A nutrient-dependent division antagonist is regulated post-translationally by the Clp proteases in Bacillus subtilis

Abstract

Background: Changes in nutrient availability have dramatic and well-defined impacts on both transcription and translation in bacterial cells. At the same time, the role of post-translational control in adaptation to nutrient-poor environments is poorly understood. Previous studies demonstrate the ability of the glucosyltransferase UgtP to influence cell size in response to nutrient availability. Under nutrient-rich medium, interactions with its substrate UDP-glucose promote interactions between UgtP and the tubulin-like cell division protein FtsZ in Bacillus subtilis, inhibiting maturation of the cytokinetic ring and increasing cell size. In nutrient-poor medium, reductions in UDP-glucose availability favor UgtP oligomerization, sequestering it from FtsZ and allowing division to occur at a smaller cell mass.

Results: Intriguingly, in nutrient-poor conditions UgtP levels are reduced ~ 3-fold independent of UDP-glucose. B. subtilis cells cultured under different nutrient conditions indicate that UgtP accumulation is controlled through a nutrient-dependent post-translational mechanism dependent on the Clp proteases. Notably, all three B. subtilis Clp chaperones appeared able to target UgtP for degradation during growth in nutrient-poor conditions.

Conclusions: Together these findings highlight conditional proteolysis as a mechanism for bacterial adaptation to a rapidly changing nutritional landscape.

Keywords: Cell cycle; Cell division; Cell size; ClpP; UDP-glucose; UgtP.

Fig. 1

UgtP accumulates only in nutrient-rich conditions. Semi-quantitative immunoblot measuring native UgtP-His levels in a range of growth conditions. PL2265 (P_ugtP-ugtP-his) was cultured in LB (τ = 22′), minimal glucose (τ = 39′), minimal glycerol (τ = 58′), or minimal sorbitol (τ = 78′). FtsZ is shown as a loading control. Protein levels in LB are set as the reference in the relative expression below (n = 3, error = SD)

Fig. 2

UgtP accumulation is subject to post-translational control. lacZ encoding β-galactosidase fused to either (a) 700 bp upstream of the ugtP start site (P_ugtP) to generate a transcriptional fusion (PL1967) or (b) an additional 90 bases downstream of the ugtP start codon to generate a translational fusion (PL2034). Both strains were cultured in a range of nutrient conditions to generate four different growth rates. Bars indicate mean ± SD of specific β-galactosidase activity (n = 3) and an unpaired T-test was used to access significance (***p < 0.001, ** p < 0.01, *p < 0.05, ns p > 0.05). (c) qRT-PCR measurements of ugtP expression levels. Expression in three defined media is normalized to expression in LB. Values are mean ± SD (n = 3). An unpaired T-test was applied to the ∆Ct values to access significance (*p < 0.05, ns p > 0.05)

Fig. 3

UgtP is subject to nutrient-dependent, post-translational regulation by the Clp proteases. Semi-quantitative immunoblot of UgtP-His expressed from a xylose-inducible promoter (P_xyl-ugtP-his) in the absence of (a) 5 proteases, YluC, CptA, ClpP, YvjB, and Lon (PL2022, PL2028, PL2102, PL2032, and PL2033, BH10 as WT) or (b) single/combinatorial deletions of the Clp chaperones (ClpC, ClpE, and/or ClpX) (BH127, BH128, BH130, PL2102, BH135, BH136, BH137, BH138). Cells were cultured in either LB + 0.5% xylose or minimal sorbitol + 0.5% xylose, and immunoblotted against His and FtsZ. Samples shown in (a) and also for (b) were run on the same blot, but cropped during image processing. (c) Fold change in UgtP-His levels for P_xyl-ugtP-his (BH10) and P_xyl-ugtP-his + ∆clpP (BH129) after adding spectinomycin to inhibit translation. Cells were cultured in minimal sorbitol + 0.5% xylose, sampled every 30 min after spectinomycin addition, and subjected to immunoblotting against His antibody. Values are mean ± SD (n = 3). A two-way ANOVA was used to assess differences in UgtP-His levels ± clpP over time (P < 0.0001) and a Bonferroni multiple comparisons test (shown) was used to determine significance between the two strains at specific time intervals (***p < 0.001, **p < 0.01). (d) qRT-PCR measurements of clpC, clpE, clpX, and clpP expression in minimal sorbitol versus LB. Values are mean ± SD (n = 4). An unpaired T-test was applied to the ∆Ct values to access significance (***p < 0.001, **p < 0.01, ns p > 0.05)

Fig. 4

Mutations in UgtP’s putative hexose-binding site enhance susceptibility to proteolysis in vivo. (a) A schematic representation of YFP-UgtP localization and cell size for wild type, uracil-binding (URA-), hexose-binding (HEX-), and self-oligomerization (OLI-) mutants in either LB or minimal sorbitol (Additional file 3:Figure S3 & [20]). UgtP-His variants BH736 (WT), BH742 (URA-), BH752 (HEX-), BH740 (OLI-) were cultured in either (b) LB + 0.5% xylose or (c) minimal sorbitol + 0.5% xylose and subjected to semi-quantitative immunoblotting against His and FtsZ (loading control) antibodies. Relative expression compared to WT (BH736) UgtP-His is shown (n = 3, error = SD)

Fig. 5

ClpXP targets UgtP for proteolysis in vitro independent of UDP-glucose. (a) Immunoblot of purified His-UgtP after incubation with purified ClpXP. Reactions consisting of 3 μM ClpX, 6 μM ClpP, 3 μM His-UgtP, and 5 mM ATP were incubated for 45 min at room temperature. ClpXP substrate controls are shown in Additional file 6: Figure S6. (b) In vitro ClpXP cleavage assay ± UDP-glucose or glucose-6P. The assay used 3 μM ClpX, 6 μM ClpP, 3 μM His-UgtP, 5 mM ATP, and 2 mM of either UDP-glucose or glucose-6P. α-His was used to visualize His-UgtP levels by immunoblot. Each cognate set was used to gauge relative degradation (%) as shown below (n = 3, error = SD)

Fig. 6

UgtP levels do not significantly affect cell length or diglucosyl-diacylglycerol levels in carbon-poor media. (a) Cell length distributions of strains BH736 (∆ugtP; P_xyl-ugtP-his) and BH12 (P_ugtP-ugtP-his; P_xyl-ugtP-his) cultured in minimal sorbitol ±0.5% xylose, determined by measuring the distance between the mid-points of adjacent cell wall septa (n = 600, error = SD). (b) Relative diglucosyl-diacylglycerol concentrations of lipid extracts from strains PL522 (WT), PL2102 (∆clpP), BH10 (P_xyl-ugtP-his), BH129 (P_xyl-ugtP-his; ∆clpP) cultured in minimal sorbitol, determined by thin layer chromatography and subsequent densitometric analysis of separated lipids (n = 3, error = SD)