Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of ≈15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti-activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.
Type III secretion (TTS) systems are widely spread among Gram-negative bacteria pathogenic for humans, animals and plants. They comprise a TTS apparatus (TTSA) spanning the inner and outer membranes, translocators and effectors transiting through this apparatus, specific chaperones and transcription activators (Hueck, 1998). Translocators are proposed to insert into the membrane of host cells to form a pore allowing effectors to reach the cell cytoplasm where they affect a variety of cellular functions (Galan, 2001; Cornelis, 2002). Production and secretion of translocators and effectors are uncoupled and specific chaperones associate with these proteins to maintain them in a secretion-competent state (Parsot et al., 2003). The TTSA activity is induced upon contact of bacteria with host cells (Ménard et al., 1994a; Zierler and Galan, 1995; Pettersson et al., 1996; Vallis et al., 1999). Activation of the TTSA increases transcription of either most genes of the TTS system in Yersinia and Pseudomonas spp. or a set of genes encoding effectors in Shigella spp. (Straley et al., 1993; Demers et al., 1998; McCaw et al., 2002).
Control of transcription by TTSA activity involves AraC family activators containing a conserved C-terminal DNA binding domain and a variable N-terminal domain that presumably modulates their activity. Mechanisms by which secretion activity is sensed and transmitted to regulated promoters are poorly understood (Miller, 2002). In Yersinia spp., regulation by TTSA activity involves the activator VirF and TTSA substrates LcrQ and YopD and their chaperones SycH and SycD (Francis et al., 2001; Wulff-Strobel et al., 2002). In Pseudomonas aeruginosa, activity of the activator ExsA requires sequestration of the anti-activator ExsD by the anti-anti-activator ExsC, presumably following secretion of unidentified TTSA substrates titrating ExsC (Dasgupta et al., 2004). In Salmonella enterica serovar Typhimurium, the chaperone SicA binds to and is required for activity of the activator InvF, but there is no evidence that InvF activity is modulated by secretion activity (Darwin and Miller, 2001).
Bacteria of Shigella spp. are responsible for shigellosis in humans, a disease characterized by inflammation and destruction of the colonic epithelium. They use a TTS system to enter epithelial cells and trigger apoptosis in macrophages. This system is encoded by a 200-kb virulence plasmid and includes the Mxi-Spa TTSA, the IpaB and IpaC proposed translocators, over 20 effectors, four chaperones and three transcription activators (Buchrieser et al., 2000). The TTSA assembled during growth of bacteria in broth is very weakly active, with < 5% of IpaB and IpaC being secreted. TTSA activity is induced upon contact of bacteria with epithelial cells and exposure to the dye Congo red and is deregulated, i.e. constitutively active, in ipaB and ipaD mutants (Ménard et al., 1994a; Parsot et al., 1995; Bahrani et al., 1997). Transcription of ≈15 genes encoding effectors, including virA, osp and ipaH genes, but not genes encoding TTSA components and translocators is regulated by TTSA activity (Demers et al., 1998; Mavris et al., 2002a; Le Gall et al., 2005).
Genes regulated by TTSA activity are controlled by the AraC family activator MxiE (Mavris et al., 2002a). MxiE is encoded by the same operon as TTSA components (Allaoui et al., 1993) and is produced by transcriptionnal frameshifting (Penno et al., 2005). MxiE activity requires the coactivator IpgC that is also the chaperone of IpaB and IpaC (Ménard et al., 1994b; Page et al., 1999; 2001; Mavris et al., 2002a). Under conditions of non-secretion, MxiE is proposed to be inactive because IpgC is sequestered by IpaB and IpaC that behave as anti-coactivators. However, increasing IpgC production in a non-secreting strain did not induce IpaH production to the same extent as that observed in a secretion-deregulated strain (Mavris et al., 2002a), suggesting the existence of another regulatory mechanism controlling MxiE-regulated promoters. We used a genetic screen to identify a gene whose product is involved in a negative control of promoters regulated by TTSA activity under conditions of non-secretion. We present evidence that OspD1 associates with the chaperone Spa15 and MxiE and acts as an anti-activator until it is secreted.
Inactivation of ospD1 increases transcription of TTSA activity-regulated genes
To identify a gene negatively controlling transcription of TTSA activity-regulated genes, SF1001 carrying a virA–lacZ transcriptional fusion was subjected to Tn5 mutagenesis and β-galactosidase activity was assayed in ≈400 mutants. Whereas SF1001 produced 20 Miller units of β-galactosidase activity, mutants SF1041, SF1042 and SF1043 produced 450, 300 and 80 Miller units respectively. IpaB and IpaD that are required to maintain the TTSA inactive during growth in broth were not produced by SF1042 and SF1041, respectively, suggesting that increased expression of virA–lacZ in these mutants was a consequence of a deregulated secretion activity. In contrast, SF1043 had no defect in production and secretion of IpaB and IpaD. The Tn5 insertion site in this mutant corresponds to nucleotide 21 358 of the virulence plasmid pWR100, i.e. to codon 130 of ospD1 encoding a 225-residue TTSA substrate whose expression is not regulated by TTSA activity (Buchrieser et al., 2000; Le Gall et al., 2005). P1 transduction of ospD1::Tn5 from SF1043 to wild-type strain M90T-Sm gave rise to strain SC586 (ospD1-1).
In addition to virA, genes regulated by TTSA activity include members of the ipaH family comprising five chromosomal and five virulence plasmid-borne genes (Buysse et al., 1995; Buchrieser et al., 2000). To investigate whether the ospD1-1 mutation affects IpaH production, crude extracts and culture supernatants of wild-type, ospD1-1 and ospD1-1 harbouring pEAD1 (encoding OspD1) strains were analysed by SDS-PAGE and Coomassie blue staining and immunoblotting (Fig. 1). IpaB and IpaC were produced and secreted in similar amounts by these strains. The ospD1-1 strain produced larger amounts of IpaHs than the wild-type strain. This effect was complemented by the plasmid encoding OspD1. Reporter plasmids encoding lacZ transcriptional fusions under the control of ipaH7.8, ipaH9.8, ospF and ospC1 promoters (all regulated by TTSA activity) were introduced into wild- type and ospD1-1 strains, as well as the secretion-deregulated ipaD-2 strain. Expression of each fusion was increased ≈15 times in the ipaD-2 strain and ≈5 times in the ospD1-1 strain compared with the wild-type strain (Table 1). Accordingly, insertion of Tn5 within ospD1 has little effect on TTSA activity and increases transcription of TTSA activity-regulated genes.
Table 1. β-Galactosidase activitiesaproduced by bacteria harbouring plasmids encoding lacZ transcriptional fusions under the control of TTSA activity-regulated promoters.
.ß-Galactosidase activities are expressed in Miller units. Standard deviations (not shown) were within 25% of reported values.
Increased transcription of ipaH and osp genes is independent of TTSA activity in the ospD1 mutant
IpaHs were more secreted by the ospD1-1 strain than the wild-type strain, probably as a consequence of their increased production (Fig. 1). To test the role of TTSA activity in IpaH production by the ospD1-1 strain, we inactivated mxiD (encoding a TTSA component) in wild-type, ospD1-1 and ipaD-2 strains to construct mxiD-4 strains. Analysis of crude extracts and culture supernatants of wild-type, ipaD-2, ospD1-1, mxiD-4, ipaD-2 mxiD-4 and ospD1-1 mxiD-4 strains (Fig. 2A) indicated that (i) as expected, the TTSA was not functional in mxiD-4 strains; (ii) IpaH amounts in the ospD1-1 strain were intermediate between those in wild-type and ipaD-2 strains, consistent with results obtained using ipaH–lacZ transcriptional fusions; (iii) IpaH amounts were reduced in the ipaD-2 mxiD-4 strain compared with the ipaD-2 strain and (iv) IpaH amounts were similar in ospD1-1 and ospD1-1 mxiD-4 strains. Expression of lacZ transcriptional fusions encoded by reporter plasmids was reduced in the ipaD-2 mxiD-4 strain compared with the ipaD-2 strain and was similar in ospD1-1 mxiD-4 and ospD1-1 strains (Table 1). Therefore, in contrast to the ipaD-2 strain, increased IpaH production and increased expression of TTSA activity-regulated promoters in the ospD1-1 strain are independent of the TTSA activity.
IpaH production is dependent upon both MxiE and IpgC in the ospD1 mutant
To investigate the role of MxiE and IpgC in IpaH production in an ospD1 background, we inactivated ospD1 in wild-type, mxiE-2 and ipgC-2 strains to construct ospD1-2 strains. Crude extracts and culture supernatants of wild-type, ospD1-2, ospD1-2 mxiE-2 and ospD1-2 ipgC-2 strains were analysed by SDS-PAGE and Coomassie blue staining and immunoblotting using anti-IpaH and anti-IpaD antibodies (Fig. 2B). IpaD is encoded by the same operon as IpaB and IpaC and was used here as a control because IpaB and IpaC are degraded in an ipgC mutant (Ménard et al., 1994b). Like the ospD1-1 strain, the ospD1-2 strain produced more IpaHs than the wild-type strain and was not affected in production and secretion of IpaA-D. IpaH production was strongly decreased in ospD1-2 mxiE-2 and ospD1-2 ipgC-2 strains compared with the ospD-2 strain, indicating that both MxiE and IpgC are necessary for increased IpaH production in an ospD1 background.
OspD1 inhibits MxiE activity
To test whether OspD1 alone, i.e. independently of other virulence plasmid-encoded proteins, is sufficient to control negatively MxiE activity, we used virulence plasmid-cured strains harbouring plasmids encoding OspD1, MxiE and IpgC. Production of chromosome-encoded IpaHs was used to monitor MxiE activity (Mavris et al., 2002b; Penno et al., 2005). SDS-PAGE analysis of extracts of strains containing combinations of the three recombinant plasmids and corresponding vectors (Fig. 3A) indicated that (i) IpgC production was not influenced by MxiE or OspD1; (ii) OspD1 production was not influenced by IpgC or MxiE; (iii) MxiE production was not influenced by IpgC; (iv) IpaH production was dependent upon both MxiE and IpgC, consistent with the lack of activity of MxiE alone and the role of IpgC as a coactivator; (v) in strains producing MxiE and IpgC, IpaH production was decreased in the presence of OspD1; and (vi) MxiE amounts were increased in the presence of OspD1, both in the presence and in the absence of IpgC. As mxiE, ipgC and ospD1 are under the control of lac promoters, increased MxiE amounts in the presence of OspD1 do not result from increased transcription of mxiE but rather from stabilization of MxiE by OspD1. To test the effect of OspD1 on production of MxiE encoded by the virulence plasmid, crude extracts of wild-type, mxiE-2, ospD1-1, ospD1-1 harbouring pEAD1 (OspD1) and ipaD-2 strains were analysed by SDS-PAGE and immunoblotting using anti-MxiE antibodies (Fig. 3B). Decreased amounts of MxiE were present in the ospD1-1 mutant compared with the wild-type strain and this effect was complemented by the plasmid encoding wild-type OspD1. Accordingly, OspD1 has a positive effect on the steady-state amount of MxiE and a negative effect on its activity.
OspD1 interacts with MxiE
To test whether OspD1 interacts with MxiE, pEAD2 encoding OpsD1-His (OspD1 carrying a C-terminal His tag) was introduced into the wild-type strain and the recombinant protein was purified by affinity chromatography. Proteins present in each fraction of the purification procedure were analysed by SDS-PAGE (Fig. 4A). OspD1-His bound the resin and was eluted by imidazole and most MxiE molecules present in the extract were eluted together with OspD1-His. As MxiE does not bind the resin in the absence of OspD1-His (data not shown), this suggests that MxiE interacts with OspD1-His. IpgC did not bind the column, indicating that IpgC is not a chaperone for OspD1 and that the MxiE:OspD1-His complex does not contain IpgC.
Spa15 is the chaperone of OspD1
Spa15 is the chaperone for several TTS effectors, including IpaA, IpgB1, OspC3 and OspB (Page et al., 2002). To investigate whether Spa15 is also a chaperone for OspD1, proteins present in each fraction of the OspD1-His purification were analysed using anti-Spa15 antibodies (Fig. 4A). Spa15 was eluted together with OspD1-His, suggesting that Spa15 interacts with OspD1. This was confirmed by analysing proteins copurified with His-Spa15 (Spa15 carrying an N-terminal His tag) produced in a spa15 mutant harbouring pHSpa15 (Fig. 4B). MxiE and OspD1 were eluted in the same fractions as His-Spa15, suggesting that MxiE interacts with a complex containing both OspD1 and His-Spa15.
To test the potential effect of MxiE and Spa15 on stability and secretion of OspD1, crude extracts and culture supernatants of wild-type, spa15-2 and mxiE-2 strains and proteins secreted by these strains in response to exposure to Congo red (to induce TTSA activity) were analysed by SDS-PAGE (Fig. 5). A small proportion of OspD1 was present in the culture supernatant of the wild-type strain and secretion of OspD1 was induced upon exposure to Congo red. Reduced amounts of OspD1 were present in the spa15-2 strain compared with the wild-type strain, indicating that Spa15 is required for stability of OspD1, and OspD1 was not secreted by this mutant during growth in broth and upon exposure to Congo red. In contrast, inactivation of mxiE had no effect on production and secretion of OspD1. As in ospD1 mutants, decreased amounts of MxiE and increased amounts of IpaHs were present in the spa15-2 mutant compared with the wild-type strain. Thus, by stabilizing OspD1, Spa15 is involved in the control of MxiE-regulated promoters. Although IpaH production was increased in the spa15 mutant, IpaHs were not secreted upon exposure of this mutant to Congo red, suggesting that Spa15 is also required for secretion of stored IpaH molecules, as described for IpaA (Page et al., 2002).
Secretion of OspD1 is required for activation of MxiE-regulated genes
OspD1 inhibits MxiE activity and is secreted in response to TTSA activation, suggesting that OspD1 must be secreted for MxiE activation. Accordingly, a non-secretable OspD1 protein should prevent activation of MxiE-regulated promoters in conditions of deregulated secretion. Hypothesizing that the OspD1 N-terminal part contains the secretion signal, as described for TTSA substrates stored prior to being secreted (Lloyd et al., 2001), we constructed pEAD3 encoding OspD1Δ1-30-His, lacking the first 30 residues of OspD1-His. Purification of OspD1Δ1-30-His indicated that this protein still binds MxiE and Spa15 (data not shown). pEAD3 (OspD1Δ1-30-His), pEAD2 (OspD1-His) or pKJ1 (vector) were introduced into the secretion-deregulated ipaD-2 strain. Crude extracts and culture supernatants of ipaD-2 strains harbouring pKJ1, pEAD2, or pEAD3 were analysed by SDS-PAGE (Fig. 6). Production and secretion of IpaB and IpaC were not affected in the presence of OspD1-His or OspD1Δ1-30-His. As expected, OspD1-His was secreted and did not affect production and secretion of IpaHs. The observation that only a fraction of OspD1-His was present in the culture supernatant does not mean that most OspD1-His molecules were not secreted by the ipaD-2 strain, as TTSA substrates aggregate in the culture medium and are recovered in the pellet following centrifugation of bacterial cultures (Parsot et al., 1995). In contrast to OspD1-His, OspD1Δ1-30-His was not secreted and IpaHs were no longer produced by the strain expressing this recombinant protein, indicating that secretion of OspD1 is required for activation of MxiE-regulated promoters.
We presented evidence that OspD1 is involved in the mechanism regulating transcription in response to TTSA activation: (i) inactivation of ospD1 increases transcription of genes regulated by TTSA activity, independently of TTSA activity; (ii) OspD1 binds MxiE and inhibits its activity; (iii) secretion of OspD1 is required for activation of MxiE-regulated promoters. Therefore, OspD1 is an anti-activator that associates with MxiE and prevents it from being activated by its coactivator IpgC. The chaperone Spa15 associates with and is involved in stability of OspD1 and therefore acts as a co-anti-activator.
When produced in the virulence plasmid-cured strain, OspD1 had a positive effect on MxiE stability, indicating that Spa15 is not required for the interaction of OspD1 with MxiE. Spa15 exhibits structural similarities with Yersinia spp. SycE and S. enterica SicP chaperones, suggesting a similar mode of binding of these chaperones to their partner(s) (Stebbins and Galan, 2001; Birtalan et al., 2002; van Eerde et al., 2004). As SycE and SicP bind residues 17–85 and 35–139 of YopE and SptP, respectively, Spa15 might bind residues ≈30–100 of OspD1 and the region of interaction between OspD1 and MxiE might correspond to the OspD1 C-terminal part consisting in two repeats of a 44-residue motif (Buchrieser et al., 2000). Although OspD1 could be produced in the virulence plasmid-cured strain, it was degraded in the spa15 mutant. This suggests that degradation of OspD1 in the spa15 mutant is promoted by the interaction of unprotected OspD1 with virulence plasmid-encoded protein(s), possibly other Spa15 partners such as IpgB1 that is also degraded in the spa15 mutant (Page et al., 2002).
The identification of an anti-activator for MxiE indicates that TTSA activity is transmitted to the transcription apparatus by two sensors, OspD1 and IpgC, which has several implications. (i) The activity of MxiE-controlled promoters in ospD1 mutants is evidence that some IpgC molecules are not titrated by IpaB and IpaC under conditions of non-secretion. Production of an excess of IpgC is probably a result of the constraint imposed by IpaB and IpaC instability in the absence of IpgC (Ménard et al., 1994b; Page et al., 1999). (ii) Steady-state amounts of MxiE were reduced in the absence of OspD1, suggesting that MxiE is stabilized by its association with OspD1; paradoxically, a TTSA substrate (OspD1) acts as a chaperone for a cytoplasmic protein (MxiE). (iii) MxiE-regulated genes are induced upon entry into epithelial cells and no longer transcribed in intracellular bacteria, suggesting the TTSA is not active during intracellular growth (Demers et al., 1998). TTSA components and translocators are expressed by intracellular bacteria and involved in escape of bacteria from protrusions during intercellular dissemination (Page et al., 1999). Following closure of the TTSA, de-activation of MxiE can not rely on titration of IpgC by newly synthesized IpaB and IpaC, because new IpgC molecules are produced at the same time as IpaB and IpaC. In contrast, neosynthesized OspD1 can titrate MxiE in the presence of free IpgC. (iv) As translocators are proposed to form the pore through which effectors transit, these proteins should be addressed first to the TTSA, liberating IpgC. However, it is only after transit of OspD1 that MxiE is liberated. Dependency of MxiE activity upon secretion of both the anti-coactivators and the anti-activator ensures that late effectors (encoded by genes controlled by MxiE) are produced and addressed to the TTSA only after translocators and early effectors (encoded by genes not controlled by MxiE) have transited through the TTSA.
In conclusion, the mechanism by which TTSA activity is sensed and transmitted to the transcription apparatus involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the opening of the TTSA (Fig. 7). All these proteins are produced in conditions of non-secretion and their production is not increased in conditions of secretion (Le Gall et al., 2005). In conditions of non-secretion, IpaB and IpaC are associated independently with IpgC, which prevents premature interactions between the two translocators and titrates, although not completely, IpgC. OspD1 is associated with Spa15, which stabilizes OspD1 and probably maintains it in a secretion competent state. MxiE is associated with the Spa15–OspD1 complex, which both stabilizes MxiE and prevents it from being activated by IpgC. Upon TTSA activation, transit of IpaB and IpaC liberates IpgC, however, this is not sufficient to activate MxiE as long as OspD1 is present in the cytoplasm. If the TTSA remains active, transit of OspD1 occurs, liberating MxiE. The ensuing, not yet demonstrated, interaction between MxiE and IpgC allows MxiE to bind DNA target sites and activate transcription at MxiE-regulated promoters. Late effectors such as IpaHs are then produced and transit through the TTSA. Following closure of the TTSA, neosynthesized OspD1 in association with Spa15 sequesters MxiE again, leading to de-activation of MxiE-regulated genes. In the S. enterica flagellar system, secretion of the anti-sigma factor FlgM upon completion of the hook-basal body (HBB) complex liberates the sigma factor σ28 to transcribe flagellin genes (Hughes et al., 1993). Secretion of FlgM is a check point signalling completion of the HBB complex, the activity of which is not regulated by external signals. The more complex regulatory mechanism used by S. flexneri to sense TTSA activity is likely resulting from the constraint of signalling the opening and closure of the TTSA in response to external signals.
Strains and plasmids
Shigella flexneri strains were grown in trypticase soy broth under aerobic conditions at 37°C. M90T-Sm (wild type), SF1001 (virA–lacZ), SF1060 (mxiE-2), SF619 (ipgC-2), RM89 (ipaD-2), SF1601 (spa15–2) and BS176 (virulence plasmid-cured) have been described, as well as the pBBR1 mcs2 derivative encoding IpgC (pKH128), the pUC19 derivative encoding MxiE (pKH126), reporter plasmids encoding ipaH7.8–, ipaH9.8–, ospC1– and ospF–lacZ fusions and pHSpa15 encoding His-Spa15 (Allaoui et al., 1992; Ménard et al., 1993; 1994b; Demers et al., 1998; Mavris et al., 2002a,b; Page et al., 2002).
For Tn5 mutagenesis, SF1001 (virA–lacZ) was used as a recipient for conjugation with an Escherichia coli strain harbouring an F′ carrying Tn5. Transconjugants were screened using the β-galactosidase substrate Xgal, leading to the isolation of SF1041, SF1042 and SF1043. P1 transduction of ospD1::Tn5 from SF1043 to M90T-Sm gave rise to SC586 (ospD1-1). pSW23-T containing the R6K origin of replication and the RP4 origin of transfer was used to construct pKH123 carrying a XbaI-EcoRI fragment encompassing codons 84-275 of mxiD and pOD1 carrying a BamHI-EcoRI fragment encompassing codons 17-145 of ospD1. Integration of pKH123 into mxiD of M90T-Sm (wild type), RM89 (ipaD-2) and SC586 (ospD1-1) gave rise to SF1127 (mxiD-4), SF1139 (ipaD-2 mxiD-4) and SF1131 (ospD1-1 mxiD-4) and integration of pOD1 into ospD1 of M90T-Sm (wild type), SF1060 (mxiE-2) and SF619 (ipgC-2) gave rise to SF1214 (ospD1-2), SF1206 (mxiE-2 ospD1-2) and SF1209 (ipgC-2 ospD1-2). pEAD1 encoding OspD1 was constructed by cloning a polymerase chain reaction (PCR) fragment encompassing ospD1 and 19 and 27 bp of its 5′ and 3′ regions between SalI and KpnI sites of pUC19. The SalI-KpnI fragment encompassing ospD1 in pEAD1 was cloned into pSU19 to construct pKH130. pKJ1 and pKJ3 were constructed by cloning a PCR fragment carrying lacI and 222 and 246 bp of its 5′ and 3′ regions between NheI and XbaI sites of pQE60 and pQE30 respectively. The ospD1 coding sequence and ospD1 codons 31-225 were amplified by PCR and cloned between NcoI and BglII sites of pKJ1 to construct pEAD2 encoding OspD1-His and pEAD3 encoding OspD1Δ1-30-His respectively.
Purification of OspD1-His and His-Spa15 and protein analysis
Approximately 1011 bacteria of M90T-Sm harbouring pEAD1 or SF1601 harbouring pHSpa15 were grown in the presence of 100 µM IPTG at 37°C for 5 h, harvested by centrifugation, resuspended in 4 ml of PBS, pH 7.5, containing 10 mM imidazole and disrupted by sonication. The extract was clarified by centrifugation, the supernatant was incubated in the presence of 500 µl of cobalt-containing resin for 2 h at 4°C and the mixture was loaded onto a column. The flow through was collected, the resin was washed with the same volume of buffer and elution was performed in the presence of 100 mM imidazole. OspD1-His produced by the E. coli strain DH5a harbouring pEAD2 was solubilized in 6 M guanidium chloride, purified by affinity chromatography and used to immunize a rabbit. For analysis of production and secretion of proteins, bacteria in the exponential phase of growth were harvested by centrifugation. Culture supernatants were filtered using 0.4 µm filters and filtrates were concentrated 100 times by TCA precipitation. Bacterial pellets were resuspended in 1/10 vol of PBS and incubated at 37°C in the presence of Congo red (50 µg ml−1) for 20 min to induce TTSA activity. Samples were centrifuged and supernatants were concentrated 10 times by TCA precipitation. Extracts were analysed by SDS-PAGE and stained using Coomassie blue or transferred to nitrocellulose membranes. Horseradish peroxidase-labelled secondary antibodies were visualized by enhanced chemiluminescence. β-Galactosidase activity was assayed using the substrate 0-nitrophenyl-β- d-galactopyranoside on bacteria harvested during the exponential phase of growth.
We are pleased to acknowledge John Rohde and Tony Smith for a critical reading of the manuscript. This work was supported in part by Grants from the Medical Research Council of Canada (to B. D), a TMR program from the European Community and a GIP involving Hoechst-Marion-Roussel and the Pasteur Institute.