitivity assays were performed as previously described (7, 16).

Western blotting. Extracts were analyzed by SDS-PAGE under denaturing conditions (37). Proteins were transferred to nitrocellulose filters and reacted with mouse monoclonal anti-PhoA antibodies (Tebu, Le Perray-en-Yvelines, France) according to Sambrook et al. (55). Bound antibodies were detected with horseradish peroxidase-coupled goat anti-mouse immunoglobulin antibodies, diaminobenzidine, and hydrogen peroxide.

Computer methods. Computer analysis of DNA was done using the DNA Strider program (39). Homologies and similarities of gene parts were sought with the FASTA computer program (47). RNA folding was obtained using the MulFold program (34, 66).

Nucleotide sequence accession number. The GenBank accession number for the sequence shown in Fig. 2 is AF272839.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Selection of multicopy suppressors of the toxicity provoked by unfoldable DsbA'-PhoA hybrid proteins. The DsbA'-PhoA hybrid proteins were originally generated by random TnphoA insertions in the dsbA gene carried on plasmid pPB2172 (5). The fusion plasmids are all derivatives of pBR322 in which the fusion is transcribed from the rather weak and constitutive dsbA proximal promoter (4, 19, 48). Attempts to transform cells of E. coli K-12 degP derivatives with pAG2694 (encoding DsbA_1-136-PhoA, the longest unfoldable hybrid protein) systematically failed. Bacteria could not form colonies on LB plates with AMP at as little as 10 µg/ml, while transformants with pAG2993 (encoding DsbA_1-160-PhoA, the shortest foldable hybrid protein) grew well with AMP at as much as 500 µg/ml. Furthermore, a derivative plasmid of pAG2694 carrying a deletion of the dsbA_1-136::TnphoA gene fusion could be efficiently introduced into degP bacteria by transformation. These results indicate that pAG2694 toxicity is due to the constitutive expression of the DsbA_1-136-PhoA fusion.

Analysis of the cloned regions. The recombinant suppressor phagemids were analyzed by restriction mapping or by sequencing the bordering DNA fragment of the cloned region. Among the 18 isolated phagemids, 11 carry the degP gene and are supposed to be protective by restoring the proteolytic activity of DegP in the periplasmic space. Five other recombinant phagemids contain the recG gene, which was previously shown to reduce the plasmid copy number of pBR322 derivative plasmids (32). We found that the coexpression of recG on a pACYC184-derived plasmid and of dsbA_1-136::TnphoA on pAG2694 in a phoA degP⁺ strain roughly halves both the PhoA and the -lactamase activities (A. Guigueno, unpublished data). This suggests a protective effect of recG coexpression simply by reducing the plasmid copy number and hence reducing the expression of the toxic fusion protein.

A small RNA is the effector of protection. The nucleotide sequence of the 195-bp active DNA fragment contains ⁷⁰-dependent 10 and 35 putative promoter sequences (respectively TATTAT and TGCACA; Fig. 2). It also carries a typical Rho-independent termination signal. The transcription of this DNA fragment was demonstrated by primer extension analysis, which indicated the presence of an RNA initiated 7 nucleotides downstream from the 10 promoter box (Fig. 3). While plasmid pAG3284 confers resistance to DsbA_1-136-PhoA toxicity, pAG3284-derived plasmids carrying either the 35 box deletion (see Fig. 1) or the 10 box mutagenesis (plasmid pPB3602) are totally unable to suppress the toxicity.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence of the toxicity-suppressing DNA cloned in pAG3284. The nucleotide sequence is presented in lowercase letters. The SspI site (Ss) is indicated. A putative promoter is shown in bold type (35 promoter box and 10 promoter box), and the transcription termination signal is indicated (term.). Numbering starts from the origin of transcription, which is indicated (+1) according to Fig. 3. The DNA corresponding to an RNA is translated in the three open reading frames (uppercase letters; *, stop codons).

View larger version (84K): [in this window] [in a new window]   FIG. 3.   Origin of transcription of the uptR gene. The primer extension experiment was performed as indicated in Materials and Methods. Runs 1, 2, and 3 correspond to experiments done with 25, 5, and 0.5 µg, respectively, of RNA extracted from cells of strain SBS2146/pAG3284, and run 4 corresponds to an experiment done with 25 µg of RNA obtained from SBS2146/pHSG575. Lanes T, G, C, and A correspond to dideoxy sequencing reactions initiated by the same oligonucleotide used to initiate primer extension in runs 1, 2, 3, and 4. The nucleotide sequence deduced from lanes T, G, C, and A is indicated from the bottom to the top.

Analysis of the physiological role of UptR RNA. The 195-bp nucleotide sequence carrying uptR was found to be identical to that of a DNA region of unknown function and described only in data banks (Desiraju and Lu, GenBank accession number M86632). This DNA region was described as part of the episomal F factor. Recently, Mizobuchi and coworkers determined the nucleotide sequence of the F factor (cited in reference 40). Mizobuchi confirmed to us that uptR is actually located on the F factor in a previously unstudied region between the RepFIB region and the pifA gene (K. Mizobuchi, personal communication). The DNA sequence corresponding to UptR RNA is described as a "multicopy suppressor of the mutator phenotype of mutT mutants of E. coli." We were interested to determine whether pAG3284, our toxicity suppressor plasmid encoding uptR, could also be a multicopy suppressor of the mutator phenotype of mutT mutants. The function of the mutT gene product is to hydrolyze mutagenic nucleotides 8-oxo-dGTP and 8-oxo-GTP, formed by oxidation, into the corresponding nucleotide monophosphate (6, 25). In a mutT strain, the accumulation of 8-oxo-dGTP induces A:T  C:G transversions (43). The mutT mutator phenotype can be easily detected by using a strain carrying a mutated -galactosidase gene with an amber codon at position 461 (17). We then compared the appearance of Lac⁺ colonies on lactose minimal medium of mutT derivatives of CC101 (lacZ[GAG₄₆₁TAG₄₆₁]) (17) carrying either pHSG575 or pAG3284 (uptR⁺). We did not find any substantial difference between the two strains. Furthermore, we found that pAG3284 does not modify the rate of spontaneous mutagenesis (as indicated by scoring rifampin-resistant colonies; data not shown). These results suggest that our suppressor plasmid pAG3284 does not suppress the mutator phenotype of the mutT strain. The difference from the results of Desiraju and Lu may be due in particular to the strains used in the two studies. The mutT allele used by Desiraju and Lu is an insertion mutation that results in an elevated rate of spontaneous mutations and that may not be a null mutation (A.-L. Lu, personal communication).

UptR-mediated toxicity suppression does not activate the Cpx pathway or the ^E pathway. Suppression of envelope-associated toxicity has been shown to involve activation of at least one of the Cpx or ^E envelope stress response pathways (14, 16, 58). We considered the possibility that DsbA_1-136-PhoA toxicity suppression by UptR involves activation of at least one of these pathways. Therefore, we tested the capacity of the suppressor plasmid pAG3284 to induce either the Cpx pathway or the ^E regulon. For this purpose, we used MC4100 derivatives strains, TR50 and SP887, which carry cpxP-lacZ and fkpA-lacZ chromosomal operon fusions, respectively (18, 19). Induction of the cpxP-lacZ fusion is dependent on activation of the Cpx pathway, while -galactosidase activity originating from the fkpA-lacZ fusion is a reporter of ^E pathway activation. Plasmids pHSG575 and pAG3284 (uptR⁺) were introduced into strains SBS3648 (TR50 degP) and SBS3649 (SP887 degP), and -galactosidase activity was determined. Figure 4 shows that the presence of pAG3284 does not change induction of either cpxP-lacZ or fkpA-lacZ, indicating that multicopy UptR activates neither the Cpx pathway nor the ^E regulon.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Multicopy uptR does not induce fkpA-lacZ and cpxP-lacZ operon fusions. (A) -Galactosidase activities of strains SP887 (MC4100 RS88[fkpA-lacZ]) transformed with pHSG575 (lane 1) and SP887 degP transformed with either pHSG575 (lane 2) or pAG3284 (uptR+; lane 3) were determined. Cells were grown in LB medium containing CHL. Assays were performed on three independent cultures. (B) -Galactosidase activities of strains TR50 (MC4100 RS88[cpxP-lacZ]) transformed with pHSG575 (lane 1) and TR50 degP transformed with either pHSG575 (lane 2) or pAG3284 (uptR+; lane 3) were assayed. Bacteria were grown in LB containing 100 mM phosphate buffer (pH 7.0) and CHL. Values are the means of three independent measurements.

Expression of the toxic hybrid protein is unchanged by the suppressor plasmid pAG3284. Toxicity associated with DsbA'-PhoA unfoldable hybrid protein is dependent on the expression level of the toxic protein (31). One possible means of reducing toxicity is that pAG3284 reduces production of the toxic hybrid protein to below the lethal threshold. As shown above with recG, this is possible by reducing the plasmid copy number. However, in degP⁺ bacteria, the global alkaline phosphatase activity of the DsbA_1-136-PhoA hybrid protein made from pAG2694 was little affected by multicopies of pAG3284 (424 ± 31 U/mg of protein for MC4100 phoA carrying pAG2694 plus pHSG575 versus 398 ± 42 U/mg of protein for MC4100 phoA carrying pAG2694 plus pAG3284, obtained from four independent experiments). In addition, the shock fluid of K10 cells carrying pBR322 plus either the empty vector pHSG575 or pAG3284 showed the same -lactamase specific activity (6,100 ± 250 and 5,900 ± 200 U/mg of periplasmic protein, respectively; obtained from triplicate cultures). Therefore, the presence of pAG3284 in bacteria does not significantly influence the production of the toxic hybrid protein and cannot explain toxicity suppression.

Suppressor plasmid pAG3284 reduces the retention of unfoldable hybrid proteins by the membrane and its consequences. A sign following the expression of a DsbA'-PhoA hybrid protein with an unfolded DsbA' domain is its abnormal retention in the membrane fraction, which is suspected to lead to the reduced kinetics of maturation of several exported proteins (31). In order to further elucidate the role of pAG3284 in toxicity suppression, we analyzed the cell localization of unfolded DsbA'-PhoA hybrid protein (31). We examined the influence of the presence of pAG3284 in degP⁺ bacteria on the partition of the DsbA_1-136-PhoA hybrid protein (Fig. 5, lanes 1 and 2). When pAG3284 was present, a greater proportion of PhoA activity was found in the periplasmic fraction (approximately 25%; Fig. 5A). At the same time, the amount of DsbA_1-136-PhoA bound to the membrane was substantially reduced (Fig. 5B). Clearly, the presence of pAG3284 in bacteria leads to a diminution of hybrid protein bound to the membrane. This diminution could be due either to a more efficient release of DsbA_1-136-PhoA from the membrane or to a more efficient splitting of PhoA degradation fragments from the membrane-bound hybrid. However, we observed that the amount of free PhoA fragments in the periplasmic space is little affected by the presence of pAG3284 (Fig. 5C), suggesting that UptR acts by releasing the entire hybrid protein from the membrane rather than by degrading the toxic hybrid. We analyzed the periplasmic fraction and the total membrane fraction obtained from cultures of degP bacteria carrying pAG3284 and expressing the toxic hybrid protein DsbA_1-136-PhoA (Fig. 5, lane 3). The membrane fraction contains high PhoA activity (70%, Fig. 5A), which is found exclusively in the hybrid protein form (Fig. 5B). In the periplasmic fraction, no PhoA degradation fragments were observed, unlike in the degP⁺ bacteria. This indicates that these PhoA degradation fragments are probably due to the proteolytic activity of DegP. Their absence in degP bacteria, in which UptR-mediated toxicity suppression occurs efficiently, reinforces the hypothesis of toxicity suppression through release of the entire hybrid protein from the membrane.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Influence of pAG3284 on partition of the DsbA1-136-PhoA hybrid protein and of its PhoA degradation fragments between the membrane fraction and the periplasm. Lysozyme-EDTA-treated spheroplasts were prepared from exponentially growing bacteria transformed with the toxic plasmid pAG2694 and either pHSG575 or the suppressor plasmid pAG3284: SBS2027 (degP+)/pAG2694 + pHSG575 (lane 1), SBS2027 (degP+)/pAG2694 + pAG3284 (lane 2), and SBS2603 (degP)/pAG2694 + pAG3284 (lane 3). Spheroplasts were then centrifuged and resuspended in sucrose medium in the presence of protease inhibitors (see Materials and Methods). (A) The PhoA activity in the supernatant, the resuspended pellet, and a corresponding volume of the supernatant of the centrifuged growth medium was measured in each case and is expressed as a percentage of the total PhoA activity (sum). Assays were done in triplicate, and in all cases the sum of activities represented 87 to 98% of that in intact bacteria. (B) Western blot analysis with a monoclonal anti-PhoA antibody of the hybrid protein and of its PhoA degradation fragments in membrane fractions prepared from spheroplasts as indicated in Materials and Methods. The position of the hybrid protein is indicated. (C) Western blot analysis with a monoclonal anti-PhoA antibody of the periplasmic fractions.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Influence of pAG3284 on the kinetics of MBP processing. Bacteria of strain K10/pAG2694 (DsbA1-136-PhoA) transformed with either pHSG575 or pAG3284 were pulse labeled with [35S]Promix (methionine plus cysteine) for 30 s and chased. Proteins were immunoprecipitated with polyclonal anti-MBP antiserum and analyzed by SDS-PAGE (10% acrylamide) and autoradiography. p, preprotein; m, mature protein.

UptR shows complementarity with the 5' end of yjiE, an E. coli gene of unknown function. Several small RNAs have already been described in E. coli (for a review, see reference 64). From its predicted structure and size, UptR RNA is very similar to other small RNAs which act as riboregulators (Fig. 7A). Furthermore, searches for homology between UptR and the whole E. coli genome revealed complementarity over a 35-nucleotide stretch with the coding strand of one presumed gene, referred to as yjiE (10). yjiE is a 909-nucleotide open reading frame located in the 98-min region and transcribed counterclockwise (kb 4556 to 4555 of the physical map according to reference 54). The complementarity involves nucleotides 37 to 3 of the uptR RNA and nucleotides 1 to 35 of the yjiE putative coding sequence (Fig. 7B). From these observations, we considered the possibility that modification of yjiE expression may be involved in toxicity suppression.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   (A) Predicted secondary structures of UptR RNA. RNA folding was done with the MulFold program. Putative stem-loops 1 and 2 are indicated. (B) Nucleotide complementarity between segments of UptR RNA and the putative yjiE mRNA. The upper sequence corresponds to uptR RNA and is written from right to left (5' end to 3'). Part of the yjiE putative mRNA is shown in the lower sequence. The reversed-out AUG codon corresponds to the start codon of the most probable open reading frame following a Shine-Dalgarno-like sequence (10, 26). Bars indicate nucleotide complementarity.

Does uptR overexpression modify the physiology of other aberrant envelope proteins? In order to understand the role of UptR in toxicity suppression, we determined whether multicopy uptR could affect the physiology of certain well-studied aberrant envelope proteins. We chose proteins provoking four types of misfunctions in the envelope and for which suppressors of these misfunctions have been isolated. First, a set of strains (MM1 to MM5) have been described which can produce MBPs with signal sequences altered in the hydrophobic core (3). These modifications interact with MBP export to various extents, resulting in different growth rates on maltose minimal medium. Second, strain JHC285Kan carries the lamBA23D mutation, which specifies a protein with a modified signal peptide cleavage site (16). Its induction is toxic to the cell, probably due to the cell's attempt to target a protein tethered in the inner membrane by its signal peptide to the outer membrane (15). Third, strain NJH101 carries the lamB-lacZ gene fusion, whose expression results in toxicity due to jamming of the hybrid protein in the inner membrane (57). This jamming is presumed to be due to an early folding of the LacZ part of the fusion in the cytoplasm before exportation. Fourth, strains WBS1106 and WBS164 carry the lamB-lacZ-phoA tripartite fusion and the lamB-lacZX90 fusion, respectively (59). Their induction is also toxic to the cell, but the hybrid proteins are exported to the periplasmic space, where they exert a toxicity of unknown cause. Bacteria of each of the preceding strains were transformed with pHSG575 and pAG3284, and the effects of uptR overexpression on toxicity were determined.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Coupling between folding and export has previously been demonstrated in E. coli with several DsbA'-PhoA hybrid proteins differing only in the folding ability of their DsbA' fragment. High-level expression to the envelope of unfoldable DsbA'-PhoA hybrid protein leads to toxicity. The origin of toxicity is suspected to be the association of DsbA'-PhoA with the inner membrane via its DsbA' unfoldable fragment, which for unknown reasons would lead to blockage of the exportation process. This toxicity can be alleviated by the DegP periplasmic proteolytic activity, highlighting its role in the housecleaning functions in the envelope. At the beginning of this work, we were interested in determining if bacteria could overcome this toxicity without the help of the DegP protease. For this purpose, we established DsbA_1-136-PhoA-mediated toxicity conditions in strains lacking DegP. Such conditions were used to select multicopy suppressors that would keep the bacteria alive. Characterization of their mode of action is the subject of this work, which sheds light on the toxicity of unfoldable DsbA'-PhoA proteins and on a new means of extracytoplasmic toxicity suppression.

In addition to the degP gene, two genes were isolated as multicopy suppressors of DsbA'-PhoA hybrid protein toxicity. Cloning of the recG gene corroborates the result of Hong et al. showing that recG overexpression reduces ColE1 plasmid copy number (32). Indeed, the toxic plasmid bearing the dsbA'::phoA gene fusion is a derivative of pBR322, and the presence of recG in multicopy reduces the plasmid copy number. The expression of the DsbA'-PhoA hybrid was also reduced to below the lethal threshold, so that growth was allowed under nonpermissive conditions.

The third multicopy suppressor isolated was named uptR, for unfolded protein toxicity-relieving factor. The uptR gene is approximately 130 bp long, including the promoter sequences and the transcription terminator. We demonstrated that the effector of toxicity suppression is the untranslated UptR small RNA. According to its origin of transcription and secondary-structure predictions, this small RNA is 92 nucleotides long. It belongs to the F episome and is located in a region that has not been studied previously (K. Mizobuchi, personal communication). The physiological role of uptR in bacteria is unknown. As for its role in toxicity suppression, we found that in contrast to recG, overexpression of uptR does not modify the copy number of the pBR322-derived toxic plasmids. It does not significantly change the expression level of the toxic DsbA_1-136-PhoA hybrid protein. The main consequences of multicopy uptR in cells producing toxic DsbA'-PhoA are a reduction in the membrane-associated hybrid protein and a diminution in exportation deficiency. These phenotypes are also observed when DegP affords toxicity suppression. However, the two suppressors involve different mechanisms. Multicopy degP suppression leads to degradation of the unfolded part of the hybrid protein and the appearance of free PhoA fragments in the periplasm (see Fig. 5B, lane 1) (31). The proteolytic activity of DegP is the effector of toxicity suppression, since expression of DegP_S210A, a variant of DegP without proteolytic activity, cannot suppress toxicity (31). Multicopy uptR, on the other hand, does not involve an increase in proteolytic activity degrading the unfolded DsbA' part in the periplasm. Several observations support this conclusion. First, in a degP⁺ strain, the presence of the multicopy suppressor does not increase the degradation of the periplasmic DsbA'-PhoA hybrid (Fig. 5B, compare lanes 1 and 2). Second, in a degP mutant strain carrying the multicopy suppressor, the periplasmic fraction does not contain PhoA degradation fragments (Fig. 5B, lane 3). From these observations, we propose that multicopy uptR reduces toxicity by diminishing the amount of hybrid protein associated with the inner membrane without involving its degradation.

This result raises the question of how toxicity suppression takes place in the envelope. Several examples of toxicity factors and associated multicopy suppressors have been described and characterized in E. coli (14, 16, 58). Suppression is often obtained by activation of the Cpx pathway or the ^E regulon. We demonstrated that multicopy uptR does not induce the Cpx pathway or the ^E regulon. This means that multicopy uptR does not alleviate toxicity by using the known stress response pathways. The origin of toxicity involves the unfolded DsbA' part of the hybrid protein and the inner membrane. In order to alleviate this toxicity, bacteria can act either on the unfolded DsbA' part or on the inner membrane. Hence, we can envisage two types of suppression provoked by multicopy uptR. First, the inner membrane characteristics may be modified, so that toxicity would be reduced. Let us consider that the toxic hybrid protein associates with the inner membrane by sequestering one of the members of the exportation machinery and hence leads to jamming. Modification of the amount or the characteristics of this component may alleviate toxicity, as already described for a LamB-LacZ hybrid and the PrlA/SecY secretory machinery component (8). Second, the hybrid protein may be efficiently released from the inner membrane and then excluded from misinteraction with the membrane, for instance, by shielding the DsbA' part from the inner membrane or by allowing it to adopt a conformation preventing association. Proteins with such activities have been described in the envelope but are dedicated to the specific transport of outer membrane protein from the inner membrane to the outer membrane (35, 41). Recently, Silhavy's group hypothesized the existence of a new stress response pathway that could be implicated more specifically in protein trafficking in the bacterial envelope (15). They isolated and described a multicopy suppressor of toxicity due to the expression of processing-defective outer membrane protein LamB. This suppressor could "clear defective membrane proteins, with the processing-defective LamB being one such substrate." The mechanism described by the authors, the clearing of defective membrane protein, is very similar to that observed with UptR-mediated toxicity suppression. However, we found that the multicopy suppressor of LamBA23D toxicity does not alleviate DsbA_1-136-PhoA toxicity (J. Dassa and P. Belin, unpublished data) and that UptR overexpression does not change LamBA23D toxicity. Furthermore, we demonstrated that uptR overexpression does not suppress the toxicity associated with different toxic envelope proteins. These results suggest that (i) DsbA_1-136-PhoA toxicity is a novel type of toxic envelope protein, and (ii) UptR-mediated toxicity suppression may not be a general method of envelope stress response. Identification of the specific factors involved in DsbA'-PhoA toxicity suppression will need further investigation of the nature of the elements underlying toxicity.

However, understanding of the basis of this toxicity suppression may also be improved by analyzing the role of multicopy uptR in the cytoplasm. Our results raise the following question of how overexpression of a small RNA induces changes in the periplasmic space that lead to relief of extracytoplasmic toxicity. The mechanisms of action of small RNAs described so far in E. coli belong to three distinct groups: catalytic RNAs, RNA active by association with a protein, and RNA active by RNA-RNA base-pairing (64). From its size and secondary-structure predictions, UptR RNA is very much like small riboregulatory RNAs rather than catalytic RNAs or those acting by association with a protein. These similarities led us to envisage the possibility of riboregulatory activity for UptR. We identified a gene of unknown function, yjiE, whose RNA sequence around the translation initiation region displays complementarities with UptR RNA. However, we found that inactivation of yjiE or multicopies of the yjiE gene chromosomal region do not modify the sensitivity of bacteria to the toxic plasmid. We cannot exclude that yjiE expression may be regulated by UptR, but if this is the case, variations in yjiE expression are not sufficient to explain toxicity suppression, suggesting that another factor(s) may be involved.

For the moment, the function of UptR RNA in DsbA'-PhoA toxicity suppression remains obscure. Elucidating its structure-function relationship will probably help in understanding its role in toxicity suppression and cellular physiology. DsbA'-PhoA and UptR are an additional pair of toxic protein suppressors described in E. coli. They appear to be a useful new tool for gaining further insights into protein secretion and the folding of envelope proteins.