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

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.

Cell wall (5 mg) was dissolved in 3 ml hydrofluoric acid at 4˚C for 48 h with stirring. Next, the sample was centrifuged (90000 rpm/ 30 min/ 4˚C). The supernatant was discarded and the pellet was washed twice with H2O MilliQ, once with 100 mM Tris/HCl pH 7.0 and twice with icecold H2O MilliQ, respectively. The murein was resuspended in H2O MilliQ and stored with sodium azide (0.05%) at 4˚C.
Muropeptides were generated from the digestion of peptidoglycan with cellosyl (Hoechst, Germany) following an established protocol 3 . PG was digested with 8 μg of cellosyl in cellosyl buffer (20 mM NaH2PO4, pH 4.8) at 37°C with shaking. Samples were incubated at 100˚C for 7 min and centrifuged at 14,000 rpm for 10 min. An equal volume of sodium borate (0.5 M, pH 9.0) was added to samples in addition to a full small spatula of solid sodium borohydride and centrifuged at 4000 rpm for 30 min. The pH was adjusted between 3 and 4 with 20% phosphoric acid.

Metabolomics
Sampling of intracellular metabolites: for intracellular metabolite samples, 20 OD units of cells were harvested via vacuum-dependent fast-filtration system as described by Meyer et al., (2014) 4 with modifications. In brief, the main culture was transferred into a falcon tube and cooled by dipping it periodically in liquid nitrogen for 10 s maximum (around 1 s each time). During this in/out of liquid nitrogen cycle, the sample was carefully shaken to avoid freezing and metabolite leakage caused by cell lysis. The cooled cell culture was filtered (regenerated cellulose membrane filter, 0.45 µm pore size, 100 mm diameter, RC55 Whatman) and washed 2 times with isotonic 0.9% sodium chloride solution at 4⁰C. The filter was immediately transferred to a falcon tube containing 5 mL of ice-cold extraction solution (60% w/v of ethanol absolute 99.8%) and internal standard (ISTD) constituted of 2.5 nmol of camphorsulphonic acid (CSA) for HPLC-MS and 20 nmol of ribitol for GC-MS analysis. The metabolites were quenched by freezing the sample immediately in liquid nitrogen. The falcon tube was stored at -80°C until extraction.
For cell disruption and metabolites extraction, 10 freeze/thaw cycle was performed by alternately thawing on ice, vortexing, and shaking the sample. Afterwards, the sample was centrifuged for 5 min at 4°C and 13000 rpm. The supernatant was collected to a new falcon tube and left on ice. A second extraction was carried out with 5 mL of deionized water. The two supernatants were combined and distilled water was added to get a final organic solution concentration of 10%. The sample was frozen and stored at -80°C. The sample was lyophilized with a Christ Alpha 1-4 LSC lyophilizer at -52°C and 0.25 mbar. The sample was stored at -20°C until analytical analyses.
GC-MS measurement and data analysis of intracellular metabolites. The dried samples were derivatized firstly with 60 µL of methoxyamine hydrochloride (20 mg/ml solution in pyridine) for 90 min at 37°C and secondly with 120 µL of N-methyl-Ntrimethylsilyltrifluroacetamide (Chromatographie-Service GmbH) for 30 min at 37°C. Samples were centrifuged for 2 min at room temperature and the supernatant was transferred into GC-vial for injection. GC-MS analysis was performed with an Agilent 6890N GC system with an autosampler G2614A model coupled to a mass selective detector 5973N model (Agilent Technologies, USA). A 2 µL sample was injected (G2613A model series injector) with a split 1:10 at 250°C using helium as the carrier gas (split flow of 10 mL/min and 8.8 Psi). The chromatographic run was performed as described by Dörries et al., (2013) 5 . Using a 30 m DB 5-column (JW Scientific, Folsom, USA) with 0.25 mm inner diameter and 2.5 µm film thickness, and a constant gas flow of 1 mL/min -1 . The oven program started with an initial temperature hold at 70°C for 1 min and continued with a heating rate of 1⁰C/min up to 76⁰C, 5⁰ C/min up to 220°C, and 20°C/min up to 330°C, with a hold for 3 min followed by a 10 min isothermal cool-down to 70°C. The analytes were transferred to a quadrupole mass analyser operated in the EI ionization mode with an ionization energy of 70 eV. Data acquisition was done in 40 min runtime. A full scan mass spectrum were acquired from 50 to 500 m/z at a rate of 2 scans/s and with a 6 min solvent delay.
The qualitative analysis of the detected compounds was performed using ChromaTOF software  Relative intracellular concentrations of peptidoglycan precursors in BSB1, pgcA and ugtP mutants in LB medium. Data are presented as mean values ± SD of biological three biological replicates.