ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria

Abstract

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Keywords: DNA replication; cell cycle; mycobacteria; protein degradation.

FIG 1

ClpX is essential for cell cycle completion. (A) Quantitative RT-PCR of clpX transcript from cultures of M. smegmatis carrying pTet_ON-clpX in the presence or absence of inducer (+aTc and −aTc) at 24 h. Data are fold change relative to sigA (means and standard errors of the means [SEM]; n = 5 per condition). **, P = 0.0079 by Mann-Whitney. (B) Growth curves of M. smegmatis carrying pTet_ON-clpX in the presence or absence of inducer (+aTc and −aTc). Data are means and SEM; n = 3 per condition. (C) Representative images of wild-type cells (i), isotype control cells (ii and iii), and cells depleted of clpX (iv). Bar, 5 μm. (D) Counts of division events in M. smegmatis carrying pTet_ON-clpX in the presence or absence of inducer. Data show the ultimate fate of each cell, either a division event or branched cell. (E) Quantification of cell lengths for M. smegmatis carrying pTet_ON-clpX in the presence or absence of inducer (+aTc and −aTc). Length of cells were measured using ImageJ. All cells are plotted (means and SEM). Significance was determined by a Mann-Whitney test. ****, P < 0.0001.

FIG 2

Identification of interactors of ClpX. (A) Volcano plot of total spectral counts as identified by MS/MS. Adjusted P values (determined by a G test with multiple-comparison correction) and fold changes are shown. Representative nucleotide-related proteins are indicated by blue highlighting. Representative cell wall synthesis or cell division proteins are indicated by green highlighting (proteins are listed in Table S2). (B) KEGG-based enrichment analysis with significance cutoffs of a fold change of ≥3.5 and an FDR of <0.05. Blue indicates KEGG pathways related to nucleotide binding. Green indicates KEGG pathways related to cell wall synthesis or cell division.

FIG 3

SSB activates ClpX and ClpXP1P2 via its C-terminal sequence. (A) SDS-PAGE gel of proteins retained on the filter or recovered in the eluate (flowthrough) in a filtration assay. (B) Rate of ATP hydrolysis for full-length proteins. Data are percent hydrolysis with ClpX alone. (C) Rate of ATP hydrolysis for full-length proteins. Data are percent hydrolysis with ClpC1 alone. (D) Degradation extent for SSB or GFP-SsrA at time zero or after 5 h of incubation with ClpXP1P2 at 37°C. (E) Rate of GFP-SsrA degradation. Data are percent degradation with ClpXP1P2 (left) or ClpC1P1P2 (right). (F) Rate of ATP hydrolysis for full-length SSB and short peptides. Data are percent hydrolysis with ClpX alone. Peptide sequences are listed beneath the graph. Data are representative of three biological replicates. P values are based on three technical replicates and are internally controlled. *, P < 0.05; **, P < 0.01; ***, P <0 .001; ns, not significant.

FIG 4

ClpX is required for proper DNA replication. (A) Representative images of DnaN-eGFP in cells of M. smegmatis carrying pTet_ON-clpX or pAmi_ON-ftsZ with or without inducer at 15 h after removal. Bar, 5 μm. Arrowheads indicate dead cells. Arrows indicate DnaN-eGFP foci. (B) Schematic of qPCR probes used in this assay. (C) qPCR data for the indicated cell types. Fold changes are shown relative to dnaA and inducer-containing controls. P values were determined by a t test. *, P < 0.05; **, P < 0.01.