Cell morphology maintenance in Bacillus subtilis through balanced peptidoglycan synthesis and hydrolysis

Abstract

The peptidoglycan layer is responsible for maintaining bacterial cell shape and permitting cell division. Cell wall growth is facilitated by peptidoglycan synthases and hydrolases and is potentially modulated by components of the central carbon metabolism. In Bacillus subtilis, UgtP synthesises the glucolipid precursor for lipoteichoic acid and has been suggested to function as a metabolic sensor governing cell size. Here we show that ugtP mutant cells have increased levels of cell wall precursors and changes in their peptidoglycan that suggest elevated DL-endopeptidase activity. The additional deletion of lytE, encoding a DL-endopeptidase important for cell elongation, in the ugtP mutant background produced cells with severe shape defects. Interestingly, the ugtP lytE mutant recovered normal rod-shape by acquiring mutations that decreased the expression of the peptidoglycan synthase PBP1. Together our results suggest that cells lacking ugtP must re-adjust the balance between peptidoglycan synthesis and hydrolysis to maintain proper cell morphology.

Figure 1

The ugtP mutant has altered cell surface and PG composition. (A) Teichoic acid glycolipids pathway of the cell wall in B. subtilis. DAG, diacylglycerol. (B) Volcano plot for data generated by metabolomics analysis for BSB1 ΔugtP and BSB1 ΔpgcA showing increased levels of several lipid II precursors (C) Transmission electron microscopy (TEM) images showing a rougher cell surface structure in cells lacking UgtP compared to wild type cells. Cyt cytoplasm, CM cytoplasmic membrane, CW cell wall. Arrows indicate the rough cell surface. (D) HPLC analysis of muropeptides isolated from BSB1 ΔugtP and BSB1 ΔpgcA mutants showed increased levels of Di (muropeptide 5) and Tri-Ala-mDap(NH₂)₂ (muropeptide 8) muropeptides compared to BSB1 suggesting increased endopeptidase activity. (E) Diagram representing the relative quantification of the two muropeptides in BSB1, BSB1 ΔugtP and BSB1 ΔpgcA. *p ≤ 0.05. (F) The scheme represents the structure of the two muropeptides and the cleavage site of the LytE and CwlO endopeptidases.

Figure 2

The ugtP mutant requires LytE to maintain rod-shape. (A) Spot plate assay showing the lethality of the ugtP lytE double knockouts when cells were grown on PAB media. The ectopic expression of UgtP or supplementing the plates with magnesium sulphate allowed BSB1 ΔugtP ΔlytE to grow on PAB. (B) Fluorescence microscopy using brightfield and membrane stain to study the morphology of the mutants. BSB1 ΔugtP ΔlytE showed severe shape defects during exponential phase when grown in LB media. The complementation of ugtP recovered this phenotype. I− without IPTG, I+ with IPTG. Scale bar 3 µm. (C) TEM analysis of the mutants revealed the severity of the ugtP lytE morphology defects (arrows) where cells showed loss of rod-shape and membrane integrity. Arrows indicate ruptures in the cell wall (D) HPLC analysis of muropeptides isolated from BSB1 ΔugtP ΔlytE and BSB1 ΔugtP ΔcwlO showed alterations in the amidation patterns compared to BSB1. These alterations were mainly detected in the muropeptides Tri-(NH₂) (muropeptide 3), TetraTri-(NH₂) (muropeptide 15) and TetraTri-(NH₂)₂ (muropeptide 21). (E) Diagram representing the difference in the level of selected muropeptides in mutants compared to BSB1. 100% indicates the level of a muropeptide in BSB1. *p ≤ 0.05.

Figure 3

The overexpression of CwlO improved the viability and rod morphology of the ugtP lytE mutant. (A) Spot plate assay for cells with an additional cwlO gene under the control of a xylose promoter. BSB1 ΔugtP ΔlytE cells overexpressing CwlO (+xylose) were viable on PAB. (B) Fluorescence microscopy for BSB1 ΔugtP ΔlytE cells overexpressing CwlO recovered partially the rod-shape morphology however cells were still bent. X− without xylose, X+ with xylose. Scale bar 3 µm.

Figure 4

The deletion of ugtP in cells lacking MreB homologues. (A) Spot plate assay for single or double mutants lacking UgtP and/or MreB homologues. Only the ugtP mreB double mutant showed lethality when cells were grown on nutrient agar supplemented with 0.2% glucose. Media supplemented with magnesium recovered the phenotype of the mutants. (B) Fluorescence microscopy of mutants grown in LB with 20 mM magnesium sulphate showed a loss of the rod-shape and severe shape defects only for BSB1 ΔugtP ΔmreB cells. Scale bar 3 µm. (C) The two proposed distinct pathways for the lateral PG hydrolysis during cell elongation in wild type and BSB1 ΔugtP cells.

Figure 5

Changes in the PBP1 activity levels allowed growth of the ugtP lytE mutant. (A) The scheme represents the ponA operon. P1 and P2 represent the 2 promoters in the operon while St represents the stop codon. WT, the codon composition in wild type cells. S1 and S3 represent the two isolated suppressors with alterations in the recU sequence. S2 represents the third suppressor with a G141A point mutation in the PBP1 encoding gene ponA. (B) Bocillin binding assay shows smaller band corresponding to PBP1 for the suppressors S1 and S3 compared to wild type. Bands corresponding to other PBPs were comparable for the three suppressors and wild type cells. (C) Western blot against cell lysates from suppressor mutants and wild type cells using PBP1 polyclonal antibody and PBP3 polyclonal antibody as a loading control. The S1 and S3 samples showed smaller bands corresponding to the levels of PBP1 compared to the S2 mutant and WT cells suggesting lower levels of PBP1 in the cells. (D) Spot plate assay for mutants with deletions in the ugtP, lytE and ponA genes, and with ectopic expression of PBP1, PBP1 E115A or PBP1 G141A. UgtP lytE ponA triple mutants expressing low levels of PBP1 or PBP1 G141A, showed better phenotype compared to both BSB1 ΔugtP ΔlytE and BSB1 ΔugtP ΔlytE ΔponA. (E) Fluorescence microscopy for the aforementioned mutants showed that BSB1 ΔugtP ΔlytE ΔponA recovered the rod-shape, however, cells were thin and bent. BSB1 ΔugtP ΔlytE ΔponA expressing low levels of PBP1 or PBP1 G141A, showed amelioration in the cell morphology where mutants looked mostly like the ugtP single mutant previously described as short cells (Supplementary Fig. S2). Scale bar 3 µm.

Figure 6

Schematic representation of the cell wall changes occurring in the ugtP mutant. (A) The genetic evidence in this work suggests that in comparison to wild type cells (left panel), cells lacking UgtP (right panel) showed lower CwlO activity, an increase in the PG precursor levels resulting in an upturn in PG synthesis and an increase in the LytE endopeptidase activity. These results support the idea that the ugtP mutant maintain its cell wall integrity and rod-shape by balancing the increased PG synthesis (PBP1) and hydrolysis (LytE). A representation of the proteins involved in peripheral PG synthesis or hydrolysis in wild type cells and the mutants studied in this work are shown in panels (B–E). Dark coloured boxes represent functioning proteins and light coloured boxes represent malfunctioning proteins. (B) BSB1 cells had functional proteins resulting in balanced PG synthesis and hydrolysis. (C) ΔugtP cells exhibited a malfunction in several proteins involved in PG synthesis and/or hydrolysis, such that the cell maintains a healthy balance between PG synthesis and hydrolysis. (D) ΔugtP ΔlytE cells and (E) ΔugtP ΔponA cells showed a malfunction in several proteins however, these mutants exhibited a misbalance in PG synthesis and/or hydrolysis resulting in loss of rod-shape and cell death. Therefore, a balanced synthesis and hydrolysis, potentially by the same cytoskeleton system, is important for PG synthesis and cell shape.