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Supplementary Material Material and Methods Bacterial strains and plasmids B. subtilis strains, E. coli strains and plasmids used in this study are listed in Supplementary Table 1. General methods Liquid cultures of B. subtilis strains were grown in Difco Antibiotic Medium 3 (PAB), CH medium (Nicholson and Setlow, 1990), or S-medium (Karamata and Gross, 1970) at 37°C. Nutrient agar (Oxoid) plates were used for growth on solid medium, Modified Salts Medium plates with defined Mg2+ concentrations were prepared as described previously (Carballido-López et al., 2006). Mg2+ was supplemented in the form of MgSO4 to the medium to final concentrations as indicated. DNA manipulations and E. coli DH5α transformations were carried out using standard methods (Sambrook, 1989). B. subtilis strains were transformed according to the method of Anagnostopoulos and Spizizen (1961) as modified by Jenkinson (1983). Selection for B. subtilis transformants was carried out on nutrient agar (Oxoid), supplemented with kanamycin (5 mg ml-1) chloramphenicol (5 mg ml-1), erythromycin (1 mg ml-1), lincomycin (25 mg ml-1) and/or spectinomycin (50 mg ml-1) as required. Xylose was added to a final concentration of 0.2 or 0.5% as indicated. 1 To test the sensitivity to cations, cultures were grown to mid-exponential growth phase in PAB medium, then resuspended in PBS to an OD600 of 1.0. 10 μl of dilutions 101 to 10-6 in PBA were spotted on NA plates containing MnSO4 or MgSO4 in the concentrations as indicated. Construction of deletion and depletion strains Genes were deleted by replacing the coding sequence with antibiotic resistance markers. Therefore, approx. 2500 bp up- and downstream of the target genes were amplified, ligated to the desired resistance cassette and then B. subtilis 168 was transformed with the ligation product, transformants were selected on the appropriate antibiotic and verified by PCR. Resistance cassettes were derived by either restriction or PCR amplification from plasmids [cat from pCotC-GFP (Veening et al., 2006); erm from pMUTIN4 (Vagner et al., 1998); neo from pBEST501 (Itaya et al., 1989); spc from pLOSS* (Claessen et al., 2008); tet from pBEST307 (Itaya, 1992)]. The first 300 bp of ltaS were cloned into pSG4902 (Wu and Errington, 2003) removing the gfp gene. B. subtilis 168 was transformed with the resulting plasmid pSG5925, giving rise to strain 4622 with ltaS under control of a xylose inducible promoter at its native locus. Construction of fusion proteins GFP-LtaS and GFP-YqgS Both ltaS and yqgS were inserted into pSG1729 (Lewis and Marston, 1999) giving rise to plasmids pSG5916 and pSG5917. Transformation of B. subtilis 168 with these plasmids resulted in strains 4606 and 4608 carrying gfp-ltaS or gfp-yqgS respectively under control 2 of a xylose-inducible promoter at the amyE locus. The native gene ltaS or yqgS respectively was deleted to generate strains 4607 and 4609 with the fusion protein as the only copy when inducing expression. Construction of point mutations in ltaS Point mutations in ltaS were constructed based on the protocol of the QuikChange® SiteDirected Mutagenesis Kit (Stratagene). Oligonucleotides encoding the desired point mutations were designed (5’CGGGACAAGGTAAAGCATCTGATGCTGAATTTATGATGG-3’ and 5’CCATCATAAATTCAGCATCAGATGCTTTACCTTGTCCCG-3’ for the T297A mutation, 5’-GTGATGTACGGAGCCCACTACGGCATCTCTG-3’ and 5’CAGAGATGCCGTAGTGGGCTCCGTACATCAC-3’ for the D471A mutation) and a PCR was performed using pSG5916 as template. The PCR product was digested with DpnI, then transformed into E. coli DH5α. The plasmids were purified and the point mutations verified by sequencing. Deletion of ltaS and yqgS in strains carrying the mutated gene under control of a xylose inducible promoter at the amyE locus then gave rise to strains 4626 and 4627. Determination of expression levels Strains 4601, 4602, 4603, and 4604 were obtained by integration of pMUTIN4 derived plasmids (Vagner et al., 1998) in the chromosome, resulting in the lacZ gene being fused to the promoter of ltaS, yfnI, yqgS and yvgJ respectively. Therefore, approx. 250 bp upstream of the target gene were cloned in pMUTIN4 giving rise to plasmids pSG5921, 3 pSG5922, pSG5923 and pSG5924 respectively. To determine expression levels, activity of β-galactosidase activity was measured as described previously (Daniel et al., 1996). In short, cultures were grown at 37ºC in PAB or Schaeffer’s Medium as indicated, at the desired time points samples were taken and immediately frozen. Then cells were lysed, incubated with the substrate ONPG (time recorded) and centrifuged for 5 minutes before β-galactosidase activity was determined by measuring the OD420 of the supernatant, normalising for cell growth and calculating the activity according to the method of Miller (1972). Sporulation experiments Sporulation efficiency was determined by microscopic counting of cells derived from cultures growing for 24 h in Schaeffer’s medium at 37ºC. Alternatively, the cultures were treated with 10% chloroform or incubated for 20 min at 80ºC and dilutions were plated on Nutrient Agar plates. Colony forming units of untreated cultures were used as a reference to calculate the percentage of spores in the culture. Induction of expression from spoIIA and spoIIQ promoters was examined by assaying βgalactosidase activity of strains 4614, 4615, 4616, and 4617 after inducing sporulation by the starvation method in Schaeffer’s medium at 37ºC. The alkaline phosphatase assay was performed as described previously (Errington and Mandelstam, 1983; Glenn and Mandelstam, 1971). 4 Phase contrast and fluorescence microscopy For microscopy, cells from an overnight liquid or solid culture were diluted into PAB medium supplemented with 20 mM MgSO4 when required and grown at the temperature indicated. Samples were mounted on microscope slides covered with a thin film of 1% agarose in minimal medium (Glaser et al., 1997). Staining of the membrane was achieved by mixing 2 μl of Nile Red (Molecular Probe) solution (12.5 mg ml-1) with 600 μl agarose on the slide or by addition of FM5-95 (200 μg/ml) to the culture (end concentration 1 μg/ml). Nucleoids were stained by mixing 8 μl of the cell suspension with 2 μl of DAPI (Sigma) solution (1 mg ml-1 in 50% glycerol) before mounting the sample on the agarose covered slide. Images were acquired with a Sony CoolSnap HQ cooled CCD camera (Roper Scientific) camera attached to a Zeiss Axiovert M135 microscope or to a Zeiss Axiovert 200M microscope. ImageJ (http://rsb.info.nih.gov/ij/) was used to analyse the images, manipulation was limited to altering brightness and contrast. Transmission electron microscopy Strains were grown in PAB medium to mid-exponential phase or until initiation of sporulation as indicated, samples were taken and fixed in 2% glutaraldehyde. Samples were processed by the Electron Microscopy Research Service of Newcastle University. Briefly, cell pellets were fixed overnight in 2% glutaraldehyde in Sorenson’s phosphate buffer (TAAB Laboratory Equipment), pH 7.4, then in 1% osmium tetroxide (Agar Scientific) for one hour. Samples were then dehydrated in an acetone graded series before being impregnated with a graded series of epoxy resin (TAAB Laboratory Equipment) in 5 acetone and finally embedded in 100% resin and set at 60°C for 24 h. The pellets were sectioned and counter stained with 2% uranyl acetate and lead citrate (Leica) before being imaged on a Philips CM100 Compustage Transmission Electron Microscope (FEI) with an AMT CCD camera (Deben). Images were analysed using ImageJ (http://rsb.info.nih.gov/ij/). Protein purification The ORF encoding the C-terminus of LtaS (residues 215-649) was cloned in pET11a (Invitrogen), and E. coli BL21 was transformed with the resultant plasmid pSG5918. Expression of the protein was induced by addition of 1mM IPTG final concentration to exponentially growing cells. The cells were harvested after 3-4 h of induction, then the pellet was resuspended in buffer A (20 mM Tris·HCl, pH 8.0) and sonicated. The supernatant after centrifugation at 15,000 rpm for 1 h was filtered (pore size 0.45 μm) and loaded onto a 50 ml Q-Sepharose anion exchange column that had been equilibrated with buffer A. A gradient of buffer A and Buffer B (1M NaCl, 20 mM Tris·HCl, pH 8.0) eluted bound proteins from the column. Fractions containing LtaS215-649 were concentrated and further purified on a Superdex 75 gel filtration column (GE Healthcare) using Buffer C (20 mM Tris·HCl, pH 8.0, 200 mM NaCl). The fractions containing LtaS215-649 were then dialysed against buffer A, and as a final purification step loaded onto a MonoQ HR5/5 (GE Healthcare) column. Bound proteins were eluted with a 50 ml gradient of buffers A and B. Fractions containing LtaS215-649 (1 mg/ml) were stored in 500 μl aliquots at -20ºC. 6 A derivative containing selenomethionine (Se-Met) was purified from the met deficient E. coli B834(DE3) (Novagen). Se-Met was provided as the only methionine variant available, expression was induced in mid-exponential phase and cells were harvested after overnight induction at 16-18ºC. LtaS215-649 containing Se-Met was purified as described above with the addition of 1mM DTT and 4 mM MgCl2 to all buffers. Crystallisation and structure solution Crystals of a Se-Met labelled derivative of LtaS were grown in a hanging drop experiment set up at 21 °C. The protein was concentrated to 7 mg ml-1 and 1 μl of protein was added to 1 μl of well solution containing 150 mM MgCl2, 100 mM bicine, pH 8.0 and 25 % (w/v) PEG 1500. A single crystal was taken from the crystallization drop using a litho-loop and transferred to cryoprotectant solution containing the crystallization mother liquor supplemented with 20 % (v/v) PEG 300 and flash cooled in liquid nitrogen. A total of 1440 images were collected on Diamond beamline IO4, at a fixed wavelength of 0.9763 Å, with an oscillation angle of 0.55º and exposure time of 1.7 seconds. Images were processed using MOSFLM (Leslie, 2006) and intensities were scaled using SCALA (Evans, 2006). SOLVE (Wang et al., 2004) was used to phase the data by single-wave anomalous dispersion. A total of 22 selenium atoms were located, half of which were related to the others by two-fold non-crystallographic symmetry. Heavy atom positions were input into RESOLVE (Wang et al., 2004) to break the phase ambiguity with solvent 7 flattening and this was followed by automated model building. The resulting model had two NCS-related chains and 50 % of the residues were assigned to the correct sequence. The rest of the model was manually built using Coot (Emsley and Cowtan, 2004) and refined until convergence with REFMAC5 (Murshudov et al., 1997). The final model comprised residues 215-635 in two protein chains, two phospho-threonine residues, two magnesium ions, 289 water atoms and two PEG chains. Data collection and refinement statistics are shown in Table 3. Determination of MIC values Determination of the minimal inhibitory concentration (MIC) for susceptibility to various antibiotics was done essentially by the broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS) guidelines. (NCCLS. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically — fifth edition. Approved standard M7-A5. NCCLS, Wayne, Pa.). Cultures were grown and diluted to an OD600 of 0.001 in PAB medium and dilutions of the antibiotics in PAB were prepared. 100 μl of antibiotic solution were mixed with 100 μl of bacterial suspension in wells of a 96 well microtiter plate, which was then incubated over night (16-20 h) at 37°C with shaking. The MIC was defined as the lowest concentration inhibiting visible growth as scored by eye. 8 Supplementary Table 1 Strains and plasmids. Strain / Relevant genotype plasmid source / construction / comment B. subtilis 168 trpC2 laboratory stock 3728 trpC2 Ωneo3427 ∆mreB (Formstone and Errington, 2005) 2505 trpC2 Ω(mbl::spc) (Jones et al., 2001) 2536 trpC2 Ω(mreBH::cat) (Carballido-López et al., 2006) 4261 trpC2 ∆mbl::cat Schirner and Errington, in press 4262 trpC2 ∆mreBH::erm Schirner and Errington, in press 2020 trpC2 amyE::(spc Pxyl-gfpmut1-ftsZ) J. Sievers, unpublished JWV048 trpC2 sacA::(kan PspoIIA-mCherry) J.-W. Veening, unpublished 1302 trpC2 Ω(spoIIE::erm) A. Feucht, unpublished 1305 trpC2 spoIIE::pSG1902 (spoIIE-gfp cat) (Wu et al., 1998) CW314 spoIIE::pCW28 (spoIIE-gfp aph-A3) (Arigoni et al., 1995) 1809 trpC2 Ω(spoIIAB'-'lacZ cat)700 (Thomaides et al., 2001) MO2051 trpC2 amyE::(cat PspoIIQ-lacZ) (Londoño-Vallejo et al., 9 1997) 4282 trpC2 ∆tagO::erm derived from EB1451, (D'Elia et al., 2006) 4283 trpC2 ∆ltaS::neo this work 4284 trpC2 ∆ltaS::spc this work 4285 trpC2 ∆ltaS::cat this work 4286 trpC2 ∆ltaS::erm this work 4287 trpC2 ∆yfnI::spc this work 4288 trpC2 ∆yfnI::cat this work 4289 trpC2 ∆yfnI::erm this work 4290 trpC2 ∆yfnI::(cat::tet pECE85) this work 4291 trpC2 ∆yqgS::neo this work 4292 trpC2 ∆yqgS::spc this work 4293 trpC2 ∆yqgS::cat this work 4294 trpC2 ∆yqgS::erm this work 4295 trpC2 ∆yvgJ::spc this work 4296 trpC2 ∆yvgJ::cat this work 4297 trpC2 ∆yvgJ::erm this work 4298 trpC2 Ω(mbl::spc) ∆ltaS::neo this work 4299 trpC2 Ωneo3427 ∆mreB ∆ltaS::neo this work 4300 trpC2 Ω(mreBH::cat) ∆ltaS::neo this work 4601 trpC2 PltaS lacZ-lacI-bla-erm insertion of pMUTIN4 upstream of ltaS, this work 10 4602 trpC2 PyfnI lacZ-lacI-bla-erm insertion of pMUTIN4 upstream of yfnI, this work 4603 trpC2 PyqgS lacZ-lacI-bla-erm insertion of pMUTIN4 upstream of yqgS, this work 4604 trpC2 PyvgJ lacZ-lacI-bla-erm insertion of pMUTIN4 upstream of yvgJ, this work 4605 trpC2 ∆ltaS::neo amyE::(spc Pxyl-gfpmut1- this work ftsZ) 4606 trpC2 amyE::(spc Pxyl-gfpmut1-ltaS) this work 4607 trpC2 ∆ltaS::neo amyE::(spc Pxyl-gfpmut1-ltaS) this work 4608 trpC2 amyE::(spc Pxyl-gfpmut1-yqgS) this work 4609 trpC2 ∆yqgS::cat amyE::(spc Pxyl-gfpmut1- this work yqgS) 4610 trpC2 ∆ltaS::spc ∆yfnI::erm this work 4611 trpC2 ∆ltaS::spc ∆yqgS::erm this work 4612 trpC2 ∆ltaS::spc ∆yvgJ::erm this work 4613 trpC2 ∆yfnI::cat ∆yqgS::spc yvgJ::erm this work 4614 trpC2 ∆ltaS::spc Ω(spoIIAB'-'lacZ cat)700 this work 4615 trpC2 ∆ltaS::erm ∆yqgS::spc Ω(spoIIAB'-'lacZ this work cat)700 4616 trpC2 ∆ltaS::neo amyE::(catPzpoIIQ-lacZ) this work 4617 trpC2 ∆ltaS::spc ∆yqgS::erm this work 11 amyE::(catPzpoIIQ-lacZ) 4618 trpC2 ∆ltaS::spc sacA::(kan PspoIIA-mCherry) this work spoIIE::pCW28 (spoIIE-gfp aph-A3) 4619 trpC2 ∆ltaS::spc ∆yqgS::erm sacA::(kan this work PspoIIA-mCherry) spoIIE::pCW28 (spoIIE-gfp aph-A3) 4620 trpC2 ∆ltaS::neo ∆yfnI::cat ∆yqgS::spc this work ∆yvgJ::erm 4621 trpC2 ∆ltaS::neo ∆tagO::erm this work 4622 trpC2 ltaS::(cat pSG902 Pxyl-ltaS) this work 4623 trpC2 ltaS::(cat pSG902 Pxyl-ltaS) this work ∆yfnI::(spc::tet) ∆yqgS::neo ∆yvgJ::spc 4624 trpC2 ltaS::(cat pSG902 Pxyl-ltaS) ∆tagO::erm this work 4625 trpC2 ltaS::(cat pSG902 Pxyl-ltaS) this work ∆yfnI::(spc::tet) ∆yqgS::neo ∆yvgJ::spc ∆tagO::erm 4626 trpC2 Ω(spoIIE::erm) amyE::(spc Pxyl-gfpmut1- this work yqgS) 4627 trpC2 ∆ltaS::cat ∆yqgS::erm amyE::(spc Pxyl this work gfpmut1-ltaS) 4628 trpC2 ∆ltaS::cat ∆yqgS::erm amyE::(spc Pxyl this work gfpmut1-ltaS T297A) 4629 trpC2 ∆ltaS::cat ∆yqgS::erm amyE::(spc Pxyl this work 12 gfpmut1-ltaS D471A) E. coli DH5α laboratory stock BL21 (DE3) F- ompT hsdSB(rB-mB-)gal dcm (DE3) Novagen B834 (DE3) F- ompT hsdSB(rB-mB-)gal dcm met (DE3) Novagen plasmids pBEST501 bla neo (Itaya et al., 1989) pBEST307 bla tet (Itaya, 1992) pMUTIN4 bla erm Pspac-lacZ lacI (Vagner et al., 1998) pLOSS* bla spc Pspac-mcs PdivIVA-lacZ lacI reppLS20 (Claessen et al., 2008) (GA→CC) pCotC-GFP bla cat PcotC-cotC-gfp (Veening et al., 2006) pSG1729 bla amyE3’ spc Pxyl-gfp’ amyE5’ (Lewis and Marston, 1999) pSG4902 bla Pxyl -gfp cat (Wu and Errington, 2003) pET11a Apr, expression vector with T7 lac promoter Novagen pSG5916 bla amyE3’ spc Pxyl-gfp-ltaS amyE5’ this work pSG5917 bla amyE3’ spc Pxyl-gfp-yqgS amyE5’ this work pSG5918 pET11a yflE aa215-649 this work pSG5919 bla amyE3’ spc Pxyl-gfp-ltaS T297A amyE5’ this work pSG5920 bla amyE3’ spc Pxyl-gfp-ltaS D471A amyE5’ this work pSG5921 bla erm Pspac PltaS-lacZ lacI pMUTIN4 with the promoter region of ltaS 13 pSG5922 bla erm Pspac PyfnI-lacZ lacI pMUTIN4 with the promoter region of yfnI pSG5923 bla erm Pspac PyqgS-lacZ lacI pMUTIN4 with the promoter region of yqgS pSG5924 bla erm Pspac PyvgJ-lacZ lacI pMUTIN4 with the promoter region of yvgJ pSG5925 bla PxylltaS’ cat this work 14 Supplementary Table 2 Minimal inhibitory concentration (MIC) in g/ml of antibiotics against B. subtilis wild-type and strains carrying deletions of ltaS and homologues strain kanamycin spectinomycin ampicillin tetracycline vancomycin cephalexin penicillinG wild-type 2.5 40-50 0.5-0.6 2 0.4-0.5 0.3 0.6 4285 (∆ltaS) 0.75 30 0.2 2 0.2 0.2 0.1 4288 (∆yfnI) 2.5 40-50 0.5-0.6 2 0.4-0.5 0.3 0.6 4293 (∆yqgS) 2.5 40-50 0.5-0.6 2 0.4-0.5 0.3 0.6 4296 (∆yvgJ) 2.5 40-50 0.5-0.6 2 0.4-0.5 0.3 0.6 15 References Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. 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Wu LJ, Feucht A, Errington J (1998) Prespore-specific gene expression in Bacillus subtilis is driven by sequestration of SpoIIE phosphatase to the prespore side of the asymmetric septum. Genes Devel 12: 1371-1380. 17 Supplementary Figure 1 Effects of mutations in ltaS paralogues on growth and cell morphology (A) Measurements of β-galactosidase activity in strains 4601 (PltaS, ), 4602 (PyfnI, ), 4603 (PyqgS, ), and 4604 (PyvgJ, ), where the lacZ gene is expressed under control of the promoter regions of the four ltaS paralogues. Open symbols indicate the growth curves of the strains, closed symbols show the respective β-galactosidase activity. (B) Growth curves of wild-type (168, ) and the four single mutants in ltaS paralogues ΔltaS (4283, ), ΔyfnI (4289, ), ΔyqgS (4294, ) and ΔyvgJ (4297, ) in PAB medium at 37ºC. (C) Growth curve of wild-type (168, ) and LTA mutant (4620, ) grown in PAB medium at 37ºC. (D-I) Phase contrast microscopic images of the single mutants in ltaS (strain 4283, panel E), yfnI (strain 4289, panel F) , yqgS (strain 4294, panel G) and yvgJ (strain 4297, panel H) and LTA mutant (strain 4620, panel I) compared to a wild-type strain (168, panel A) grown in PAB medium at 37ºC. Scale bar 5 µm. Supplementary Figure 2 (A) The potential interactions of glycerol phosphate with LtaS. The potential position of glycerol phosphate in the active site of LtaS was inferred from the presence of two crystallographic waters – that may correlate to the hydroxyls of a covalently-bound glycerol phosphate. The active site of LtaS is shown as a semitransparent surface, with key active site residues drawn as ‘stick’ models. For clarity, the protein backbone is shown as a cartoon, and the active site magnesium is drawn as a sphere. (B) A comparison of the active sites of LtaS and the Pseudomons aeruginosa aryl sulphatase, PDBid 1HDH. The proteins were aligned only on common atomic positions of the phospho-threonione and magnesium of LtaS, in comparison to the equivalent 18 atoms in 1HDH. The active site residues are rendered as sticks, with LtaS coloured green and 1HDH blue. The bound metal ions are drawn as spheres, coloured green for the Mg2+ in LtaS and red for the Ca2+ in 1HDH. The residue labelling is for 1HDH. 19