FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

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1 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::p spank -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::p spank (erm)]. pjm030 [saca::p veg -mcherry (spc)] was generated by ligation with an EcoRI-BamHI P veg -mcherry fragment from per069 and pkm070 (saca::spc) cut with the same enzymes. per069 [saca::p veg -mcherry (phleo)] was generated by ligation with an EcoRI-HindIII P veg PCR product (amplified with oligonucleotide primers ojm73 and ojm74 and PY79 genomic DNA). pjm031 [his 6 -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 [his 6 -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 [his 6 -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.

2 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::p spoiie -lacz (cat)] was generated by ligation with an EcoRI-BamHI P spoiie PCR product (amplified with oligonucleotide primers ojm318 and ojm319 from PY79 genomic DNA) and pdg1661 [amye::lacz (cat)] (Guerout-Fleury et al., 1996). pjm063 [his 6 -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 Meisner et al. 2

3 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::p spank -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::p spank (erm)] cut with SpeI and SphI. BJM362 [ycgo::p spank -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: Guerout-Fleury, A. M., N. Frandsen & P. Stragier, (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180: Meisner et al. 3

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 (P veg ) were grown in CH medium at 37 C to midexponential 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, P spank -cwlo, P veg -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 ) (10 µm) was incubated with (A) or without (B) His-tagged FtsX loop1 (residues ) (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. Meisner et al. 4

5 Meisner_Fig. S1 percentage WT (BJM172) ΔlytE (BJM186) ΔcwlO (BJM188) ΔftsEX (BJM190) ΔftsEX ΔcwlO (BJM212) cell length (µm)

6 Meisner_Fig. S2 ΔlytE, CwlO cytoplasm merge 0 min

7 Meisner_Fig. S3 A L S W1 W2 E CwlOΔSS His-FtsX(loop1) B L S W1 W2 E CwlOΔSS

8 Meisner_Table S1 Table S1. B. subtilis strains Strain BJM1 BJM76 BJM58 BJM54 BJM96 BJM68 BJM70 BJM148 BJM72 BJM104 BJM550 BJM949 BJM552 BJM386 BJM495 BJM493 BJM526 BJM491 BJM125 BJM133 BJM135 BJM137 BJM570 BJM172 BJM186 BJM188 BJM190 BJM212 BJM318 BJM754 BJM372 BJM433 BJM406 BJM967 BJM969 BJM874 BJM898 BJM902 BJM900 BJM955 BJM957 BJM827 BJM912 BJM861 BJM857 BJM863 BJM929 BJM663 Genotype prototrophic wild-type (PY79) lyte::cat lyte::kan cwlo::cat cwlo::kan ftsex::kan ftse::kan ΔftsE ftsx::kan lytf::cat cwls::cat sigi::cat mrebh::cat lyte::cat, ycgo::pspank-lyte (erm) lyte::cat, ycgo::pspank-lyte (erm), cwlo::kan lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan lyte::cat, ycgo::pspank-lyte (erm), ΔftsE lyte::cat, ycgo::pspank-lyte (erm), ftsx::kan ftsex::kan, ycgo::pspank-ftsex (erm) ftsex::kan, ycgo::pspank-ftsex (erm), lyte::cat ftsex::kan, ycgo::pspank-ftsex (erm), cwlo::cat ftsex::kan, ycgo::pspank-ftsex (erm), lytf::cat ftsex::kan, ycgo::pspank-ftsex (erm), cwls::cat swra+ saca::pveg-mcherry (spc) swra+ saca::pveg-mcherry (spc), lyte::cat swra+ saca::pveg-mcherry (spc), cwlo::cat swra+ saca::pveg-mcherry (spc), ftsex::kan swra+ saca::pveg-mcherry (spc), ftsex::kan, cwlo::cat swra+ saca::pveg-mcherry (spc), ftsex::kan ycgo::pspank-ftsex (erm) swra+ saca::pveg-mcherry (spc), ftsex::kan ycgo::pspank-ftsex (erm) lyte::cat cwlo::cat, ycgo::pspank-cwlo (erm) cwlo::kan, ycgo::pspank-cwlo (erm) cwlo::cat, ycgo::pspank-cwlo (erm), lyte::kan cwlo::kan, ycgo::pspank-cwlo (erm), sigi::cat cwlo::kan, ycgo::pspank-cwlo (erm), mrebh::cat amye::pspoiie-lacz (cat) amye::pspoiie-lacz (cat), lyte::kan amye::pspoiie-lacz (cat), cwlo::kan amye::pspoiie-lacz (cat), ftsex::kan ftsex::kan, ycgo::pspank-ftsex (erm), sigi::cat ftsex::kan, ycgo::pspank-ftsex (erm), mrebh::cat lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan amye::ftsex lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan amye::ftse(k41m) ftsx lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan amye::ftse(k41a) ftsx lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan amye::ftse(d162n) ftsx lyte::cat, ycgo::pspank-lyte (erm), ftsex::kan amye::ftse(d162a) ftsx cwlo::cat, ycgo::pspank-cwlo coiled-coil ( ) (erm) amye::pxyla-gfp-mbl (spc)

9 BJM1007 BJM837 BJM839 BJM435 BJM692 BJM667 BJM677 amye::pxyla-gfp-mbl (spc), lyte::cat amye::pxyla-gfp-mbl (spc), ftsex::kan, ycgo::pspank-ftsex (erm) amye::pxyla-gfp-mbl (spc), ftsex::kan, ycgo::pspank-ftsex (erm), lyte::cat swra+ saca::pveg-mcherry (spc), cwlo::kan ycgo::pspank-cwlo (erm) swra+ saca::pveg-mcherry (spc), cwlo::kan ycgo::pspank-cwlo (erm) lyte::kan amye::pxyla-gfp-mbl (spc), cwlo::cat, ycgo::pspank-cwlo (erm) amye::pxyla-gfp-mbl (spc), cwlo::cat, ycgo::pspank-cwlo (erm), lyte::kan

10 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 ( )

11 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

12 ojm232 ojm318 ojm319 ojm321 ojm307 ojm308 ojm309 ojm133 ojm117 ojm114 ojm115 ojm156 ojm157 ojm320 CGCGGATCCTCACTGGAACGGCTTTTTCAC CGCGAATTCATCCTAACAAATCGGTTTCTC CGCGGATCCACCTGTTATATTCGTTGCCTG GGTCCGAGCGGAGCAGGTATGTCTACTTTTATTAAAATGATTTAC GGTCCGAGCGGAGCAGGTGCATCTACTTTTATTAAAATGATTTAC ACCCTGATGTTGTCATTGCTAACGAACCGACAGGAAACCTTGA ACCCTGATGTTGTCATTGCTGCAGAACCGACAGGAAACCTTGA CTCACAGAGAACAGATTGGTGGTGAAACATTAGATGAAAAGAAACAAAAAATCG CTTCCTGCAGTCACCCGGGCTTATTGAACAACACGTCTTACAACAC GCTCTTCTGGTATGATTAAAATTCTCGGGCGCCAC CGGCTCGAGTTATACTCGCAGAAACTTGCGGA CTCACAGAGAACAGATTGGTGGTATGATAGAGATGAAGGAAGTATATAAAGC CTTCCTGCAGTCACCCGGGCGAATTTTAATCATATGAACCATACTCC CTCACAGAGAACAGATTGGTGGTGGCACTGTTATCAGCAACTC