Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis - PubMed (original) (raw)
Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis
Richard A Festa et al. J Bacteriol. 2007 Apr.
Abstract
In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA (proteasome accessory factor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions in pafB and pafC. In contrast to the pafA mutant, the pafB and pafC mutants were not severely sensitized to RNI, but pafB and pafC were nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein in M. tuberculosis. Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.
Figures
FIG. 1.
Complementation of a pafA transposon mutation. (A) Top, schematic of the pMV-pafABC and pMV-pafA complementation plasmids. Bottom, assay for M. tuberculosis RNI resistance in vitro, showing CFU/ml of WT M. tuberculosis containing pMV306 (vector), the pafA mutant with pMV306, and the pafA mutant with pMV-pafA or pMV-pafABC after exposure to acidified medium (pH 5.5) (gray bars) or acidified medium with 3 mM nitrite (black bars) for 6 days. White bars indicate starting CFU/ml. One experiment representative of three independent experiments, each done in triplicate, is shown. Error bars indicate standard deviations. (B) Immunoblot analysis of PafA, PafB, and PafC in total cell lysates without exposure to RNI. DlaT (dihydrolipoamide acyltransferase) was used as a loading control.
FIG. 2.
Organization of the pafABC operon in the Actinomycetales. (A) Schematic showing the organization of the pafABC operon in selected Actinomycetales. The percent identity of each protein orthologue to the M. tuberculosis protein is noted. Between pafA and pafB, Nocardia farcinica encodes a hypothetical protein and a putative transcriptional regulator and Streptomyces coelicolor encodes a peptidyl-prolyl cis-trans isomerase (fkb) and a hypothetical protein. (B) PCR analysis of cDNA made from WT M. tuberculosis RNA. The genetic organization of this region is shown above. Black and gray bars indicate the amplified regions.
FIG. 3.
pafB and pafC mutants are susceptible to RNI. (A) RNI survival assay, as described for Fig. 1A, of a pafB mutant and two pafC mutants. This experiment represents one of three independent experiments, each done in triplicate. Error bars indicate standard deviations. (B) Total cell lysates of WT, pafA, pafB, and two pafC strains were tested for the presence of PafA, PafB, PafC, and DlaT by immunoblotting. (C) Detection of PafB and PafC in WT, pafB, pafC, and _pafC_-complemented strains. Antibodies against DlaT were used for the loading control. A schematic of the pMV-pafB complementation plasmid is also shown.
FIG. 4.
PafB and PafC interact. (A) BTH interactions were quantified by β-galactosidase assay. Constructs used are denoted beneath the bars, where pafB (“B”) or pafC (“C”) was fused to the T18 or T25 domain of Cya in pUT18C or pKT25, respectively. Each assay was done in triplicate using three independent samples per assay that were then averaged. These results are representative of two independent experiments. Error bars indicate standard deviations. (B) PafB coelutes with PafC-His6 from nickel-agarose. Immunoblot analysis was performed on the soluble lysates (“S”), flowthrough (“F/T”), two washes (“W”), and the first three elutions (“E”) using polyclonal antibodies to PafA, PafB, and PafC. Paf proteins were not detected in the fourth elution (not shown).
FIG. 5.
PafA, but not PafB or PafC, is required for maintaining WT steady-state levels of M. tuberculosis proteasome substrates. (A) Immunoblot analysis of Mpa in the WT and a pafA mutant complemented with empty vector and in the pafA mutant with pMV-pafA or pMV-pafABC. (B) Immunoblot analysis of Mpa, FLAG-FabD-His6, and FLAG-PanB-His6 in WT, pafA, pafB, and pafC strains. Proteins were detected using antibodies to Mpa or the FLAG epitope. Antibodies to DlaT were used for the loading control.
Similar articles
- Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response.
Fudrini Olivencia B, Müller AU, Roschitzki B, Burger S, Weber-Ban E, Imkamp F. Fudrini Olivencia B, et al. Sci Rep. 2017 Oct 25;7(1):13987. doi: 10.1038/s41598-017-14410-z. Sci Rep. 2017. PMID: 29070902 Free PMC article. - The transcription of pafA, encoding the prokaryotic ubiquitin-like protein ligase, is regulated by PafBC.
Korman M, Elharar Y, Fishov I, Gur E. Korman M, et al. Future Microbiol. 2019 Jan;14:11-21. doi: 10.2217/fmb-2018-0278. Epub 2018 Dec 14. Future Microbiol. 2019. PMID: 30547686 - The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide.
Darwin KH, Ehrt S, Gutierrez-Ramos JC, Weich N, Nathan CF. Darwin KH, et al. Science. 2003 Dec 12;302(5652):1963-6. doi: 10.1126/science.1091176. Science. 2003. PMID: 14671303 - The Mycobacterium tuberculosis proteasome: more than just a barrel-shaped protease.
Cerda-Maira F, Darwin KH. Cerda-Maira F, et al. Microbes Infect. 2009 Dec;11(14-15):1150-5. doi: 10.1016/j.micinf.2009.08.003. Epub 2009 Aug 9. Microbes Infect. 2009. PMID: 19671445 Free PMC article. Review.
Cited by
- Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis.
Adefisayo OO, Dupuy P, Nautiyal A, Bean JM, Glickman MS. Adefisayo OO, et al. Nucleic Acids Res. 2021 Dec 16;49(22):12805-12819. doi: 10.1093/nar/gkab1169. Nucleic Acids Res. 2021. PMID: 34871411 Free PMC article. - Survival in Hostile Conditions: Pupylation and the Proteasome in Actinobacterial Stress Response Pathways.
von Rosen T, Keller LM, Weber-Ban E. von Rosen T, et al. Front Mol Biosci. 2021 Jun 7;8:685757. doi: 10.3389/fmolb.2021.685757. eCollection 2021. Front Mol Biosci. 2021. PMID: 34179091 Free PMC article. Review. - The Pup-Proteasome System Protects Mycobacteria from Antimicrobial Antifolates.
Guzzo MB, Li Q, Nguyen HV, Boom WH, Nguyen L. Guzzo MB, et al. Antimicrob Agents Chemother. 2021 Mar 18;65(4):e01967-20. doi: 10.1128/AAC.01967-20. Print 2021 Mar 18. Antimicrob Agents Chemother. 2021. PMID: 33468462 Free PMC article. - Potential Role of Proteasome Accessory Factor-C in Resistance against Second Line Drugs in Mycobacteria.
Narain A, Dubey RK, Verma AK, Srivastava A, Kant S. Narain A, et al. J Lab Physicians. 2020 Dec;12(4):250-262. doi: 10.1055/s-0040-1722552. Epub 2020 Dec 30. J Lab Physicians. 2020. PMID: 33390674 Free PMC article. - Population Genomics of Mycobacterium leprae Reveals a New Genotype in Madagascar and the Comoros.
Avanzi C, Lécorché E, Rakotomalala FA, Benjak A, Rapelanoro Rabenja F, Ramarozatovo LS, Cauchoix B, Rakoto-Andrianarivelo M, Tió-Coma M, Leal-Calvo T, Busso P, Boy-Röttger S, Chauffour A, Rasamoelina T, Andrianarison A, Sendrasoa F, Spencer JS, Singh P, Dashatwar DR, Narang R, Berland JL, Jarlier V, Salgado CG, Moraes MO, Geluk A, Randrianantoandro A, Cambau E, Cole ST. Avanzi C, et al. Front Microbiol. 2020 May 11;11:711. doi: 10.3389/fmicb.2020.00711. eCollection 2020. Front Microbiol. 2020. PMID: 32477280 Free PMC article.
References
- Baumeister, W., J. Walz, F. Zühl, and E. Seemüller. 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92:367-380. - PubMed
- Berks, B. C., T. Palmer, and F. Sargent. 2005. Protein targeting by the bacterial twin-arginine translocation (Tat) pathway. Curr. Opin. Microbiol. 8:174-181. - PubMed
- Chong, S., and G. A. Garcia. 1994. A versatile and general prokaryotic expression vector, pLACT7. BioTechniques 17:686, 688, 690-691. - PubMed
- Chu-Ping, M., J. H. Vu, R. J. Proske, C. A. Slaughter, and G. N. DeMartino. 1994. Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20 S proteasome. J. Biol. Chem. 269:3539-3547. - PubMed
- Cole, S. T., K. Eiglmeier, J. Parkhill, K. D. James, N. R. Thomson, P. R. Wheeler, N. Honoré, T. Garnier, C. Churcher, D. Harris, K. Mungall, D. Basham, D. Brown, T. Chillingworth, R. Conner, R. M. Davies, K. Devlin, K. Duthoy, T. Feltwell, A. Fraser, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, C. Lacroix, J. Mclean, S. Moule, L. Murphy, K. Oliver, M. A. Quail, M.-A. Rajandream, K. M. Rutherford, S. Rutter, K. Seeger, S. Simon, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, K. Taylor, S. Whitehead, J. R. Woodward, and B. G. Barrell. 2001. Massive gene decay in the leprosy bacillus. Science 409:1007-1011. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases