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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
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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
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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.
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