Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function

Abstract

We present an analysis of the cellular phenotype and biochemical activity of a conserved bacterial GTPase of unknown function (YloQ and YjeQ in Bacillus subtilis and Escherichia coli respectively) using a collection of antibiotics of diverse mechanisms and chemical classes. We created a yloQ deletion strain, which exhibited a slow growth phenotype and formed chains of filamentous cells. Additionally, we constructed a conditional mutant in yloQ, where growth was dependent on inducible expression from a complementing copy of the gene. In phenotypic studies, depletion of yloQ sensitized cells to antibiotics that bind at the peptide channel or peptidyl transferase centre, providing the first chemical genetic evidence linking this GTPase to ribosome function. Additional experiments using these small-molecule probes in vitro revealed that aminoglycoside antibiotics severely affected a previously characterized ribosome-associated GTPase activity of purified, recombinant YjeQ from E. coli. None of the antibiotics tested competed with YjeQ for binding to 30 or 70 S ribosomes. A closer examination of YloQ depletion revealed that the polyribosome profiles were altered and that decreased expression of YloQ led to the accumulation of ribosomal subunits at the expense of intact 70 S ribosomes. The present study provides the first evidence showing that YloQ/YjeQ may be involved in several areas of cellular metabolism, including cell division and ribosome function.

Figure 1. Growth of yloQ deletion strains on solid media

(A) Strains were grown overnight at 30 °C in the presence of 2% (w/v) xylose (left panel) and in its absence (right panel). (B) Growth of the B. subtilis yloQ deletions after 48 h. The plates from (A) were left to grow for a total of 48 h at 30 °C in the presence of xylose (B, left panel) and in its absence (B, right panel).

Figure 2. Growth of the B. subtilis yloQ deletion strains in liquid media

(A) Cells were grown in LB medium at 30 °C. Closed symbols indicate samples grown with 2% xylose and open symbols indicate samples grown in the absence of xylose: (●, ○) strain EB6, (▼, ▽) strain EB611 and (■, □) strain EB1256. (B) Strain EB611 was grown in LB medium at 30 °C with different xylose concentrations: 2% (●), 0.2% (○), 0.063% (▼), 0.02% (▽), 0.002% (■) and 0% xylose (□).

Figure 3. Characterization of conditional B. subtilis yloQ deletion strains by microscopy

Strains EB611 (A) and EB1256 (B) were grown in the absence of xylose and visualized by light microscopy. Scale bars, 10 μm.

Figure 4. Sensitivity of B. subtilis YloQ depleted cells to various antibacterial agents

Fold sensitization in MIC is calculated with respect to the complemented deletion strain (0.2%). Bars from left to right: 0.2, 0.063, 0.02, 0.002 and 0% xylose. (A) Antibacterial agents targeting cellular functions other than protein synthesis. (B) Protein synthesis inhibitors. A-site drugs indicate agents that block the A-site by direct binding or by preventing tRNA release. Peptide channel or peptidyl transferase centre drugs bind at or near these locations on the ribosome.

Figure 5. Impact of protein synthesis inhibitors on the ribosome-stimulated GTPase activity of YjeQ in vitro

(A) Impact of aminoglycosides on the stimulation of YjeQ GTPase activity by 70 S ribosomes. The level of GTPase stimulation resulting from interaction of YjeQ (500 nM) with the 70 S ribosome (500 nM) in the absence of antibiotic [6] was set as a reference (0% inhibition) for reactions tested with three concentrations of antibiotic: 20 μM (dark-grey bar), 40 μM (white bar) and 80 μM (black bar). (B) Impact of aminoglycosides on the stimulation of YjeQ GTPase activity by 30 S ribosomes. The level of GTPase stimulation resulting from interaction of YjeQ (500 nM) with the 30 S ribosome (500 nM) in the absence of antibiotic [6] was set as a reference (0% inhibition) for reactions tested with three concentrations of antibiotic: 80 μM (dark-grey bar), 160 μM (white bar) and 320 μM (black bar).

Figure 6. In vitro ribosome-binding assays to probe the impact of protein synthesis inhibitors on YjeQ association with 70 and 30 S ribosomes

1, No antibiotic; 2, neomycin; 3, streptomycin; 4, spectinomycin; and 5, tetracycline. Supernatant (S) and pellet (P) fractions were Western blotted and probed with an anti-YjeQ antibody. (A) Association with 70 S ribosomes tested at 80 μM drugs and (B) association with 30 S ribosomes tested at 320 μM drugs.

Figure 7. Polyribosome profiles of B. subtilis YloQ depleted cells at different inducer concentrations

(A) EB611, 2% xylose; (B) EB611, 0.2% xylose; (C) EB611, 0.063% xylose; (D) EB611, 0.02% xylose; (E) EB611, 0.0063% xylose; and (F) EB611, 0.002% xylose.