Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Huet, G. Articles by Saves, I. Search for Related Content PubMed PubMed Citation Articles by Huet, G. Articles by Saves, I.

 Previous Article  |  Next Article 

Journal of Bacteriology, May 2006, p. 3412-3414, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3412-3414.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Protein Splicing of SufB Is Crucial for the Functionality of the Mycobacterium tuberculosis SUF Machinery

Gaëlle Huet, Jean-Philippe Castaing, Didier Fournier, Mamadou Daffé, and Isabelle Saves^*

Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et Biologie Structurale (UMR5089), CNRS/Université Paul Sabatier Toulouse III, 205 Route de Narbonne, F-31077 Toulouse Cedex, France

Received 29 December 2005/ Accepted 15 February 2006

Top Abstract Text References

 
The SufBCD complex is an essential component of the SUF machinery of [Fe-S] cluster biogenesis in many organisms. We show here that in Mycobacterium tuberculosis the formation of this complex is dependent on the protein splicing of SufB, suggesting that this process is a potential new target for antituberculous drugs.

Top Abstract Text References

 
The worldwide recrudescence of tuberculosis has been associated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis, its causative agent. This alarming situation has reinforced the need for the urgent development of new antituberculous drugs targeting novel and specific mycobacterial functions.

In a recent work (7), we have identified the M. tuberculosis SUF (mobilization of sulfur) machinery as the unique and essential system of [Fe-S] cluster assembly in mycobacteria. This system is required for the maturation of physiologically important metalloproteins and plays an important role in the resistance to iron limitation and oxidative stress. It is encoded by a mycobacterial operon of seven genes, Rv1460 to Rv1466 (according to the M. tuberculosis genome annotation [5]), among which Rv1461 encoding the highly conserved SufB protein (7) is interrupted by an intein coding sequence (15).

In the present study, we show the inability of the unspliced SufB protein to play its role in the SUF machinery owing to its inability to interact with some of its Suf partners. This highlights the prerequisite of protein splicing in SufB maturation and validates the SufB protein splicing as a specific molecular target for the development of novel antituberculous drugs since blocking the protein splicing process of essential proteins was proposed as a singular way to efficiently kill mycobacteria (1, 3, 6, 14).

Construction of a sufB mutant and expression of unspliced SufB. The Rv1461 open reading frame (ORF), encoding the M. tuberculosis SufB protein, was cloned in pGADT7 and pGBKT7 vectors (Clontech) for yeast two-hybrid assays (7). In these constructs, the Rv1461 gene was mutated in order to block the protein splicing process of the SufB precursor peptide: the asparagine residue at the C-terminal extremity of the intein sequence (position 611) and the adjacent cysteine (i.e., the first residue of the C-extein at position 612) were replaced by an aspartic acid and a valine, respectively. Site-directed mutagenesis was done using complementary oligonucleotide pairs (5'-TTGTAGATCGGTGCGGTGACGTCGTGCACGGCGAACCCGT-3' and 5'-ACGGGTTCGCCGTGCACGACGTCACCGCACCGATCTACAA-3'). To verify the protein splicing of the recombinant wild-type mycobacterial SufB protein when expressed in yeast and the blockage effect of the mutation, Saccharomyces cerevisiae strain AH109 (Clontech) was electrotransformed with the wild-type and mutated pGADT7 and pGBKT7 derivatives, plated, and grown at 30°C in minimal DOBA (dropout base with agar; BIO101) medium containing amino acid complement (Complete Supplement Mixture; BIO101) devoid of leucine (Leu) or tryptophan (Trp), to select transformed yeast cells. Two milliliters of a 1-day culture were harvested, yeast cells were lysed by boiling in 20 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer, and proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the wild-type and mutant SufB proteins, in fusion either with the hemagglutinin epitope tag (pGADT7 derivatives) or with the c-Myc epitope tag (pGBKT7 derivatives) was monitored by Western blotting using anti-hemagglutinin (Fig. 1) or anti-c-Myc (data not shown) antibodies (Sigma), respectively, and anti-mouse peroxidase-linked antibodies for immunodetection. We first ascertained the efficient protein splicing of the recombinant wild-type mycobacterial SufB protein since its apparent molecular weight confirmed the excision of the intein from the SufB precursor (Fig. 1). Secondly, it was confirmed that, as expected from the mechanism described by Xu and Perler (16), the mutation at the C-terminal border of the intein hinders the transesterification step of the protein splicing. Effectively, yeast cells transformed with the mutated plasmids produced a protein with an apparent molecular weight consistent with that of the full-length SufB precursor.

View larger version (24K): [in this window] [in a new window]

FIG. 1. Expression of the wild-type (WT) and unspliced (Mut) SufB protein in yeast.

Interactions between wild-type and unspliced SufB and other Suf proteins. Protein-protein interactions were studied using the MATCHMAKER GAL4 Two-Hybrid System 3 from Clontech as previously described (7). In addition to the ORF Rv1461, encoding SufB protein and its mutant, ORFs Rv1462 to Rv1466, encoding the other Suf proteins, i.e., SufDCS, a NifU-like protein, and a hypothetical Suf protein (Hyp), were cloned in the pGADT7 and pGBKT7 vectors, respectively. That allowed the expression of each mycobacterial protein fused in the C terminus to either the DNA binding domain or the transcriptional activator domain of the GAL4 transcription factor (4). The yeast strain AH109 was electrotransformed with all pairs of the pGADT7 and pGBKT7 derivatives such that one plasmid encoded the SufB protein or its mutant and the other one encoded a potential partner, allowing each protein-protein interaction to be assayed in both ways. Double transformants were selected in minimal medium devoid of Leu and Trp at 30°C. Interactions were revealed by the expression of three different reporter genes, i.e., HIS3, ADE2, and MEL1. The expression of the HIS3 and ADE2 reporter genes permits the growth of yeast in medium lacking histidine (His) and adenine (Ade), respectively. For its quantitative measurement, 3 to 6 double transformant colonies were grown overnight in the liquid medium lacking Leu and Trp only; dilutions of these cultures were plated in parallel on selective (deficient in His and Ade) and nonselective media. The percentage of the number of colonies formed (CFU) under selective versus nonselective conditions was recorded (Table 1). The MEL1 reporter encodes -galactosidase. Its expression was estimated by the blue intensity of the colonies grown in the presence of 20 µg/ml X--Gal (5-bromo-4-chloro-3-indolyl -D-galactopyranoside; Clontech) in selective medium (Table 1). In each case, these parameters were compared with those of yeast cells transformed with positive and negative control vectors. Except for yeast cells expressing the binding domain-fused Rv1466, which intrinsically acted as a transcription activator, the expression of the reporters was null in all control transformants.

View this table: [in this window] [in a new window]

TABLE 1. Interactions between wild-type or unspliced SufB protein and other Suf proteins

As described in other organisms (10, 12, 18), the M. tuberculosis SufB protein forms homotypic interactions; it interacts with SufC and SufD proteins, forming the SufBCD complex, and it interacts with SufS in an apparently less efficient way (Table 1). The unspliced SufB retains the capacity to interact with SufS and is still able to form homotypic interactions, even if weaker (the decrease was found to be significant based on the loss in blue intensity of the colonies formed when 12 different liquid cultures were plated). In contrast, the unspliced SufB is not able to interact with SufC and SufD (Table 1). This suggests that the SufB protein domains involved in its dimerization and in the interactions with SufS are correctly folded in the SufB precursor. In addition, the domains implied in the interactions with SufC and SufD are either embedded by the intein or misfolded in the presence of the intein. As expected, the NifU-like protein and the hypothetical Suf protein did not appear to interact with the unspliced SufB, as was the case with the wild-type protein. By showing that the formation of the SufBCD complex is dependent upon SufB splicing, these results clearly demonstrated the crucial role of protein splicing in the maturation of the M. tuberculosis SufB protein.

Even if the function of SufBCD complex is not yet fully understood (2, 8), several studies brought to light its major role in the SUF machinery. The high conservation of these three proteins and the phenotype analysis of sufB, sufC, and sufD mutants in several organisms (2, 9, 11, 13, 17, 18) firmly argue for the fundamental function of the three proteins forming the complex in [Fe-S] cluster biogenesis.

Since the SUF system is essential in mycobacteria, the M. tuberculosis SufBCD complex is undoubtedly a vital element. Furthermore, the mycobacterial SufBCD complex could also play a crucial function in the virulence of the human pathogen, as is the case for the plant pathogen Erwinia chrysanthemi (11). Effectively, phenotypic and functional analyses pointed to a specific role of SufBCD in the survival of Escherichia coli and E. chrysanthemi under iron starvation and oxidative stress (11, 13), i.e., the stressful conditions that are encountered by M. tuberculosis during infection.

In toto, the present work suggests that the maturation of an M. tuberculosis intein-containing protein is essential, validating the concept of the inhibition of protein splicing as a way to fight tuberculosis.

 
* Corresponding author. Mailing address: Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et Biologie Structurale (UMR5089), CNRS/Université Paul Sabatier Toulouse III, 205 Route de Narbonne, F-31077 Toulouse Cedex, France. Phone: 33 561 175 470. Fax: 33 561 175 994. E-mail: Isabelle.Saves{at}ipbs.fr.

Top Abstract Text References

 

1
1. Adam, E., and F. B. Perler. 2002. Development of a positive genetic selection system for inhibition of protein splicing using mycobacterial inteins in Escherichia coli DNA gyrase subunit A. J. Mol. Microbiol. Biotechnol. 4:479-487.[CrossRef][Medline]
2
2. Barras, F., L. Loiseau, and B. Py. 2005. How Escherichia coli and Saccharomyces cerevisiae build Fe/S proteins. Adv. Microb. Physiol. 50:41-101.[CrossRef][Medline]
3
3. Belfort, M. August 1998. Inteins as antimicrobial targets: genetic screen for intein function. U.S. Patent 5,795,731.
4
4. Chien, C. T., P. L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88:9578-9582.[Abstract/Free Full Text]
5
5. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry III, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, B. G. Barrell, et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544.[CrossRef][Medline]
6
6. Colston, M. J. 1996. The molecular basis of mycobacterial infection. Mol. Aspects Med. 17:385-454.[CrossRef]
7
7. Huet, G., M. Daffé, and I. Saves. 2005. Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen's survival. J. Bacteriol. 187:6137-6146.[Abstract/Free Full Text]
8
8. Johnson, D. C., D. R. Dean, A. D. Smith, and M. K. Johnson. 2005. Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74:247-281.[CrossRef][Medline]
9
9. Law, A. E., C. W. Mullineaux, E. M. Hirst, J. Saldanha, and R. J. Wilson. 2000. Bacterial orthologues indicate the malarial plastid gene ycf24 is essential. Protist 151:317-327.[Medline]
10
10. Loiseau, L., S. Ollagnier-de-Choudens, L. Nachin, M. Fontecave, and F. Barras. 2003. Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase. J. Biol. Chem. 278:38352-38359.[Abstract/Free Full Text]
11
11. Nachin, L., M. El Hassouni, L. Loiseau, D. Expert, and F. Barras. 2001. SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase. Mol. Microbiol. 39:960-972.[CrossRef][Medline]
12
12. Ollagnier-de-Choudens, S., D. Lascoux, L. Loiseau, F. Barras, E. Forest, and M. Fontecave. 2003. Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE. FEBS Lett. 555:263-267.[CrossRef][Medline]
13
13. Patzer, S. I., and K. Hantke. 1999. SufS is a NifS-like protein, and SufD is necessary for stability of the [2Fe-2S] FhuF protein in Escherichia coli. J. Bacteriol. 181:3307-3309.[Abstract/Free Full Text]
14
14. Paulus, H. 2003. Inteins as targets for potential antimycobacterial drugs. Front. Biosci. 8:S1157-S1165.[Medline]
15
15. Saves, I., L. A. Lewis, F. Westrelin, R. Warren, M. Daffé, and J. M. Masson. 2002. Specificities and functions of the recA and pps1 intein genes of Mycobacterium tuberculosis and application for diagnosis of tuberculosis. J. Clin. Microbiol. 40:943-950.[Abstract/Free Full Text]
16
16. Xu, M. Q., and F. B. Perler. 1996. The mechanism of protein splicing and its modulation by mutation. EMBO J. 15:5146-5153.[Medline]
17
17. Xu, X. M., and S. G. Moller. 2004. AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis. Proc. Natl. Acad. Sci. USA 101:9143-9148.[Abstract/Free Full Text]
18
18. Xu, X. M., S. Adams, N. H. Chua, and S. G. Moller. 2005. AtNAP1 represents an atypical SufB protein in Arabidopsis plastids. J. Biol. Chem. 280:6648-6654.[Abstract/Free Full Text]

---

Journal of Bacteriology, May 2006, p. 3412-3414, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3412-3414.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Huet, G. Articles by Saves, I. Search for Related Content PubMed PubMed Citation Articles by Huet, G. Articles by Saves, I.