and its receptor, as well as with two proteins ofEscherichia coli, FtsY and Ffh, which have been proposed to be a part of a signal recognition particle-like ...
Vol. 174, No. 18
JOURNAL OF BACTERIOLOGY, Sept. 1992, p. 5978-5981
0021-9193/92/185978-04$02.00/0 Copyright © 1992, American Society for Microbiology
NOTES Role of pilA, an Essential Regulatory Gene of Neisseria gonorrhoeae, in the Stress Response MUHAMED-KHEIR TAHA,* MIREILLE LARRIBE, BRUNO DUPUY, DARIO GIORGINI, AND CHRISTIAN MARCHAL
Unite des Neissena, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedexc 15, France Received 16 April 1992/Accepted 29 June 1992
Sequence analysis has shown that PilA, a transcriptional regulator of pilin gene expression in Neisseria gonorrhoeae, has extensive homology with the 54-kDa protein of the signal recognition particle of eukaryotes and its receptor, as well as with two proteins of Escherichia coli, FtsY and Ffh, which have been proposed to be a part of a signal recognition particle-like apparatus. We tested the putative role of PilA in protein export in N. gonorrhoeae and did not find any effect. However, we did observe induction of a heat shock response and a previously described slow-growth phenotype when PilA function was impaired. We also examined the interference of pilA expression in E. coli with the function of the products of ftsY and il and observed an accumulation of pre-13-lactamase. We argue against a direct role for PiIA in protein export in gonococci and propose instead that PiLA is involved in the modulation of cell growth rate in response to different environmental conditions.
analysis has shown that two proteins of E. coli (FtsY and Ffh) share extensive homology with SRP54 and its receptor (1, 19). The ftsY and ffh genes are essential in E. coli (3, 8). Another essential gene in E. coli, ffs, encodes a 4.5S RNA which was proposed to be the homolog of the 7S RNA in the SRP, and it has been shown to coimmunoprecipitate with Ffh (15, 18). We have previously isolated apilA +/pilAa heterodiploid in N. gonorrhoeae MS11-A, which is wild type for the pilE, pitA, and pilB genes and in whichpila encodes a truncated PilA which has a negative transdominant effect on the wild-type allele (23). To determine the role, if any, ofpiUA in protein localization, we used this heterodiploid to study the export of the opacity (Opa) protein (an outer membrane gonococcal protein). We found that Opa was normally localized in the membrane fraction (data not shown). We studied next the maturation of Opa by pulse-chase labelling with [35Slmethionine and immunoprecipitation with anti-Opa antiserum. Gonococci were grown to the ear%y exponential phase of growth in defined medium (12), and [3 S]methionine (20 ,Ci/ml; 1,000 Ci/mmol; Amersham) was added for 90 s. Samples were immunoprecipitated after a chase period of 0, 2, or 6 min with cold methionine (6). Opa protein maturation
Pili are a major virulence factor of Neisseria gonorrhoeae, an exclusively human pathogen. Pili are composed primarily of the subunit, pilin, which is encoded by the chromosomal gene pilE. Expression of pilE is controlled transcriptionally by the products of two linked chromosomal genes, pilA and pilB, that act as a two-component regulatory system (21). BothpiUA and pilB are conserved in the genus Neisseria (22). PilA is a transcriptional regulator which binds to the pilE promoter (20), and it has a potential DNA-binding motif near its N terminus (23). Moreover, PilA has sequence similarities in its N-terminal and central domains with other bacterial transcriptional regulators (21, 23). pilU is an essential gene, as its inactivation by transposon insertion is lethal, although piUA+/piLU::mTn3 heterodiploids are viable (21). Heterodiploids corresponding to one particular transposon insertion, called pitA 1pilAa, exhibited reduced pilE transcription and grew slowly (21, 23). The C-terminal region of PilA has extensive homology with the products of two Escherichia coli genes, ftsYandffh, as well as with SRP54, a component of the eukaryotic signal recognition particle (SRP), and the a subunit of its receptor. In particular, these proteins have the same putative GTPbinding site (21). This sequence homology raises the possibility that PilA participates in protein export in gonococci as a part of an SRP-like apparatus. In this study, we attempted to determine whether PiLA has a role in protein export. Export of newly synthesized protein in E. coli involves a soluble chaperone, such as SecB, and a membrane-bound translocase (SecA, SecY/E) (24). This mechanism is quite different from translocation across the endoplasmic reticulum membrane in most eukaryotic cells, where translocation occurs cotranslationally and requires SRP, a cytoplasmic 11S particle containing six different proteins and a 7S RNA (4; see reference 16 for a review). Recently, sequence *
O'
Pre -Opa
A 2'
6'
0'
..
(N)a 0.
OT
B 2'
6'
Pre-OpaOpa
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel autoradiography after immunoprecipitation of pulse-chase [3 S]methioninelabelled proteins with anti-Opa antiserum in wild-type strain MS11-A (A) and a pil4+/pilAa heterodiploid (B). Samples were immunoprecipitated after 0, 2, or 6 min of chase.
Corresponding author. 5978
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VOL. 174, 1992
o'
A
B
1'
1'
5'
0'
-mp
4M~4 NU
C O'
1'
_
._*
membrane protein, since the serum used for immunoprecipitation cross-reacted with a number of gonococcal membrane proteins (data not shown). FtsY and Ffh have been proposed to function in an SRP-like apparatus in E. coli (15, 18). Overproduction of Ffh protein or a transdominant mutation of the ifs gene expressed by a mutant allele delays maturation of pre-flactamase in E. coli but has no effect on the maturation of other exported proteins (15, 18). The homology of PilA with these FtsY and Ffh proteins suggested that the synthesis of PilA in this bacterium would interfere with the stoichiometry of this putative apparatus; i.e., piUA would act as a transdominant allele of ftsY and ffh. Therefore, we studied the effect of piUA expression on the maturation of pre-p-lactamase encoded on pUC18 in E. coli. E. coli CAG1139 (9) harboring pUC18 or pNG60, a pUC18-derived recombinant plasmid expressing piUA (23), was grown in minimal M9 medium (13) supplemented with suitable amino acids. The two plasmids have an intact 1-lactamase gene. In the early exponential phase of growth, bacteria were radiolabelled with [ S]methionine (20 iCi/ml; 1,000 Ci/mmol; Amersham) for 45 s and chased with cold methionine. After 0, 1, or 5 min of chase, samples were immunoprecipitated with anti-,B-lactamase antibodies. An accumulation of pre-3-lactamase was observed in E. coli expressingpilA compared with the same strain harboring the vector only (compare Fig. 2A with Fig. 2B). However, we did not observe any accumulation of alkaline phosphatase (PhoA) precursor, another periplasmic protein, by the same type of analysis with anti-PhoA antiserum in E. coli PAP152 (21) (Fig. 2C and D). piUA is an essential gene, and the pitA lpilAa gonococcal heterodiploid exhibits a pleiotropic phenotype (21, 23). For
5'
--
_.4 Pre-B-lacarxnase .4
B-lactamase
D 5'
_
O'
1
_
_
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P
5979
-_ PhoA
FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel autoradiography after immunoprecipitation of pulse-chase [3 S]methioninelabelled proteins with anti-p-lactamase (A and B) or anti-PhoA (C and D) antisera. Samples of E. coli harboring pUC18 (A and C) or pNG60 (B and D) were immunoprecipitated after 0, 1, or 5 min of chase.
in the heterodiploid was observed to be the same as in the control (Fig. 1). A band which migrated just above the pre-Opa was observed during the chase in both wild-type and mutant cells. This protein is most likely the result of the degradation and coimmunoprecipitation of an unknown A
B
kDa
4 97 _
69w ,4
4
-
46 _
30
_
FIG. 3. Autoradiography of two-dimensional polyacrylamide gel of pulse-chase [35S]methionine-labelled proteins from wild-type strain MS11-A (A) and a pitA /pilAa heterodiploid (B). Molecular weight markers are indicated. Horizontal arrows indicate pH gradient change from basic to acidic. Proteins which are made at a higher level in each case are underlined. The 68-kDa protein which is overproduced in the heterodiploid is indicated by an arrowhead.
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A
1
2
1
2
_116 _
97w
.._66
FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel autoradiography of pulse-chase [35S]methionine-labelled proteins from apiLA / piAa heterodiploid (lanes 1) and wild-type strain MS11-A (lanes 2). (A) Samples of labelled cells were loaded directly onto the gel; (B) samples were immunoprecipitated with anti-GroEL antiserum before being loaded on the gel. Positions of molecular weight markers are indicated in thousands.
example, many proteins are made at lower levels and others made at higher levels in the mutant, as is shown by two-dimensional gel electrophoresis (Fig. 3) (14). One protein whose expression is particularly affected migrates with an apparent molecular mass of 68 kDa and immunoprecipitates with antibodies directed against the GroEL protein of E. coli (Fig. 4). The 68-kDa protein was also produced in are
large amounts after wild-type strain MS11-A was grown at 42°C or under anaerobic conditions (data not shown), indicating a stress response. This protein is most likely the same 68-kDa protein that has been reported previously by others to be expressed in response to thermal stress in gonococci (5, 11, 25). These data indicate that PilA is implicated directly or indirectly in stress response induction in gonococci. The heat shock response in E. coli was also induced under the conditions of overproduction of Ffh protein, depletion of the 4.5S RNA, or a transdominant mutation of theffs gene expressed by a mutant allele. Indeed, it preceded the accumulation of the pre-p-lactamase (2, 15, 18). The possible accumulation of misfolded proteins was proposed to induce the heat shock response in these conditions. Our results show that pre-f3-lactamase also accumulates when pilA is expressed in E. coli. A heat shock response was also observed in apil4+IpilAa heterodiploid of N. gonorrhoeae, but no effect on Opa protein localization or maturation was observed. Moreover, others have shown that no effect was observed on pilus assembly in a pilA mutant of N. gonorrhoeae whenpilE was expressed under the control of the P, promoter, indicating that there is no posttranscriptional control of PilA on pilE expression (10). Gonococci are continually exposed to stress conditions, including phagocytosis and a change to anaerobiosis, inside the host. Gonococci have reportedly been isolated from urogenital infections in association with strict anaerobes (7). Moreover, polymorphonuclear neutrophil killing of gonococci is mediated mainly by oxygen-independent mechanisms (17). We propose that PilA is a pleiotropic regulator that, in conjunction with PilB, controls cell growth under different environmental conditions present during infection. A stress response would enable the gonococcus to survive in these hostile situations. The sequence homology between Ffh and SRP54 is greatest in their C-terminal methionine-rich regions. This domain
has been proposed to bind the signal sequence as it emerges from the ribosome. If Ffh were the equivalent of the SRP54, then PilA could be the equivalent of the receptor for SRP, a rough endoplasmic reticulum-associated protein. However, PilA is likely to be a cytoplasmic protein, and it does not coimmunoprecipitate with the gonococcal homolog of the 4.5S RNA (unpublished data). All of these data, taken together, cast doubt on the presence of an SRP-like complex in gonococci, or at least the membership of PilA in such an apparatus, as no direct evidence is available to support this idea. Alternatively, PilA may regulate the transcription of some other essential gene(s) in a GTP-binding-dependent manner. In the latter case, the homology of PilA with FtsY, Ffh, SRP54, and the SRP receptor would reflect the use of GTP for different physiological purposes. We propose that PilA is important in transmitting development-specific signals during cell growth. We are grateful to Thomas Meyer for anti-Opa antiserum, to Julian Davies for anti-1-lactamase antiserum, to Cecile Wandersman for anti-PhoA antiserum, to Philippe Mazodier for anti-GroEL antiserum, and to Veronique Ribes for the 4.5S RNA-specific oligonucleotide. We are also grateful to Hilde De Reuse, Sims Kochi, and Anthony Pugsley for helpful discussions and critical reading of the manuscript and to Jean-Yves Riou for generous support. This work was supported by the INSERM (CRE 893009) and the Institut Pasteur. REFERENCES 1. Bernstein, H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, and P. Walter. 1989. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature (London) 340:482-486. 2. Bourgaize, D. B., T. A. Phillips, R. A. VanBogelen, P. G. Jones, F. C. Neidardt, and M. J. Fournier. 1990. Loss of 4.5S RNA induces the heat shock response and lambda proph4ge in Escherichia coli. J. Bacteriol. 172:1151-1154. 3. Bystrom, A. S., K. J. Hjalmarsson, P. M. Wikstrom, and G. R. Bjork. 1983. The nucleotide sequence of Escherichia coli operon containing genes for the tRNA (m1G) methyltransferase, the ribosomal protein S16 and L19 and a 21-K polypeptide. EMBO J. 2:899-905. 4. Connolly, T., and R. Gilmore. 1989. The signal recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide. Cell 57:599-610. 5. Demarco de Hormaeche, R., A. Mehlert, D. B. Young, and C. E. Hormaeche. 1991. Antigenic homology between the 65kDa heat shock protein of Mycobacterium tuberculosis, GroEL of E. coli, and proteins of Neisseria gonorrhoeae expressed during infection, p. 199-203. In M. Achtman, P. Kohl, C. Marchal, G. Morelli, and B. Thiesen (ed.), Neisseria 1990. Walter de Gruyter, Berlin. 6. Dupuy, B., M. K. Taha, A. P. Pugsley, and C. Marchal. 1991. Neisseria gonorrhoeae prepilin export studied in Escherichia coli. J. Bacteriol. 173:7589-7598. 7. Fontaine, E. A., D. Taylor-Robinson, N. F. Hanna, and E. D. Coufalik. 1982. Anaerobes in men with urethritis. Br. J. Vener. Dis. 58:321-326. 8. Gill, D. R., and G. P. Salmond. 1990. The identification of the Escherichia coli ftsY gene product: an unusual protein. Mol. Microbiol. 4:575-583. 9. Grossman, A. D., R. R. Burgess, W. Walter, and C. A. Gross. 1983. Mutations in the lon gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase. Cell 32:151-159. 10. Haas, R., D. Facius, C. P. Gibbs, T. Rudel, J. P. M. Van Putten, and T. F. Meyer. 1991. Pilin variation in Neisseria gonorrhoeae and modulation of cellular adherence, p. 585-590. In M. Achtman, P. Kohl, C. Marchal, G. Morelli, and B. Thiesen (ed.),
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