FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis Jeffrey Meisner, Paula Montero Llopis, Lok-To Sham, Ethan Garner, Thomas G. Bernhardt, David Z. Rudner

SUPPLEMENTAL MATERIAL SUPPLEMENTAL EXPERIMENTAL PROCEDURES Plasmid construction pJM026 [ycgO::Pspank-ftsEX (erm)] was generated by ligation with an SpeI-SphI ftsEX

PCR product (amplified with oligonucleotide primers oJM93 and oJM99 from PY79

genomic DNA) and pER065 [ycgO::Pspank (erm)].

pJM030 [sacA::Pveg-mCherry (spc)] was generated by ligation with an EcoRI-BamHI

Pveg-mCherry fragment from pER069 and pKM070 (sacA::spc) cut with the same

enzymes. pER069 [sacA::Pveg-mCherry (phleo)] was generated by ligation with an

EcoRI-HindIII Pveg PCR product (amplified with oligonucleotide primers oJM73 and

oJM74 and PY79 genomic DNA).

pJM031 [his6-sumo-cwlOΔss expression plasmid] was generated by isothermal

assembly with a cwlOΔss PCR product (amplified with oligonucleotide primers oJM133

and oJM117 from PY79 genomic DNA) and pTB146 (his6-sumo expression plasmid)

cut with SapI and XhoI.

pJM039 [his6-sumo-ftsE expression plasmid] was generated by isothermal assembly

with ftsE PCR product (amplified with oligonucleotide primers oJM156 and oJM157 from

PY79 genomic DNA) and pTB146 cut with SapI and XhoI.

pJM048 [his6-sumo-ftsX loop1 expression plasmid] was generated by ligation with a

SapI-XhoI ftsX loop1 PCR product (amplified with oligonucleotide primers oJM114 and

oJM115 from PY79 genomic DNA) and pTB146 cut with the same enzymes.

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pJM055 [amyE::ftsEX (spc)] was generated by ligation with an EcoRI-BamHI ftsEX PCR

product (amplified with oligonucleotide primers oJM305 and oJM232 from PY79

genomic DNA) and pDR190 (amyE::spc).

pJM058 [amyE::ftsE(K41A) ftsX (spc)] was generated by site-directed mutagenesis with

oligonucleotide primer oJM307 and pJM055 plasmid DNA.

pJM059 [amyE::ftsE(D162N) ftsX (spc)] was generated by site-directed mutagenesis

with oligonucleotide primer oJM308 and pJM055 plasmid DNA.

pJM60 [amyE::ftsE(D162A) ftsX (spc)] was generated by site-directed mutagenesis with

oligonucleotide primer oJM309 and pJM055 plasmid DNA.

pJM062 [amyE::PspoIIE-lacZ (cat)] was generated by ligation with an EcoRI-BamHI PspoIIE

PCR product (amplified with oligonucleotide primers oJM318 and oJM319 from PY79

genomic DNA) and pDG1661 [amyE::lacZ (cat)] (Guerout-Fleury et al., 1996).

pJM063 [his6-sumo-cwlOΔCC expression plasmid] was generated by isothermal

assembly with a cwlOΔcoiled-coil PCR product (amplified with oligonucleotide primers

oJM320 and oJM117 from PY79 genomic DNA) and pTB146 cut with SapI and XhoI.

pJM064 [amyE::ftsE(K41M) ftsX (spc)] was generated by site-directed mutagenesis with

oligonucleotide primer oJM321 and pJM055 plasmid DNA.

Strain construction

Gene deletion mutants were each generated by isothermal assembly (Gibson, 2011).

Each isothermal assembly reaction contained three PCR products: an antibiotic

resistance cassette and upstream and downstream sequences that flank the gene of

interest. Chloramphenicol and kanamycin resistance cassettes, flanked by lox66 and

lox71 sites, were amplified by PCR with oJM28 and oJM29 from pWX465 and pWX470,

respectively. The loxP sites allow the antibiotic resistance cassettes to be removed by

recombination with Cre recombinase. Upstream and downstream products were

amplified to delete lytE (with oligonucleotide primers oJM40, oJM41, oJM42, and

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oJM53), cwlO (oJM36, oJM37, oJM38, and oJM39), ftsEX (oJM54, oJM55, oJM56, and

oJM57), ftsE (oJM54, oJM55, oJM58, and oJM59), ftsX (oJM60, oJM61, oJM58, and

oJM59), lytF (oJM95, oJM96, oJM97, and oJM98), cwlS (oJM160, oJM161, oJM162,

and oJM163), sigI (oJM342, oJM343, oJM344, and oJM345), and mreBH (oJM209,

oJM210, oJM211, and oJM212).

BJM148 (ΔftsE) was generated by transformation of BJM70 (ftsE::kan) with pDR244

(temperature-sensitive plasmid with bacteriophage P1 cre recombinase and spc

resistance cassette). Transformants were selected on LB supplemented with 100 µg/ml

spectinomycin at 30°C, a permissive temperature for pDR244 replication.

Transformants were streaked on LB and incubated at 42°C, a non-permissive

temperature for pDR244 replication. Single colonies were then restreaked on LB, LB

with spectinomycin, and LB with kanamycin and incubated 37°C. Those that grew on

LB, but not LB spectinomycin or LB kanamycin, had lost pDR244 and the kanamycin

resistant cassette. These strains were confirmed by PCR with oligonucleotide primers

oJM54 and oJM59.

BJM154 [ycgO::Pspank-lytE (erm)] was generated by transformation of BKM424

(ycgO::spc) with an isothermal assembly reaction containing lytE PCR product

(amplified with oligonucleotide primers oJM103 and oJM104 from PY79 genomic DNA)

and pER065 [ycgO::Pspank (erm)] cut with SpeI and SphI.

BJM362 [ycgO::Pspank-cwlO (erm)] was generated by transformation of BKM424

(ycgO::spc) with an isothermal assembly reaction containing cwlO PCR product

(amplified with oligonucleotide primers oJM136 and oJM128 from PY79 genomic DNA)

and pER065 cut with SpeI and SphI.

Supplemental References Gibson, D. G., (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498: 349-361. Guerout-Fleury, A. M., N. Frandsen & P. Stragier, (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180: 57-61.  

Meisner et al. 4

SUPPLEMENTAL FIGURE LEGENDS Figure S1. Quantitative analysis of cell length distribution in cells lacking LytE, CwlO, FtsEX, or both CwlO and FtsEX. The indicated strains harboring mCherry expressed under constitutive control (Pveg) were grown in CH medium at 37°C to mid-

exponential phase and visualized by phase-contrast and fluorescence microscopy.

Cytoplasmic mCherry images were analyzed using MicrobeTracker to assess cell

length. More than 400 cells were analyzed for each strain.

Figure S2. CwlO is required for cell elongation. Time-lapse fluorescence microscopy of ∆lytE, ∆cwlO, Pspank-cwlO, Pveg-mCherry (BJM692). Cells were grown in CH medium

supplemented with 1 mM IPTG at 37°C to mid-exponential phase, washed with medium

lacking IPTG. Cells were immobillized on agarose pads in a humidified incubator at

30°C and visualized by phase-contrast and fluorescence microscopy every 5 minutes

for 3 hours. Merged images of phase contrast and cytoplasmic mCherry (top) and

mCherry (bottom) are shown. Cell division without significant elongation upon FtsEX

depletion is highlighted (yellow caret). Examples of cell lysis are indicated (white caret).

Figure S3. Interaction assay between CwlO and the large extracellular loop of FtsX. Purified CwlO (residues 31-473) (10 µM) was incubated with (A) or without (B) His-tagged FtsX loop1 (residues 44-170) (10 µM) in 20 mM Tris pH 7.5 and 150 mM

NaCl for 1 hour at 4°C. Ni-NTA agaraose (Qiagen) was added to each reaction and then

incubated with gentle mixing for 1 hour at 4°C. The reactions were subjected to

centrifugation at 5,000 rpm in a microcentrifuge. The Ni-NTA agarose was washed twice

with 20 mM Tris pH 7.5, 150 mM NaCl, 50 mM imidazole. Proteins were eluted with

SDS-PAGE sample buffer containing 100 mM EDTA. Proteins in the load (L),

supernatant (S), washes (W1 and W2) and eluate (E) were analyzed on a 15% SDS-

PAGE gel and stained with Coomassie blue.

 

0 2 4 6 8 10 120

5

10

15

20

cell length (µm)

perc

enta

ge

WT (BJM172)ΔlytE (BJM186)

ΔftsEX (BJM190)ΔcwlO (BJM188)

ΔftsEX ΔcwlO (BJM212)

Meisner_Fig. S1

mer

ge

0 min

cyto

plas

m

25 50 75 100 125 150

Meisner_Fig. S2

ΔlytE, CwlO

His-FtsX(loop1)

CwlOΔSS

EW2W1SLA

B EW2W1SL

CwlOΔSS

Meisner_Fig. S3

Meisner_Table S1 Table S1. B. subtilis strains Strain Genotype BJM1 prototrophic wild-type (PY79) BJM76 lytE::cat BJM58 lytE::kan BJM54 cwlO::cat BJM96 cwlO::kan BJM68 ftsEX::kan BJM70 ftsE::kan BJM148 ΔftsE BJM72 ftsX::kan BJM104 lytF::cat BJM550 cwlS::cat BJM949 sigI::cat BJM552 mreBH::cat BJM386 lytE::cat, ycgO::Pspank-lytE (erm) BJM495 lytE::cat, ycgO::Pspank-lytE (erm), cwlO::kan BJM493 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan BJM526 lytE::cat, ycgO::Pspank-lytE (erm), ΔftsE BJM491 lytE::cat, ycgO::Pspank-lytE (erm), ftsX::kan BJM125 ftsEX::kan, ycgO::Pspank-ftsEX (erm) BJM133 ftsEX::kan, ycgO::Pspank-ftsEX (erm), lytE::cat BJM135 ftsEX::kan, ycgO::Pspank-ftsEX (erm), cwlO::cat BJM137 ftsEX::kan, ycgO::Pspank-ftsEX (erm), lytF::cat BJM570 ftsEX::kan, ycgO::Pspank-ftsEX (erm), cwlS::cat BJM172 swrA+ sacA::Pveg-mcherry (spc) BJM186 swrA+ sacA::Pveg-mcherry (spc), lytE::cat BJM188 swrA+ sacA::Pveg-mcherry (spc), cwlO::cat BJM190 swrA+ sacA::Pveg-mcherry (spc), ftsEX::kan BJM212 swrA+ sacA::Pveg-mcherry (spc), ftsEX::kan, cwlO::cat BJM318 swrA+ sacA::Pveg-mcherry (spc), ftsEX::kan ycgO::Pspank-ftsEX (erm) BJM754 swrA+ sacA::Pveg-mcherry (spc), ftsEX::kan ycgO::Pspank-ftsEX (erm) lytE::cat BJM372 cwlO::cat, ycgO::Pspank-cwlO (erm) BJM433 cwlO::kan, ycgO::Pspank-cwlO (erm) BJM406 cwlO::cat, ycgO::Pspank-cwlO (erm), lytE::kan BJM967 cwlO::kan, ycgO::Pspank-cwlO (erm), sigI::cat BJM969 cwlO::kan, ycgO::Pspank-cwlO (erm), mreBH::cat BJM874 amyE::PspoIIE-lacZ (cat) BJM898 amyE::PspoIIE-lacZ (cat), lytE::kan BJM902 amyE::PspoIIE-lacZ (cat), cwlO::kan BJM900 amyE::PspoIIE-lacZ (cat), ftsEX::kan BJM955 ftsEX::kan, ycgO::Pspank-ftsEX (erm), sigI::cat BJM957 ftsEX::kan, ycgO::Pspank-ftsEX (erm), mreBH::cat BJM827 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan amyE::ftsEX BJM912 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan amyE::ftsE(K41M) ftsX BJM861 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan amyE::ftsE(K41A) ftsX BJM857 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan amyE::ftsE(D162N) ftsX BJM863 lytE::cat, ycgO::Pspank-lytE (erm), ftsEX::kan amyE::ftsE(D162A) ftsX BJM929 cwlO::cat, ycgO::Pspank-cwlO∆coiled-coil (∆32-293) (erm) BJM663 amyE::PxylA-gfp-mbl (spc)

BJM1007 amyE::PxylA-gfp-mbl (spc), lytE::cat BJM837 amyE::PxylA-gfp-mbl (spc), ftsEX::kan, ycgO::Pspank-ftsEX (erm) BJM839 amyE::PxylA-gfp-mbl (spc), ftsEX::kan, ycgO::Pspank-ftsEX (erm), lytE::cat BJM435 swrA+ sacA::Pveg-mcherry (spc), cwlO::kan ycgO::Pspank-cwlO (erm) BJM692 swrA+ sacA::Pveg-mcherry (spc), cwlO::kan ycgO::Pspank-cwlO (erm) lytE::kan BJM667 amyE::PxylA-gfp-mbl (spc), cwlO::cat, ycgO::Pspank-cwlO (erm) BJM677 amyE::PxylA-gfp-mbl (spc), cwlO::cat, ycgO::Pspank-cwlO (erm), lytE::kan

Meisner_Table S2 Table S2 Plasmids Plasmid Description pKM064 sacA::cat (amp) pBB283 yhdG::kan (amp) pWX465 lox66-cat-lox71 (amp) pWX470 lox66-kan-lox71 (amp) pDR244 PpagA-cre, ori(ts) from pE194 (spec) (amp) pER065 ycgO::Pspank (erm) (amp) pJM026 ycgO::Pspank-ftsEX (erm) (amp) pER069 sacA::Pveg-mCherry (phleo) (amp) pKM070 sacA::spec (amp) pJM030 sacA::Pveg-mCherry (spec) (amp) pJM067 amyE::PsigI-lacZ (cat) (amp) pJM062 amyE::PspoIIE-lacZ (cat) (amp) pJM064 ycgO::Pspank-ftsE(K41M) ftsX (erm) (amp) pJM058 ycgO::Pspank-ftsE(K41A) ftsX (erm) (amp) pJM059 ycgO::Pspank-ftsE(D162N) ftsX (erm) (amp) pJM060 ycgO::Pspank-ftsE(D162A) ftsX (erm) (amp) pJM031 His6-SUMO-CwlO∆SS (31-473) pJM048 His6-FtsX loop1 (44-170) pJM039 His6-SUMO-FtsE pJM063 His6-SUMO-CwlO CTD (334-473)

Meisner_Table S3 Table S3. Oligonucleotide primers  Primer Sequence oJM28 TTCTGCTCCCTCGCTCAG oJM29 CAGGGAGCACTGGTCAAC oJM40 AGTTGCAATCACAAGTGTATG oJM41 CTGAGCGAGGGAGCAGAATTCATATTTTCCTCCCCAAATGTT oJM42 GTTGACCAGTGCTCCCTGTAATTTTTAGAGAAAACCCGTTCATTGG oJM53 TCACCTGTGAGCATATAATAGTAG oJM36 AGAAGCGGCCGCTTATTCTG oJM37 CTGAGCGAGGGAGCAGAACTCACTTTTTATATCCTCCCTTTTAC oJM38 GTTGACCAGTGCTCCCTGTAATAAATATGACAAGGGCCTTCT oJM39 TCATCCGTCTGAAGCACAC oJM54 TGCTATCGGAGAGCATTGG oJM55 CTGAGCGAGGGAGCAGAAATCATGAAATCACCTAATCTTTTATATC oJM56 GTTGACCAGTGCTCCCTGTAAAGTGAAAAAGCCGTTCCAG oJM57 TAATGTCTCTGCAGTGCGAG oJM58 GTTGACCAGTGCTCCCTGAGAGGGGAGTATGGTTCATATG oJM59 TCGATTCTCCATTGATGCTG oJM60 TGTCTTACAGAAGATTGACAGAG oJM61 CTGAGCGAGGGAGCAGAATTAATCATATGAACCATACTCCCCTC oJM95 AAGATTCGCGGCACGTATG oJM96 CTGAGCGAGGGAGCAGAATTCATCTTAGCTCTCCTTTTTCC oJM97 GTTGACCAGTGCTCCCTGTAAAAACAGAAACTGTGCGGCCT oJM98 CTGAAATAGCGAACGGGAAAAGC oJM160 TACGTACTACGCAGTGAAGG oJM161 CTGAGCGAGGGAGCAGAATTCATTCATTCAACCTCCTAAACTG oJM162 GTTGACCAGTGCTCCCTGTAAAAATGAGTATGACAAAAAGCCT oJM163 ATTGGCCTGAACGATCAAGTC oJM342 TCGGTGAAAATTGGAAGGCAG oJM343 CTGAGCGAGGGAGCAGAATTCACCTCAGTTCCTCCCTATAAC oJM344 GTTGACCAGTGCTCCCTGTAAAGGGGTGCTGCACTCATG oJM345 ACATTCTATAGTCTGCGCCAC oJM103 ATTAAGCTTGTCCCGGGTAAGTTAACATTTGGGGAGGAAAATATG oJM104 CCACCGAATTAGCTTGCATGCAATGAACGGGTTTTCTCTAAAAATTAG oJM136 ATTAAGCTTGTCCCGGGTAACGTACATAAGGAATTAGTGTTTTTTAC oJM128 CCACCGAATTAGCTTGCATGAAAGAGCCTTCTGTTTATAGAAGG oJM314 ATAAAAGATTAGGTGATTTCATGAGTAAAGGAGAAGAACTTTTC oJM315 ATCATTCCGGAACCCTCGAGTTTGTATAGTTCATCCATGC oJM312 CATGACTTCACTAACGATGC oJM313 GAAATCACCTAATCTTTTATATC oJM316 ACAAACTCGAGGGTTCCGGAATGATAGAGATGAAGGAAGTA oJM317 TCCGTATGTCAAGTGGCTGC oJM180 ACCATAAACAACCATCATTCCTCG oJM93 CGCACTAGTAAAAGATTAGGTGATTTCATGATAG oJM99 CGCGCATGCTCACTGGAACGGCTTTTTCAC oJM73 CGCGAATTCTTGAAAACCTGCATAGGAGAG oJM74 CGCAAGCTTTCACTACATTTATTGTACAACACG oJM305 CGCGAATTCCTGGCAAGGATGTTTTTATGC

oJM232 CGCGGATCCTCACTGGAACGGCTTTTTCAC  oJM318 CGCGAATTCATCCTAACAAATCGGTTTCTC oJM319 CGCGGATCCACCTGTTATATTCGTTGCCTG oJM321 GGTCCGAGCGGAGCAGGTATGTCTACTTTTATTAAAATGATTTAC oJM307 GGTCCGAGCGGAGCAGGTGCATCTACTTTTATTAAAATGATTTAC oJM308 ACCCTGATGTTGTCATTGCTAACGAACCGACAGGAAACCTTGA oJM309 ACCCTGATGTTGTCATTGCTGCAGAACCGACAGGAAACCTTGA oJM133 CTCACAGAGAACAGATTGGTGGTGAAACATTAGATGAAAAGAAACAAAAAATCG oJM117 CTTCCTGCAGTCACCCGGGCTTATTGAACAACACGTCTTACAACAC oJM114 GCTCTTCTGGTATGATTAAAATTCTCGGGCGCCAC oJM115 CGGCTCGAGTTATACTCGCAGAAACTTGCGGA oJM156 CTCACAGAGAACAGATTGGTGGTATGATAGAGATGAAGGAAGTATATAAAGC oJM157 CTTCCTGCAGTCACCCGGGCGAATTTTAATCATATGAACCATACTCC oJM320 CTCACAGAGAACAGATTGGTGGTGGCACTGTTATCAGCAACTC  

supplemental materialMeisner et al. RESUBMISSION.pdfMeisner et al resubmission.pdfFigures+Tables (resubmission)FIGURE 1.1.pdfFIGURE 2.4(PML)FIGURE 3.33FIGURE 4.5(PML)FIGURE 5.5FIGURE 6.6FIGURE 7.7Figure 8.8FIGURE S1.1FIGURE S2.2FIGURE S3.3Tables 8