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.

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Figures

FIG. 1.

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.

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.

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.

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.

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.

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