doi:10.1038/nature10244 a O07391_MYCAV/127-243 NLPC_HAEIN/80-181 SPR_SHIFL/79-183 P74160_SYNY3/112-245 O24914_HELPY/301-437 Q51835_PORGI/68-178 DPP6_BACSH/163-263 YKFC_BACSU/185-292 YDHO_ECOLI/153-263 YAFL_ECOLI/131-241 PGDS_BACSU/178-285 LYTE_BACSU/232-333 LYTF_BACSU/385-486 CWLS_BACSU/311-412 Q9RIE1_YERPE/136-252 Q9FZU5_9VIRU/118-229 Q9MCU3_BPHK0/116-231 Q9I5S7_PSEAE/119-250 CWLO_BACSU/355-469 O07390_MYCAV/156-272 P60_LISGR/408-510 YDDH_BACSU/221-327 Q47734_ENTFA/224-330 P54_ENTFC/414-516 P60/364-466 Spr/79-183 Tse1/24-108 b G V P - Y S W G G F D C S G L M R Y G F A G V - G V L I - P R G D L I F Y H V T M Y L G M L E A S G G T R - Y R M G G I D C S A F M Q T T F S E V F G I E L - P R G D L V F F H V G V Y I G F M H A S T G V R - Y R L G G I D C S G F V Q R T F R E Q F G L E L - P R G D L V L F H V G I Y I G F V H A S T E N Y - Y L W G G Y D C S G L M Q A A F A S Q - G I W L - P R G D L I F F H V G M Y L G Y I H S S G G Q K - Y G W G G R D C S A F T R D S F A N - F G I L L - P R G T L I Y L H I M L Y L G V A H S I W G K P - Y R Y Q G M D C S G Y V A Y L Y S C - Y D I H I - P R G D L L F F H V A L L I E M L H N T N A T P - Y R W G G I D C S G L C S M A Y L L N - G V I I - F R G D L L F F H V A L Y L G Y V H A S L G L P - Y L W G G F D C S G F M Y S I F K A N - G Y S I - P R G D L L F F H V G L Y V G M L H S P K G K P - Y R W G G F D C S G L V Y Y A Y K D L V K I R I - P R G D L V F F H V G V Y V G F I Q S P R G K P - Y V W G G F D C S G L V F Y A Y N K I L E A K L - P R G D L L F F H M G V Y L G F I E S P R G V P - Y V F G G F D C S G L V Q Y V F Q Q A L G I Y L - P R G D V V Y F H A G I Y A G F I Q A S R G T P - Y K W G G F D C S G F I S W Y V L N K Q - T S V - G R G D F V F F H M G I Y I G F I H A G S G V P - Y R W G G F D C S G F I Y Y V L N K V - - T S V - S R G D F V F F H V G I Y L G F I N A N D G V P - Y R W G G F D C S G F I Y Y L I N N V - - S S I - S R G D F V F F H M G I Y L G F I H A S S G R P - Y V F G V F D C W M L C R D Y L K R E F N V E L - N P G D V F F I H C A V Y I G I L H H Q I G R V - F E Y G K T D C G A L V R D A F M L - M G L V F - P D G D V V L T H A V L Y L G M L H H A - E R P - F V L G H F D C W G L V M S Y F R Q T H G I E L - H D G D L V I M H A G I L L E L L H H L Y G R E - F A H G V L D C W S L C R D W Y R R E A G L E L - P D G D M L V M H A G I Y L G L L H H L Y G Q S P Y K F G G F D C S S F V R W A Y A S A - G V N L G P V G D L V F F H V G I Y L G F L N D N T G V P - Y S W G G F D C S G L V L Y S F A G V - G I K L - P H G D V I F Y H V T I Y L G M L E A P D G K P - Y T W G A F D C S G F T S Y V F N Q V - G L S L - S G G D L V F F H V G I Y I G M I D A Q D G Q P - Y A W G G F D C S G L V Q W S F A K A - G I T L - P R G D L V F F H V G I Y V G M F N S N D G W R - Y V Y G G F D C S G L T Q W T Y G K - A G I N L - P R G D L V F F H V G I Y L G M F H A G D G T P - Y V W G G F D C S G F T R Y V Y L Q - - - V T - - G R G D L L F W H V A I S L G Y I H A P Q G K A - Y S W G G F D C S G F T K Y V F A K S - G I S L - P R G D L V F F H V G I Y V G M I N A Q D G T P - Y R Y G G V D C S G F V V V T M R D R F D L Q L - P R G D L V F F H V G I Y D T F I H A S T D K S - Y L A G T D N C S G F V Q S V A A E - L G V P M - P R G F L V I A H V A V V I S Y R Q K Y P Motif I Motif II Motif III B2NBT5_ECOLX/79-207 MLTC_ECOLI/194-322 Q46781_ECOLX/12-138 IAGB_SALTI/20-139 IPGF_SHIFL/19-138 O07378_9ZZZZ/40-167 PBL_ECOLI/27-153 O85953_SPHAR/102-211 VP07_BPPRD/9-117 YJBJ_BACSU/62-172 A6PZ97_SALSA/65-185 O25362_HELPY/390-508 SLT_HAEIN/430-549 YQBO_BACSU/1351-1479 O03937_9CAUD/1378-1501 Q38352_BPLLH/146-268 O64282_9CAUD/1335-1457 B9TU22_GADMO/78-202 O83631_TREPA/378-493 O26092_HELPY/80-195 O67414_AQUAE/86-201 MLTD_ECOLI/102-218 LYG_CYGAT VIRB1_AGRT5 SLT70/482-602 GEWL/50-170 Tse3/220-385 A I I A I E S G G N P N A V S A I M Q T E S S F N P Y A V S S V M T V E G G - K P G S V S A I A Q Q E S A M K P G A I G A I A E K E S G F N K S A V N A I S A G E S S L R P G A I N A I A L V E S N L K K D S I G A V V A Q E S G Y K A W A I S G V V Q T E S S G N P R T T S A V I K Q E S G F N A K A V S G I I S R E S R A G S A L D H A I A R Q E S F L L P A V I S A I A R Q E S A W N P M A Q S T I A Q H E S G G N P K A I N K V I K R E S N G D P S V I N A T I Q R E S G G N P R A I N A T I Q K E S G G N P N A Q N A I I S R E S R A G N V I P P F L P V V E S G F L E R A V S F L A M A E S K F S S R A Y S Y L P I V E S M Y N P F A V S L L P I V E S A F D P H A T S G I I S R E S H A G K V L K N A I A Q V E S R F D P L A V H A I A R Q E S A W N P K V K S G I I S R E S H A G K V L K N A I I L A E Q R D Q T R D E D A I G L M Q L K A S T S A L G L M Q V V Q H T A D L G I M Q I N T H A W D L G L M Q I N S F H M D Y G I M Q I N D F H S D Y G L M Q I N S T H I D Y W L M Q I N Q M H I A M G M M Q V M P G T A A M G L M Q L M P A T A A M G L M Q L M P S T A G F G L M Q V D K R Y H 20 A L G L M Q I M P F N V A R G L M Q L L P S T A S Q G L M Q T I P S T F S K G L V Q T I Q P T F S K G L M Q T I D P T F S I G L M Q T I G R T F G F G L M Q V D K R Y H A V G I W Q F M R N S I A V G I W Q F M P S T A A A G I W Q L M P Q T A A A G I W Q I I P S T G G F G L M Q V D K R S H D V G L M Q I N S R N F A S G L M Q I M P G T A G F G L M Q V D K R S H S I G L G Q V V V S T A L V V S Y A N G V I T A Y N G G G V G R Y H S A A V G A Y N A G A V G A Y N A G C L G S Y N A G C L G T Y N A G A L A A Y N A G A L L A Y H G G A L A A Y N A G G I S A Y N A G V A Y A Y N A G I A A A Y N A G P Y V G Y A N G G P M G Y D S G A A Y G Y A K G A T H G Y A N G G I A A Y N T G A L A A Y N C G V A M A Y N Y G V L A A Y N C G T V A A Y N S G G I S A Y N A G A I S A Y N T G S S A A Y N A G G I S A Y N A G D L R A Y A - G Supplementary Figure 1. Tse1 and Tse3 contain amino acid motifs belonging to cell wall amidase and muramidase enzyme families, respectively. (a) Partial amino acid alignment of Tse1 with representative amidase family members. Motifs previously identified as conserved in this family are shown 1. Putative catalytic residues are denoted by a star. The third catalytic residue is variant, with the requirement of a polar or acidic residue. Protein names in both a and b represent Swiss-Prot 2 entries, with the exception of those proteins represented in Figure 1a. Numerical values following Swiss-Prot entries provide amino acid residue boundaries of the respective catalytic domains. (b) Partial amino acid alignment of Tse3 with goose egg white lysozyme (GEWL) and closely related representative lytic transglycosylase family 1 members. Motifs previously identified as conserved in this muramidase family are shown 3,4. The putative catalytic glutamic acid is denoted by a star. WWW.NATURE.COM/NATURE 1
kda 50 Tse1 Tse1* Tse3 Tse3* 37 25 20 15 10 Supplementary Figure 2. Gel electrophoretic analysis of purified Tse proteins. SDS-PAGE analysis of Ni 2+ -affinity purified Tse1, Tse3, and respective catalytic amino acid substitution mutants (*). Each lane was loaded with 10 μg of the indicated protein. Proteins were visualized by Coomassie Blue. 2 WWW.NATURE.COM/NATURE
200 Absorbance at 205 nm (A.U.) B A Tetra 1 2 TetraTetra 0 40 80 120 Time (min) Tetra Pellet TetraTetra no enzyme Tse1 Tse1* C GlcNAc GlcNAc Tetra, 3 (Di), 1 D-Glu 4 GlcNac TetraTetra GlcNac Supernatant GlcNAc (Tetra-- ), 2 GlcNAc MurNAc GlcNAc GlcNAc MurNAc GlcNAc MurNAc 5 no enzyme 100 GlcNAc Absorbance at 205 nm (A.U.) 4 3 5 HMW Tse3 Tse3* 0 40 80 120 Time (min) 0 40 80 120 Time (min) Supplementary Figure 3. Peptidoglycan hydrolase activity of Tse1 and Tse3. (a) E. coli peptidoglycan was incubated with Tse1, Tse1* or no enzyme followed by incubation with cellosyl. The resulting muropeptides were reduced with sodium borohydride and separated by HPLC. Tse1, but not Tse1*, fully digested TetraTetra giving rise to two new compounds (1 and 2) that were subsequently identified by mass spectrometry as GlcNAc---D-Glu (Di, 1) and GlcNAc----(-)- (Tetra--, 2), indicative of amidase (DL-endopeptidase) activity of Tse1. (b) E. coli peptidoglycan was incubated with Tse3, Tse3* or no enzyme and the sample was centrifuged. The pellet samples were digested with cellosyl to solubilize the muropeptides. Peptidoglycan fragments present in supernatant and cellosyl-digested pellet samples were reduced with sodium borohydride and separated by HPLC. Fractions 3-5 from the Tse3 supernatant sample were identified by MS/MS analysis as Tetra (3), Tetra2 (4) and Tetra3 (5), consistent with Tse3 exhibiting muramidase activity. The supernatant of the Tse3, but not the Tse3*-treated sample, contained soluble high molecular weight peptidoglycan fragments eluting in a broad series of unresolved peaks between 80-120 min (HMW). (c) Proposed structures of Tse products 1-5. Abbreviations used: GlcNAc, N-acetylglucosamine; N-acetylmuramitol (reduced N-acetylmuramic acid);, L-alanine; D-Glu, D-glutamic acid;, D-isoglutamic acid;, meso-diaminopimelic acid;, D-alanine. The molecular masses summarized in Supplementary Table 1 as well as MS/MS spectra (not shown) support peak assignments for the Tse products. The MS/MS data confirmed the Tse1-mediated cleavage to occur on the donor peptide of the crosslink, giving rise to product 2 with a nonlinear hexapeptide. WWW.NATURE.COM/NATURE 3
a b c 0.5 Cyto Peri Cyto Peri Optical density (600 nm) 0.4 0.3 0.2 0.1 control Tse1 Tse3 0 0 1 2 3 4 5 6 7 Time (hrs) Tse1* Tse3* α-his 5 α-β-lac α-his 5 α-β-lac peri-tse1* peri-tse3* α-his 5 α-β-lac α-his 5 α-β-lac d Tse1 peri-tse1 peri-tse1* α-his 5 Tse3 peri-tse3 peri-tse3* Supplementary Figure 4. Periplasmically-localized Tse1 and Tse3 promote the lysis of E. coli cells. (a) Growth in liquid media of E. coli expressing native Tse1 and Tse3 enzymes. Cultures were induced at the indicated time (arrow). (b, c) Western blot analysis of subcellular distributions of the indicated Tse* proteins in E. coli. To avoid cellular lysis during sample preparation, the catalytically inactive Tse variants (Tse*) were used in this experiment. Native proteins (b) and those containing an N-terminal PelB leader sequence (c) were analyzed. Equivalent fractions of the cytoplasmic (Cyto) and periplasmic (Peri) samples were loaded in each panel. The oligohistidine-tagged Tse proteins were detected with an α-his 5 antibody. RNA polymerase (RNAP) and β-lactamase (β-lac) enzymes were used as cytoplasmic and periplasmic fractionation controls, respectively. (d) Western blot analysis of total Tse protein levels for strains used in experiments presented in Fig. 1e and Supplementary Fig. 4a. RNA polymerase was used as a loading control. Error bars ± s.d. (n=3). 4 WWW.NATURE.COM/NATURE
peri-tse1 peri-tse3 peri-tse1(c30a) peri-tse1(e250q) vector vector Supplementary Figure 5. Periplasmic Tse1 and Tse3 promote the lysis of E. coli and produce distinct cellular morphologies. Full fields of phase contrast micrographs from Fig. 1f; additional vector controls also shown. Scale bars correspond to 10 μm. WWW.NATURE.COM/NATURE 5
parental Δtse1 ΔclpV1 + tsi1 Δtse3 ΔclpV1 + tsi3 + tsi3 SS 25 20 Growth (generations) 15 10 ** ** ** 5 0 Δtsi1 Δtsi3 Supplementary Figure 6. Quantification and complementation of intercellular self-intoxication of P. aeruginosa. Indicated strains were grown as in Fig. 3c and their growth was quantified. Asterisks denote strains that grew significantly less than the rets parental strain (P <0.01). Error bars ± s.d. (n=3).` 6 WWW.NATURE.COM/NATURE
a tsi3 tsi3 SS α-vsv G b Total Beads Tse3-His 6 IP: α-vsv G WB: α-his 5 Tse1-His 6 Supplementary Figure 7. Signal sequence-deficient Tsi3 retains function in vitro. (a) Western blot analysis indicating expression levels of Tsi3 and Tsi3 SS in P. aeruginosa rets tsi3 under induction conditions used in Supplementary Fig. 6. RNA polymerase was used as a loading control. (b) Western blot analysis of hexahistidine-tagged Tse proteins ( His 6 ) in total and bead-associated fractions of an α-vsv G immunoprecipitation of the VSV G-fused Tsi3 SS protein from E. coli. WWW.NATURE.COM/NATURE 7
Supplementary Table 1. Masses of reduced muropeptides in HPLC fractions. Muropeptide No 1 Proposed Neutral Mass (Da) of reduced Retention Product of compounds (HPLC fractions) Structure time (min) calculated determined 1 Di Tse1 + cellosyl 36.35 698.2858 698.2865 2 Tetra(Ala-Dap) Tse1 + cellosyl 36.95 1184.5296 1184.5370 3 Tetra Tse3 31.60 941.4077 941.4123 4 Tetra 2 Tse3 56.68 1862.7892 1862.8096 5 Tetra 3 Tse3 68.50 2784.1707 2784.1929 1 Numbers correspond to peak numbers in Figure 1b,d. 8 WWW.NATURE.COM/NATURE
Supplementary Table 2. Oligonucleotides used in this study. Number Sequence 1 628 5 -TCAATCAGTATCTAGACTGGAAATCGTGCCCCAGTTC-3 629 5 -AACTCGAGCCGCAAGCATGCTGAAGTCCATGTATCACCTATGCGTG-3 630 5 -TTCAGCATGCTTGCGGCTCGAGTTAGTTGATTCAGGCCGTGCTGC-3 631 5 -TGTTAAGCTAAAGCTTGTCGTTGTTGCCTTTCACGTC-3 721 5 -TCAATCAGTATCTAGACTGCTCAGGTTGAGCTGGCAG-3 722 5 -AACTCGAGCCGCAAGCATGCTGAATTTCATGGGGCGGGTTCTCCG-3 723 5 -TTCAGCATGCTTGCGGCTCGAGTTAAATGATCCGGGCCGGAGCGC-3 724 5 -TGTTAAGCTAAAGCTTCTGGCCCTGGGCAGGCTGCAAC-3 735 5 -TCAATCAGTATCTAGACATGAGCGTCGGCGACCTGCTG-3 736 5 -AACTCGAGCCGCAAGCATGCTGAAGATCAGGTCGCTGGTGGCGGTC-3 737 5 -TTCAGCATGCTTGCGGCTCGAGTTGACCCTGGAATGAGGTTCCCATG-3 738 5 -TGTTAAGCTAAAGCTTCGAAAAAGCGCTACCGCAACC-3 835 5 -TTCAGCATGCTTGCGGCTCGAGTTCAATAGAACCGCTCTCTGCGGAG-3 836 5 -TGTTAAGCTAAAGCTTGTTGCGCTGGGCGAGGTAGGCCAG-3 1289 5 -TAGAACAGCATATGGACAGTCTCGATCAATGC-3 1290 5 -ACTATTACCTCGAGACTGGCCCTGGGCAGGCTGCAAC-3 1291 5 -TAGAACAGCATATGACCGCCACCAGCGACCTG-3 1292 5 -ACTATTACCTCGAGTGGGAACCTCATTCCAGGGTC-3 1469 5 -TAATGGTACCCCCAACGGAGAACCCGCCCC-3 1470 5 -TAATTCTAGATTTCTTTGCGGTCTGGCAGAACGG-3 1472 5 -TAATGGTACCGCCACCTTCCTCGACCCTGG-3 1473 5 -TAATTCTAGATTGCAACCTGGCCCGCTCC-3 1475 5 -TAATGGATCCGGACAGTCTCGATCAATGCATCGTCAACG-3 1476 5 -TAATCTCGAGTGGGAACCTCATTCCAGGGTCGAG-3 1477 5 -TAATGGATCCGGACAGTCTCGATCAATGCATCGTCAACG-3 1478 5 -TAATCTCGAGACTGGCCCTGGGCAGGC-3 1479 5 -CGAACAAGGACAACGCTTCCGGCTTCGTC-3 1480 5 -GACGAAGCCGGAAGCGTTGTCCTTGTTCG-3 1481 5 -CGATCATCCTCGCCCAGCAGCGTGACCAG-3 1482 5 -CTGGTCACGCTGCTGGGCGAGGATGATCG-3 1485 5 -TAATTCTAGAGCCGATCAGCACGCTGTTCC-3 1486 5 -GCAACCTGGCGACTGTCTTCATGGGAACCTCATTC-3 1487 5 -GAAGACAGTCGCCAGGTTGCAATAGAACCGC-3 1488 5 -TAATGAGCTC GTGAGGCGATAGCGGGTCAG-3 1497 5 - TAGTACAGTACATATGAAACTGCTCGCCGGCAGCTTC-3 1498 5 -TAGTACAGTACATATGAAGACAGTCGCCCTGATTCTC-3 1522 5 -TCAAGTACTAGAGCTCACGGGAGGAAAGATGGGGGTGGACTTCGACAAGACC-3 1 Restriction enzyme sites are underlined. WWW.NATURE.COM/NATURE 9
Supplementary References 1 Anantharaman, V. & Aravind, L. Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes. Genome biology 4, R11 (2003). 2 Boeckmann, B. et al. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic acids research 31, 365-370 (2003). 3 Scheurwater, E., Reid, C. W. & Clarke, A. J. Lytic transglycosylases: bacterial space-making autolysins. Int J Biochem Cell Biol 40, 586-591, (2008). 4 Dijkstra, B. W. & Thunnissen, A. M. 'Holy' proteins. II: The soluble lytic transglycosylase. Current opinion in structural biology 4, 810-813 (1994). 10 WWW.NATURE.COM/NATURE