Secb-Like Chaperone Controls a Toxin–Antitoxin Stress-Responsive System in Mycobacterium Tuberculosis

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

Patricia Bordesa, Anne-Marie Cirinesia, Roy Ummelsb, Ambre Salaa, Samer Sakra, Wilbert Bitterb, and Pierre Genevauxa,1

aLaboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientiﬁque and Université Paul Sabatier, F-31000 Toulouse, France; and bDepartment of Medical Microbiology, VU University Medical Center and Department of Molecular Microbiology, VU University, 1081 BT, Amsterdam, The Netherlands

Edited by Linda L. Randall, University of Missouri, Columbia, MO, and approved April 12, 2011 (received for review January 22, 2011) A major step in the biogenesis of newly synthesized precursor aggregation in the absence of the cytosolic chaperones DnaK and proteins in bacteria is their targeting to the Sec translocon at the Trigger Factor (TF) (12). inner membrane. In Gram-negative bacteria, the chaperone SecB Because of its role in protein export, the chaperone SecB is binds nonnative forms of precursors and specifically transfers considered a proteobacterial invention associated with the them to the SecA motor component of the translocase, thus presence of an outer membrane present in α-, β-, γ-, and some facilitating their export. The major human pathogen Mycobacte- δ-proteobacteria (13, 14). Nonetheless, genes showing homol- rium tuberculosis is an unusual Gram-positive bacterium with ogy to secB are infrequently found in Planctobacteria, Spi- a well-defined outer membrane and outer membrane proteins. rochaetae, Sphingobacteria, Eurybacteria, and Endobacteria Assistance to precursor proteins by chaperones in this bacterium (13). Functions in protein translocation have not been de- remains largely unexplored. Here we show that the product of the scribed for any of these putative SecB-like proteins, and no previously uncharacterized Rv1957 gene of M. tuberculosis can SecB homologs have been found in mycobacteria. Despite their substitute for SecB functions in Escherichia coli and prevent pre- classification as Gram-positive bacteria, recent discoveries have protein aggregation in vitro. Interestingly, in M. tuberculosis, shown that there is a well-defined outer membrane in myco- Rv1957 is clustered with a functional stress-responsive higB-higA bacteria (15, 16) and that Mycobacterium tuberculosis encodes MICROBIOLOGY toxin–antitoxin (TA) locus of unknown function. Further in vivo asignificant number of putative outer membrane proteins experiments in E. coli and in Mycobacterium marinum strains that (17, 18). How these outer-membrane proteins are assisted in do not possess the TA-chaperone locus show that the severe tox- the cytoplasmic space and targeted to the inner membrane in icity of the toxin was entirely inhibited when the antitoxin and the these bacteria remains unknown. chaperone were jointly expressed. We found that Rv1957 acts di- In this work, we first show that the major human pathogen rectly on the antitoxin by preventing its aggregation and protect- M. tuberculosis encodes a SecB-like chaperone, namely Rv1957, ing it from degradation. Taken together, our results show that the which can functionally replace the Escherichia coli SecB chaperone, fi SecB-like chaperone Rv1957 speci cally controls a stress-responsive both in vivo and in vitro. We also demonstrate that Rv1957 my- TA system relevant for M. tuberculosis adaptive response. cobacterial chaperone controls the HigB-HigA stress-responsive toxin–antitoxin (TA) system of M. tuberculosis by acting directly DnaK | molecular chaperones | trigger factor | protease on its cytosolic HigA antitoxin substrate. The role of this atypical SecB-like chaperone of M. tuberculosis is discussed. n bacteria, newly synthesized proteins emerging from the ri- Ibosome are assisted by essential molecular chaperones and Results and Discussion targeting factors, which facilitate folding and/or partition them as Rv1957 from M. tuberculosis Shows Similarities to the SecB cytosolic, inner-membrane, or exported proteins. Generic mo- Chaperone. Inspection of the M. tuberculosis genome reveals the lecular chaperones such as DnaK-DnaJ (Hsp70-Hsp40), Trigger unexpected presence of a gene (Rv1957) coding for a protein Factor, GroEL-GroES (Hsp60-Hsp10), and SecB are key players with about 13% identity to the E. coli SecB chaperone. Re- in this process (1, 2). In most Gram-negative bacteria, a substan- markably, although Rv1957 is present in the other members of tial number of newly made presecretory proteins are targeted the M. tuberculosis complex (MTBC)—i.e., Mycobacterium can- posttranslationally to the Sec translocon located at the inner etti, Mycobacterium africanum, and Mycobacterium microti—it is membrane by the export chaperone SecB (3–5). SecB is a homo- not found in the most closely related species outside the MTBC, tetrameric chaperone of 17 kDa monomers assembled as a dimer M. marinum or Mycobacterium kansasii.Arefined alignment of dimers (6) that binds nonnative substrates either co- or post- based on the known E. coli SecB structures and constructed translationally with high affinity and maintains them in a trans- mostly by increasing gaps in the two adjacent β-sheets, β-1 and location-competent state. Binding to the chaperone prevents β-4, in SecB reveals a sequence that is 19% identical and 31% unproductive folding, aggregation, and degradation of the pre- similar to that of SecB (Fig. 1A). This alignment shows that protein clients. Substrate selection by SecB is strongly influenced Rv1957 has several clusters of key residues critical for SecB by the folding rate of proteins, where rapidly folding proteins oligomerization and interaction with substrates or with its spe- would escape binding to the chaperone (4, 5). This discriminative cific partner SecA at the Sec translocon (19–25). Previous work model called “kinetic partitioning” reflects the preference of SecB for slow-folding presecretory proteins (7–9). SecB specifically interacts with the SecA motor component of the Sec translocon at Author contributions: P.G. designed research; P.B., A.-M.C., R.U., A.S., S.S., W.B., and P.G. the cytoplasmic membrane (10). Such a unique and dedicated performed research; P.B., W.B., and P.G. analyzed data; and P.B. and P.G. wrote the paper. partnership between substrate-bound SecB and SecA facilitates The authors declare no conflict of interest. efficient preprotein targeting and transfer to the Sec translocon as This article is a PNAS Direct Submission. well as the subsequent translocation through the membrane (11). 1To whom correspondence should be addressed. E-mail: [email protected]. As a chaperone with generic properties, SecB also binds aggre- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gation-sensitive cytosolic protein substrates and prevents their 1073/pnas.1101189108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1101189108 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 full complementation by Rv1957 required induction with IPTG (Isopropyl β-D-1-thiogalactopyranoside), suggesting that Rv1957 may be less efficient than SecB in assisting protein export in E. coli (Fig. 1B). However, the steady-state expression level of Rv1957 was significantly lower than the one observed for SecB (Fig. S1). This could also account, at least in part, for the less efficient complementation by Rv1957. Next, we tested whether Rv1957 could directly assist the export of two well-known SecB substrates, namely OmpA and maltose-binding protein (MBP), in the absence of SecB. Plasmids encoding Rv1957 and SecB were independently transformed into the E. coli secB null strain, and 35S-met pulse-chase experiments followed by immunopre- cipitation with anti-OmpA or anti-MBP antibodies were performed. As shown in Fig. 1C, Rv1957 was capable of restoring OmpA and MBP export, even though less efficiently than the E. coli SecB. In this case, rescue by Rv1957 was to some extent more robust for OmpA than for MBP. These results clearly show that Rv1957 from M. tuberculosis exhibits SecB-like chaperone functions in vivo. TF and DnaK chaperones assist the folding of newly synthesized cytosolic proteins in E. coli. As a consequence, an E. coli strain lacking both of these major chaperones exhibits a strong temperature-sensitive phenotype and accumulates high levels of Fig. 1. A functional SecB-like chaperone in M. tuberculosis.(A) Sequence aggregated cytosolic proteins (12, 29, 30). Overexpression of alignment of the Rv1957 gene product and the E. coli SecB chaperone using SecB partially rescues such defects, thus indicating that SecB has MUSCLE (Multiple Sequence Comparison by Log-Expectation) and further a general chaperone function in the absence of these chaperones refinement using score-assisted manual alignment with Genedoc. The sec- (12). To further investigate the chaperone function of Rv1957 in ondary structure of E. coli SecB based on the crystal structure of SecB (PDB a more stringent in vivo system, we asked whether Rv1957 could entry 1QYN) is shown. β-Sheets are shown in orange and α-helices in light blue. The colored bars above amino acids indicate residues involved in in- rescue protein folding and bacterial growth in the absence of TF teraction with SecA (red) and interactions with substrates (green). Asterisks and DnaK (12). As shown in Fig. 2A, plasmid-encoded Rv1957 indicate known mutations affecting SecB–preprotein interactions by dis- partially suppressed protein aggregation in the absence of DnaK rupting the oligomeric state of SecB. (B) Midlog phase cultures of strain and TF. Nevertheless, suppression was considerably less efficient W3110 ΔsecB containing plasmids pFr, pFr-SecB, or pFr-Rv1957 were serially than that exhibited by SecB possibly due to the fact that a frac- diluted and spotted on LB–ampicillin agar plates without or with 50 μM IPTG tion of Rv1957 copurified with the aggregates. In this case, ag- and incubated at the indicated temperatures. (C) Pulse-chase analysis gregation of Rv1957 would decrease its functional concentration. 35 showing the processing of S-labeled preMBP and proOmpA in strain However, it may be that the copurification reflects a bona fide Δ W3110 secB containing plasmids pFr, pFr-SecB, or pFr-Rv1957. Cell cultures chaperone interaction. In contrast to SecB, suppression of the were grown, labeled, and chased at 30 °C for various times following a 1-h Δ Δ incubation at 30 °C in the presence of 500 μM IPTG. temperature-sensitive growth phenotype of the tig dnaKdnaJ strain by Rv1957 was very weak and only visible after prolonged incubation (2 d) at 32 °C and required expression from the high- has shown that there are several conserved interaction sites for copy-number plasmid pSE380 (Fig. 2B). This indicates that fi SecB on SecA, i.e., at the N-terminal and at the C-terminal zinc- Rv1957 is more ef cient in assuming the role of SecB in protein binding region of SecA (20, 24, 26). Remarkably, the housekeep- export than in rescuing generic chaperones in E. coli. To firmly demonstrate that Rv1957 has a bona fide SecB-like ing SecA1 from M. tuberculosis, which does not have the cysteine fi residues involved in zinc binding at the extreme C-terminal chaperone function, we puri ed Rv1957 and tested its ability to prevent protein aggregation in vitro. We noted that purified region, retains all of the conserved neighboring residues involved fi in contact with SecB (19). Whether Rv1957 is capable of in- Rv1957 migrated in SDS/PAGE at a signi cantly higher molecular weight than predicted, i.e., 27 kDa versus 20 kDa (Fig. S2A), teracting with SecA1 remains to be determined. Finally, both in agreement with our in vivo experiments shown in Fig. S1. Gel Rv1957 and SecB are highly acidic proteins with comparable filtration analysis revealed that native Rv1957 has a molecular isoelectric points (4.49 and 4.26, respectively). Such similarities weight of about 115 kDa, corresponding to that of a homotetra- raise the possibility that Rv1957 encodes a functional SecB-like meric protein, as observed for SecB (Fig. S2 A and B). The chaperone in M. tuberculosis. homotetrameric nature of Rv1957 reinforces the fact both SecB and Rv1957 might be evolutionarily related. Rv1957 Exhibits SecB-Like Chaperone Functions both in Vivo and in Purified Rv1957 was then compared with SecB for its ability Vitro. Deletion of secB in E. coli confers a SecB-dependent cold- to prevent aggregation of denatured proOmpC, a model outer sensitive growth phenotype at temperatures below 23 °C. This is membrane protein known as a SecB substrate in E. coli (28). due to a strong export defect for many proteins (27, 28). To Rv1957 efficiently prevented proOmpC aggregation in a manner examine whether Rv1957 could replace SecB in vivo, the Rv1957 comparable to that exhibited by the native E. coli SecB (Fig. 2C). gene was cloned on a low-copy plasmid under the control of a These in vitro results are in complete agreement with the pre- lac-inducible promoter and tested for its ability to complement vious in vivo experiments and clearly demonstrate that M. tu- the secB mutant phenotype in E. coli (Fig. 1B). Remarkably, berculosis possesses a bona fide SecB-like chaperone. plasmid-encoded Rv1957 fully suppressed the cold-sensitive phenotype of the secB mutant, even at the stringent temperature Rv1957 Controls a Toxin–Antitoxin Stress-Responsive System. The of 16 °C. As expected, bacterial growth at low temperature was observation that Rv1957 is not found in all Mycobacteria sug- also restored by plasmid-encoded SecB and not by the empty gests that it may not fulfill a general SecB-like chaperone func- plasmid control. Note that SecB was sufficiently expressed tion in protein export under normal growth conditions. Remark- without inducer to allow complementation (Fig. 1B). In contrast, ably, Rv1957 is clustered together with genes that are part of

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1101189108 Bordes et al. Downloaded by guest on September 24, 2021 Fig. 2. Rv1957 chaperone functions. Rv1957 partially prevents protein aggregation and rescues bacterial growth of the Δtig ΔdnaKdnaJ triple chaperone mutant. (A) Aggregates were extracted from strain MC4100 Δtig ΔdnaKdnaJ containing plasmids pSE380-ΔNcoI vector, pSE-SecB, or pSE-Rv1957 grown at 22 °C following a 2-h expression of SecB and Rv1957 with 500 μM IPTG and a temperature switch at 33 °C for 1 h. Whole-cell extracts (wce) and their corresponding aggregates (agg) are shown. The arrow indicates the fraction of Rv1957 copurifying with the aggregates. (B) Fresh transformants of MC4100 Δtig MICROBIOLOGY ΔdnaKdnaJ containing plasmids pSE380ΔNcoI, pSE-SecB, or pSE-Rv1957 were grown to midlog phase at 22 °C. IPTG inducer (0, 0.05, or 0.1 mM) was added, and cultures were incubated for 1 h at the same temperature. Cultures were then serially diluted and spotted on LB–ampicillin agar plates for 48 h at the indicated temperature. (C) Rv1957 prevents puriﬁed proOmpC aggregation in vitro. Aggregation kinetics of denatured proOmpC (2 μM) were followed at 25 °C by measuring light scattering at 320 nm in the presence of SecB (dark gray line) or Rv1957 (light gray line) or in the absence of chaperone (black line).

The SecB4 and Rv19574 concentrations (1, 2, or 4 μM) are indicated above each graph. The percentage of aggregation was normalized to proOmpC aggregation obtained when no chaperone was added.

a functional type II TA locus of unknown function, which is re- thus avoid nonnative unbalanced HigA/HigB levels, we decided lated to the higBA family (host inhibition of growth) (Fig. 3A) to keep the native higB-higA gene organization intact. We cloned (31, 32). TA loci encode two-component systems composed of higB (toxin), higA (antitoxin), or both higB-higA on a low-copy a toxin and an antitoxin, which are essential elements of the plasmid under the control of a tightly regulated pBAD promoter prokaryotic response to environmental insults (33–35). Under to minimize background toxicity of the toxin. All three constructs specific stress conditions such as nutritional stress, the antitoxin, were expressed in an E. coli wild-type strain in the presence or in which normally forms an inactive complex with its specific toxin the absence of the Rv1957 chaperone cloned on a compatible partner, is degraded by activated proteases. The resulting free plasmid under the control of a lac-inducible promoter. As shown active toxin subsequently degrades or inhibits its cellular targets in Fig. 3B, expression of the toxin HigB alone or in the presence (RNAs or proteins, respectively), thus modulating the global of either the HigA antitoxin or the Rv1957 chaperone efficiently level of translation or DNA replication (33–35). Such a TA- blocked bacterial growth. This indicates that, under the con- dependent response significantly reduces cell growth and favors ditions tested, neither the antitoxin nor the chaperone was ca- adaptation to stress and long-term survival. In this respect, it is pable of inactivating the toxin in vivo. In sharp contrast, toxicity interesting to note that TA systems are abundant in some of the toxin was totally abolished when the antitoxin and the pathogenic bacteria including Vibrio cholerae and M. tuberculosis chaperone were jointly expressed. To further demonstrate that (31, 32, 34) and thus represent a promising class of antimicrobial inhibition of the toxin by Rv1957 is associated with its SecB-like targets (34). chaperone function, we replaced Rv1957 by the E. coli SecB in Previous work has shown that transcription of the higB-higA- the same strain expressing the toxin and the antitoxin and Rv1957 operon is induced by DNA damage (36, 37), heat shock monitored antitoxin rescue in vivo as performed in Fig. 3B.In (38), nutrient starvation (39), and hypoxia (32, 40), suggesting this case, we found that overexpression of E. coli SecB also a role in an adaptive response of M. tuberculosis to stress. Fur- resulted in toxin inactivation (Fig. 3C). However, inactivation by thermore, deletion of the antitoxin gene higA alone inhibited SecB required at least a 10-fold increase in inducer concentra- bacterial growth whereas deletion of the entire higB-higA-Rv1957 tion and was thus significantly less efficient compared with operon had no apparent effect (41). Remarkably, disruption of Rv1957. Taken together, these results strongly suggest that Rv1957 resulted in a slow growth phenotype (42). Because mo- Rv1957 controls the HigB-HigA TA system in M. tuberculosis. lecular chaperones are often key players of the stress response, To ensure that toxin inactivation by both the antitoxin HigA we hypothesized that the SecB-like chaperone Rv1957 could be and the chaperone Rv1957 was not due to the heterologous involved in the modulation of such a TA system in M. tubercu- E. coli system, different fragments of the higB-higA-Rv1957 op- losis. It was previously shown that, as a functional antitoxin, eron (i.e., higB, higB-higA,orhigB-higA-Rv1957) were cloned in overexpression of HigA, separately from HigB, in a heterologous a mycobacterial shuttle vector under control of the hsp60 pro- E. coli protease-deficient strain neutralized the HigB-dependent moter and introduced into wild-type M. marinum, which is inhibition of growth (31). To investigate such a putative TA- closely related to M. tuberculosis but lacking a chromosomal chaperone (TAC) system under more stringent conditions, and ortholog of the higB-higA-Rv1957 operon. As observed in E. coli,

Bordes et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 This suggests that this strain is able to counteract the toxic effects of the toxin by chromosomally expressed antitoxin and a SecB- like chaperone. Taken together, these in vivo results therefore demonstrate that the mycobacterial chaperone speciﬁcally controls a functional stress-responsive cytoplasmic TA system.

Rv1957 Chaperone Specifically Assists the HigA Antitoxin. Antitoxins are generally unstable protease-sensitive proteins (33, 34). This suggests that Rv1957 could act directly on the antitoxin HigA to prevent its degradation and/or assist its correct folding. To shed light on such a mechanism, we tested whether Rv1957 coex- pression affected HigA expression and/or solubilization in vivo in E. coli. As shown in Fig. 4A, the antitoxin was efficiently detected in whole-cell fractions only when the Rv1957 chaperone was coexpressed, indicating that Rv1957 protects HigA from degradation by a yet unknown protease(s) (28, 34). A direct in vivo interaction between Rv1957 and HigA was confirmed by pull- down experiments performed in E. coli (Fig. S4). In addition, we found that, in the presence of the chaperone, the antitoxin was found mainly in the soluble fraction (Fig. 4A). Conversely, in the absence of Rv1957, the small fraction of HigA antitoxin that escaped proteolysis was found in the insoluble fraction (Fig. 4A), suggesting that, in the absence of the chaperone, HigA is either rapidly degraded or aggregates. These results were further confirmed using a functional strep-tagged variant of HigA and subsequent visualization with a more sensitive anti-strep anti- body (Fig. S5). To firmly demonstrate that Rv1957 efficiently stimulates HigA folding and activity by preventing its aggregation, we next performed aggregation prevention assays in vitro using purified urea-denatured HigA protein in the presence or absence of the fi chaperone (Fig. 4B). Because of its tendency to aggregate (Fig. Fig. 3. SecB-like chaperone Rv1957 speci cally controls the HigB-HigA fi toxin–antitoxin system in M. tuberculosis.(A) On-scale schematic of the ge- S5), the HigA antitoxin was puri ed in high amounts from the nomic organization of Rv1957 (546 bp) together with higB toxin (378 bp), aggregated fraction (SI Materials and Methods). As expected higA antitoxin (450 bp), and the Rv1954a (303 bp) gene of unknown func- from our in vivo results, HigA rapidly aggregated upon dilution tion. The two known promoters P1 and P2 are also shown. (B) Suppression of from the denaturant. However, in sharp contrast to proOmpC, HigB toxicity by HigA and Rv1957 in E. coli. Strain W3110 containing the aggregation of the antitoxin was very efficiently prevented by plasmid pSE380ΔNcoI or pSE-Rv1957 was transformed with pMPMK6 vector, Rv1957 (at a 1:3 chaperone:antitoxin ratio), thus emphasizing pK6-HigB, pK6-HigA, or pK6-HigB-HigA on LB–ampicillin–kanamycin agar the specific nature of the interaction between Rv1957 and HigA plates containing 0.2% glucose at 37 °C. Double transformants were grown (Fig. 4B). Remarkably, a similar chaperone:substrate ratio was in LB–ampicillin–kanamycin 0.2% glucose to midlog phase, serially diluted, fi – – suf cient for SecB to prevent aggregation of its native substrate and spotted on LB ampicillin kanamycin agar plates with or without IPTG fi inducer as indicated. Plates were incubated at 37 °C overnight. (C) Over- proOmpA (43). In contrast to Rv1957, SecB did not signi cantly expression of the E. coli chaperone SecB can replace Rv1957 in modulating rescue HigA aggregation under the same conditions (Fig. 4B). the HigB-HigA toxin–antitoxin system. Experiments were carried out as de- These results strongly support the idea that Rv1957 activates the scribed in B except that pSE-Rv1957 was replaced by pSE-SecB. (D) Suppres- antitoxin function in vivo (Fig. 3 B and D) and further demon- sion of HigB toxicity by HigA and Rv1957 in mycobacteria. Plasmids encoding strate that the chaperone specifically controls the HigB-HigA TA the toxin HigB, HigB-HigA, or HigB-HigA plus the Rv1957 chaperone under system in M. tuberculosis. A model presenting such atypical the control of the hsp60 promoter were electroporated into the M. marinum control of a TA system by a SecB-like chaperone is depicted in M strain, and plates were incubated for 2 wk at 30 °C. Fig. 4C. Conclusion only the construct containing the entire operon could be effi- Our results demonstrate that the major human pathogen M. ciently introduced in M. marinum. Attempts to introduce con- tuberculosis encodes a functional SecB-like chaperone. This is structs containing only the toxin or the toxin–antitoxin-encoding unexpected in view of the known distribution of SecB among genes resulted in only a very few viable colonies (Fig. 3D). “ ” bacterial species (13,14). However, we have shown that the my- Plasmids from four survivors were sequenced together with cobacterial chaperone has a unique and specific function in- plasmids from four colonies of the construct containing the en- volving the fine tuning of a stress-responsive TA system from the tire operon. Although all plasmids in the transformants with the higBA family. Why does M. tuberculosis possess such a unique entire operon were intact, all plasmids obtained from the survi- and sophisticated “ménage à trois”? The presence of a distinct vors without Rv1957 carried mutations (deletions of variable size outer membrane and a remarkably large number of putative covering the toxin-encoding gene and/or the hsp60 promoter outer membrane proteins in M. tuberculosis (18) suggests that region or a mutation within the hsp60 promoter region) (Fig. this bacterium could make use of a functional export chaperone S3A). Together, these experiments show that both the chaperone under certain circumstances. The fact that Rv1957 possesses key and the antitoxin are required to mitigate the toxic effects of predicted structural elements of the E. coli SecB chaperone that HigB toxin in mycobacteria as well as in the E. coli heterologous are necessary for its interaction with preproteins and with its host. Remarkably, all three constructs could be introduced effi- SecA partner at the Sec translocon (Fig. 1A) suggests that ciently into Mycobacterium bovis bacillus Calmette–Guérin, Rv1957, as part of the TAC system, could indeed be responsive which possesses orthologs of higA, higB, and Rv1957 (Fig. S3B). to export stress. In this case, the nonspecific cytoplasmic accu-

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Fig. 4. Rv1957 chaperone speciﬁcally assists the HigA antitoxin. (A) Rv1957 prevents both the aggregation and degradation of the antitoxin HigA in vivo. Steady-state levels of HigA in E. coli strain W3110 cotransformed either with the pSE-vector and pK6-HigA or with pSE-Rv1957 and pK6-HigA, grown in the presence of 0.1% arabinose and 50 μM IPTG inducers at 37 °C. Whole-cell extracts (WCE) and soluble and insoluble fractions are shown. HigA antitoxin was detected using rabbit anti-HigA antibodies. (B) Rv1957 efﬁciently protects the antitoxin HigA from aggregation in vitro. Aggregation kinetics of urea- denatured HigA (3 μM) were followed at 37 °C by measuring light scattering at 320 nm in the presence of Rv1957 (light gray line; 1 μM) or SecB (dark gray line; 1 μM) or in the absence of chaperone (black line). (C) Proposed model for Rv1957 functions based on known SecB properties. The Rv1957 chaperone (C) co- and/or posttranslationally binds nonnative HigA antitoxin (A*), thus protecting it from aggregation and degradation by proteases and maintaining it in a form competent for subsequent interaction with the toxin HigB (T). Binding of HigA to the toxin may trigger chaperone release. The possible existence of a transient tripartite complex between the Rv1957 chaperone and HigB-HigA is not known yet. Alternatively, the chaperone could release the antitoxin before its interaction with the toxin.

mulation of outer membrane precursor proteins or the synthesis were serially diluted and spotted on LB ampicillin (50 μg/mL) agar plates at of stress-specific outer membrane proteins could result in re- 22 °C and 32 °C for 2 d, with or without IPTG inducer. cruitment of the mycobacterial SecB-like chaperone to assist their efficient targeting to the Sec translocon. Such increasing In Vivo Protection from HigB Toxin. Suppression of HigB toxicity by HigA and presecretory clients of Rv1957 would be expected to compete Rv1957 was analyzed as follows. E. coli strain W3110 was transformed with plasmids pSE380ΔNcoI or pSE-Rv1957 on LB ampicillin agar plates at 37 °C. with the antitoxin for binding to the chaperone, thus facilitating Transformants were then grown in LB ampicillin at the same temperature degradation of the free antitoxin and the subsequent activation and cotransformed with pMPMK6 vector, pK6-HigB, pK6-HigA, or pK6-HigB- of the toxin until stress conditions subside. HigA on LB ampicillin kanamycin agar plates containing 0.2% glucose at 37 °C. Fresh transformants were grown in LB ampicillin kanamycin 0.2% Materials and Methods glucose to midlog phase, serially diluted, and spotted on LB ampicillin Bacterial Strains. Genetic experiments were performed in E. coli strains kanamycin agar plates with or without IPTG inducer as indicated in the W3110, W3110 ΔsecB::CmR (27), and MC4100 Δtig::CmR ΔdnaKdnaJ::KanR figure legends. Plates were incubated at 37 °C overnight. (30) as well as in M. marinum wild-type strain M (44) or M. bovis bacillus Calmette–Guérin strain Copenhagen (45). E. coli strains BL21(λDE3) and BL21 In Vitro Protein Aggregation Assay. Unfolded proOmpC [200 μM in 20 mM Tris R (λDE3) ΔdnaKdnaJ::Kan (28) were used for protein purification with the pET (pH 7.5), 200 mM NaCl, 1 mM DTT, 20% glycerol, 8 M urea] or HigA [300 μM expression system (Novagen). in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM DTT, 20% glycerol, 8 M urea] prewarmed for 10 min at 60 °C was diluted 100-fold in reaction buffer [30 In Vivo Chaperone Assays. Complementation of SecB chaperone activity in mM Hepes (pH 7.5), 40 mM KCl, 50 mM NaCl, 7 mM magnesium acetate, and vivo at a low temperature of growth was carried out as follows. Fresh 1 mM DTT] preincubated at 25 °C or at 37 °C, respectively, with or without transformants of W3110 ΔsecB::CmR containing pFr, pFr-SecB, or pFr-Rv1957 chaperones. Immediately after addition of denatured proOmpC or HigA, were grown at 37 °C to midlog phase in LB ampicillin, serially diluted, and aggregation was monitored continuously at 320 nm for 50 min at 25 °C or at spotted on LB ampicillin (50 μg/mL) agar plates in the absence or presence of 37 °C, respectively, on a Cary Scan UV-visible spectrophotometer from Var- IPTG inducer. Plates were incubated for 1 d at 37 °C, for 2 d at 20 °C, or for ian. All solutions used in this assay were filtered through a 0.22-μm filter. 5 d at 16 °C. Complementation in the absence of DnaK and TF chaperones Details on culture conditions, pulse-chase analysis, plasmid constructs, E. R R was tested in the MC4100 Δtig::Cm ΔdnaKdnaJ::Kan temperature-sensitive coli cell fractionation, pull-down assays, Western blot analysis, and protein R strain. Midlog phase cultures of fresh transformants of MC4100 Δtig::Cm purifications are given in SI Materials and Methods. ΔdnaKdnaJ::KanR containing plasmids pSE380ΔNcoI, pSE-SecB, or pSE- μ fi Rv1957 grown at 22 °C in LB ampicillin (50 g/mL) were rst diluted to an ACKNOWLEDGMENTS. We thank Elsa Perrody for help with the aggregation ∼ OD600 of 0.4 at 22 °C; IPTG inducer (0, 0.05, or 0.1 mM) was then added, and assay; Petra Langendijk-Genevaux for advice with multiple alignments; Violette cultures were incubated for an extra hour at the same temperature. Cultures Morales for her help with protein purification; Christophe Guilhot for M. tubercu-

Bordes et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 losis genomic DNA; Mamadou Daffé for helpful discussions; and Costa Georgopou- study was supported by the Centre National de la Recherche Scientiﬁque-ATIP los, Debbie Ang, and Michael Chandler for critical reading of the manuscript. This (Action Thématique et Incitative sur Programme) Microbiology program (P.G.).

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