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1 Supplementary Information Arginine-rhamnosylation as new strategy to activate translation elongation factor P Jürgen Lassak 1,2,*, Eva Keilhauer 3, Max Fürst 1,2, Kristin Wuichet 4, Julia Gödeke 5, Agata L. Starosta 1,6, Jhong-Min Chen 7, Lotte Søgaard-Andersen 4, Jürgen Rohr 7, Daniel N. Wilson 1,6, Susanne Häussler 5,8, Matthias Mann 3 & Kirsten Jung 1,2* 1 Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, D Munich, Germany 2 Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, D Martinsried, Germany 3 Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, D Martinsried, Germany 4 Max Planck Institute for Terrestrial Microbiology, Marburg 35043, Germany 5 Institute for Molecular Bacteriology, Twincore, Centre for Clinical and Experimental Infection Research, a joint venture of the Helmholtz Centre of Infection Research and the Hannover Medical School, Hannover, Germany 6 Gene Center, Department for Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany 7 Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY , USA. 8 Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany * Corresponding authors: juergen.lassak@lmu.de, jung@lmu.de

2 Supplementary Results Supplementary Fig. 1. Phylogenetic tree of sequences containing the trna-synt_2 domain. The predominant domain architectures of the sequences corresponding to the four main subfamilies are shown. Branches in red indicate sequences that are encoded within four genes of a EF-P homolog. The EpmA subfamily is highlighted in grey.

Supplementary Fig. 2. Phylogenetic tree of sequences containing the Radical_SAM domain that are encoded within a distance of four genes of efp or epma homologs.

3 Supplementary Fig. 2. Phylogenetic tree of sequences containing the Radical_SAM domain that are encoded within a distance of four genes of efp or epma homologs. Two distinct families were identifiable: the EpmB family, and a divergent clade of Radical SAM sequences that are not predicted to be associated with EF-P. The divergent Radical SAM clade exhibits long branch lengths indicative of divergence, and branches shown in red indicate sequences of the divergent Radical SAM clade that has homologs in genomes of the EpmB family. Sequences from the divergent Radical SAM clade were excluded from use as queries in further BLASTP analyses (see method section).

Supplementary Fig. 3. Phylogenetic tree of a subset of sequences that contain the Radical_SAM domain. The middle and innermost rings indicate if epmb is genetically coupled to epma or efp homologs (i.

4 Supplementary Fig. 3. Phylogenetic tree of a subset of sequences that contain the Radical_SAM domain. The middle and innermost rings indicate if epmb is genetically coupled to epma or efp homologs (i.e. encoded within a distance of four genes of each other), or if there are uncoupled epma or efp homologs encoded elsewhere in the genome associated with the given sequence. Sequences defined as EpmB homologs based on genome context and gene distribution data are indicated by black in the outermost ring.

5 Supplementary Fig. 4. Phylogenetic tree of EF-P homologs. Black in the outermost ring indicates sequences collected for more detailed analysis. The remaining rings indicate distribution of and coupling with EarP, EpmA, EpmB, and EpmC homologs. EarP, EpmA, EpmB, or EpmC are considered coupled to a given EF-P sequence if their corresponding genes are encoded within four genes of the corresponding efp gene. EarP, EpmA, EpmB, or EpmC are considered uncoupled from EF-P if their corresponding gene are encoded farther than four genes from the corresponding efp gene. Blue branches indicate a lysine at position 32/34. Black branches depict sequences used to generate Supplementary Fig. 5.

Supplementary Fig. 5. Phylogenetic subtree of EF-P homologs co-occurring either with homologs of EpmABC or EarP. Phylogenetic tree derived from the one presented in Supplementary Fig.

6 Supplementary Fig. 5. Phylogenetic subtree of EF-P homologs co-occurring either with homologs of EpmABC or EarP. Phylogenetic tree derived from the one presented in Supplementary Fig. 4 based only on the subset EF-Ps that co-occurrs either with EpmA (red), EpmB (green) or EarP (purple). Distribution of EF-P hydroxylase EpmC and dtdp-rhamnose biosynthesis protein RmlD is coloured with blue and grey squares, respectively. Evolutionary distinct branches of EF-P are marked either in purple (newly identified) or black (EpmAB(C) lysine type). Genetic coupling of efp and the genes encoding corresponding modification enzymes on the chromosome is highlighted by a dark colour shade. Genetically non-coupled genes are highlighted with a light colour shade.

7 Supplementary Fig. 6. Functional importance and specificity of EF-P E.c. /EF-P S.o. and their modification systems. (a) Growth analysis of S. oneidensis wildtype (wt), efp S.o., earp S.o. and efp S.o. / earp S.o.. Doubling times were calculated from exponentially grown cells in LB harboring either a chromosomal non-tagged or plasmid borne His 6 -tagged copy of efp S.o. (R32), earp S.o., a combination of both or EF-P S.o. -R32A/K (R32A, R32K) substitution variants. Western Blot analysis was performed using EF-P-specific antibodies. Arrows indicate the size of both native and recombinant His 6 -EF-P S.o. The genetic context of efp S.o. is schematically shown above the diagram. Data represent mean values from three independent replicates ± s.d. (b) β-galactosidase activity of E. coli P cadba -lacz reporter wildtype (wt) and efp E.c. deletion strain ( efp E.c. ). Left: cross complemented with S. oneidensis MR-1 efp (R32) and respective Arg to Ala (R32A) or Lys (R32K) substitution variants alone or in combination with EarP S.o.. Right: complemented with EF-P E.c. (K34) or the corresponding variants EF-P E.c. -K34A (K34A) or EF-P E.c. -K34R (K34R) in the presence or absence of EarP S.o.. Cells were incubated under cadba inducing conditions. Western Blot analysis was performed using His 6 -specific antibodies. Arrows indicate both the size of recombinant His 6 -EF-P E.c. and His 6 -EF-P S.o.. Data represent mean values from three independent replicates ± s.d. (c) Growth analysis of E. coli wt, and efp E.c.. Doubling times were calculated from exponentially grown cells in LB harboring a plasmid borne copy of efp E.c. or an EF-P E.c. -K34A/R substitution variant either alone or in combination with earp S.o. (EarP S.o. ). Western Blot analysis was performed using His 6 -specific antibodies. Arrows indicate both the size of recombinant His 6 -EarP S.o. and His 6 -EF-P S.o.. Data represent mean values from three independent replicates ± s.d.

8 Supplementary Fig. 7. Identification of S. oneidensis MR-1 EF-P modification. In vivo modification of recombinant C-terminally His 6 -tagged EF-P S.o. upon homologous overproduction. Dependent peptide MS analysis. (a-h) Eight high-confidence modified R32-containing dependent peptides were identified with a M mass shift of Da when compared to the corresponding unmodified base peptide. Peak colours: red/ blue/light blue y,b and a-ions, light green molecular ion, yellow neutral losses, purple internal fragments, green immonium ions, black peaks unassigned.

9 Supplementary Fig. 8. MS analysis of in vivo produced EF-P S.o. Dependent peptide analysis of EF-P, produced in the absence (a) or presence (b,) of EarP. As expected, no modified peptides were found when EF-P was produced without EarP. EF-P was found to be modified on R32 when produced in the presence of EarP, however both arginine substitution mutants were only identified with unmodified peptides. (Best scoring spectra shown). Homologous EF-P S.o. production and purification is depicted as scheme. Peak colouring as in Supplementary Fig. 7.

10 Supplementary Fig. 9. MS confirmation of R32 modification. Defining rhamnose-h 2 O (Rh) with a molecular composition of C 6 H 10 O 4 (corresponding to a mass of Da) as a variable modification on arginine resulted in the identification of several modified EF-P S.o. peptides carrying a rhamnosylation on R32. (Best scoring spectrum shown). Peak colouring as in Supplementary Fig. 7.

11 Supplementary Fig. 10. MS analysis of in vivo produced EF-P S.o. R32A and R32K substitution variants. Dependent peptide analysis of EF-P S.o. R32A (a) and R32K (b) substitution variants produced in the presence of EarP. As expected, no modified peptides were found when EF-P was produced without EarP. EF-P was found to be modified on R32 when produced in the presence of EarP, however both arginine substitution mutants were only identified with unmodified peptides. (Best scoring spectra shown). Peak colouring as in Supplementary Fig. 7.

12 Supplementary Fig. 11. MS analysis of in vitro rhamnosylation of recombinant C-terminally His 6 -tagged EF-P S.o. (a) in vitro rhamnosylation scheme and corresponding SDS Page. Arrows depict the height of EarP S.o. and EF-P S.o. (b, c, d) MS analysis defining C 6 H 10 O 4 (mass of Da, corresponding to L-rhamnose - H 2 O, abbreviated as Rh ) as a variable modification on arginine. This identified several R32-rhamnosylated EF-P S.o. peptides, when EF-P was incubated with both dtdp-l-rhamnose and EarP MS/MS spectrum of the best scoring identification is shown (b). In contrast to successful in vitro rhamnosylation of EF-P S.o. by incubation with both EarP S.o. and dtdp-rhamnose, only unmodified peptides were identified when EF-P S.o. was incubated with dtdp-rhamnose only (c) or EarP S.o. only (d). Peak colouring as in Supplementary Fig. 7.

13 Supplementary Fig. 12. EF-P and EarP are essential for Pseudomonas aeruginosa PAO1 pathogenicity. The production of Quorum Sensing-dependent virulence factors (a) rhamnolipids and (b) pyocyanin was assayed in cultures of P. aeruginosa wildtype control and the transposon mutants PA2851 (efp) and PA2852 (earp), complemented when indicated with the corresponding S. oneidensis MR-1 genes in trans.

14 Supplementary Table 1. Primers used in Name Sequence Restriction site Reference Sequencing Primers P1 T7 Promoter TAA TAC GAC TCA CTA TAG GG P2 T7 Terminator TAT GCT AGT TAT TGC TCA G P3 Seq33-Fw-100 GGC GTC CAC ACT TTG CTA TGC 1 P4 pbad-hisa-rev CAG TTC CCT ACT CTC GCA TG 1 P5 M13 (-21)uni TGT AAA ACG ACG GCC AGT P6 M13 (-29)rev CAG GAA ACA GCT ATG ACC Deletion primers P9 EpmA-us-Fw TCA CTG CCC ACC GCT GTT TGA P10 EpmA-ds-Rev GAA AAC ATA TGC TAC AGA ATG GCG C P11 efp-epmb-chk-fw GGC AAG CAA AAC AAG AAC GAT AAG G P12 efp-epmb-chk-rev CCT TAT CCG GCC TAC AGT TCA TTG C P13 EpmA-chk-Fw GCG CAA GAG GGC TAA ATT ATC AC P14 EpmA-chk-Rev TGC CCA CTG AAC TGC ACT GC P15 SO_2329-BamHI-us-Fw A GAA TAT GAC CTG CTG CTG TTC G BamHI P16 SO_2329-us-Rev GGT TTT GCA GCC CAA CAA AAG ATG TCC CAG TGG GAC G P17 SO_2329-ds-Fw CAT CTT TTG TTG GGC TGC AAA ACC GTT TAT TTG G P18 PspOMI-SO_2329-ds-Rev CGA TGG GCC CGG TTT CGG TGC CAG PspOMI AGT TAA GC P19 BamHI-dSO_2328-us-Fw GCA GGA TCC GCA TCT ATT GCC CTG BamHI CCC AGT P20 dso_2328-us-rev CGA GTA TCG ATT TCA AAC ATG ATC ACG TTA CCT GGA C P21 dso_2328-ds-fw CAT GTT TGA AAT CGA TAC TCG TAC TGG CGA ATT C P22 PspOMI-SO_2328-ds-Rev CGA TGG GCC CCC GGT TAA ATG GGG PspOMI TAT GTG AAA TGG P23 dso_ us-rev CAG TAC GAG TAT CGA TAC AAA AGA TGT CCC AGT GGG ACG P24 SO_2329-chk-Fw CGT ATA AGT GAA CTA GAC ATA AAG GAT AG P25 SO_2329-chk-Rev GAA TAA GTA CAA TCC AGA CGA TCT AGG P26 SO_2328-chk-Fw GGC AAG CAA GAG CAA AGA ACG A P27 SO_2328-chk-Rev CAA GTG CGG GTA TAG TAG TTG TCC P28 SO_3160-chk-Fw GGT GTT GGC ATG ACA GAT GAG G P29 SO_3160-chk-Rev GGG TGA ATA GCC TAC AAC TAA CTT GG P30 BamHI-SO_3160-us-Fw GCG GAT CCG TAA AGA TGG CGG CAC BamHI AGG CC P31 SO_3160-OL-us-Rev GCT AAC GGC GTT TGC CAG CTC GGT TTC AAT GCA CTT CAT P32 SO_3160-ds-OL-Fw GAA ACC GAG CTG GCA AAC GCC GTT AGC GCC TA P33 PspOMI-SO_3160-ds-Rev CGA TAT GGG CCC GCG CGA TCC CGA PspOMI TAG CGT G

15 EF-P and EarP expression plasmids P34 SacI-RBS-efp-Fw GCG ATG AGC TCA ATT AAC AAA TTT CAG AGG GCC TTA TGG P35 CGA CTC TAG ATT AGT GAT GGT GAT XbaI-efp-Eco-GS-His6- GGT GAT GGC TGC CCT TCA CGC GAG Rev AGA CGT ATT CAC P36 efp-k34r-ol-fw GTA AAA CCG GGT CGC GGC CAG GCA TTT P37 efp-k34r-ol-rev AAA TGC CTG GCC GCG ACC CGG TTT TAC P38 efp-k34a-ol-fw GTA AAA CCG GGT GCG GGC CAG GCA TTT P39 efp-k34a-ol-rev AAA TGC CTG GCC CGC ACC CGG TTT TAC P40 SacI-RBS-SO_2328-Fw GCA TAT GAG CTC GCT GCG CTG AAA P41 P42 P43 P44 P45 P46 P47 XbaI-SO_2328-GS-His6- Rev SO_2328-R32K-Fw SO_2328-R32K-Rev SO_2328-R32A-Fw SO_2328-R32A-Rev PciI-SO_2329_Fw XbaI-SO_2329-GS-His6- Rev TAG AAA ATC GAC CGT CTA GAT TAG TGA TGG TGA TGG TGA TGC GAG CCA ACG CGC TTT TTG AAT TCG CCA CTC GTT CTG GCA AAA ACG CTG CTA T ATA GCA GCG TTT TTG CCA GAA CGA G CTC GTT CTG GCG CGA ACG CTG CTA T ATA GCA GCG TTC GCG CCA GAA CGA G GTA CAT GTC AAC ATC CTC CAA CGC GTC C CGT CTA GAT TAG TGA TGG TGA TGG TGA TGC GAG CCG CTT TTT TTC ACG AAT TGA ACT AGC CG SacI XbaI SacI XbaI PciI XbaI This study

16 Supplementary Table 2. Plasmids used in Plasmid Feature / Construction comments Source Vector backbones and reporter plasmids pred/et λ-red recombinase in pbad24; Amp r GeneBridges pkd3 FRT-site flanked Cm r cassette containing plasmid, orir, Amp r 2 pnpts138-r6kt mobrp4 ori-r6k sacb; suicide plasmid for in-frame deletions; 3 Km r pbad24 Amp r -cassette, pbbr322 origin, arac coding sequence, ara 4 operator pbad33 Cm r -cassette, p15a origin, arac coding sequence, ara operator 4 pbbr1mcs-2 Kan r -cassette, pbbr broad host range origin of replication, mob 5 region for conjugative transfer pbbr1mcs-3-lacz Tet r -cassette, pbbr broad host range origin of replication, mob 6 region for conjugative transfer, lacz coding sequence p3lc-tl30 Translational CadC -LacZ fusion (sequence encodes 30 amino 1 acids of cadc) p3lc-tl30-3p p3lc-tl30 + sequence encoding 3 prolines (CCG codon) 1 Suicide plasmids to generate efp and earp deletions in Shewanella oneidensis MR1 p efp S.o. P19-P20 / P21-P22 SO_2328 Overlap deletion fragment into pnpts138-r6kt p earp S.o. P15-P16 / P17-P18 SO_2329 Overlap deletion fragment into pnpts138-r6kt p earp S.o. / efp S.o. P P15-P23 /P21-P22 SO_2329/28 Overlap deletion fragment into pnpts138-r6kt EF-P and or EarP encoding plasmids pbad33-efp E.c. -His6 P34 / P35 PCR fragment of C-terminal His 6 -Tag efp version from E. coli into pbad33 pbad33-efp E.c. -His6- P34-P39 / P35-P38 PCR Overlap fragment of C-terminal His 6 - K34A Tag efp substitution variant K34A from E. coli into pbad33 pbad33-efp E.c. -His6- P34-P37 / P35-P36 Overlap PCR fragment of C-terminal His 6 - K34R Tag efp substitution variant K34R from E. coli into pbad33 pbad33-efp S.o. -His6 P40 / P41 PCR fragment of C-terminal His 6 -Tag efp version from pbad33-efp S.o. -His6- R32A pbad33-efp S.o. -His6- R32K pbad24-earp S.o. -His6 pbbr1mcs-2-p earp - efp S.o. -His6 pbbr1mcs-2-p earp earp S.o. -His6 pbbr1mcs-2-p earp - earp S.o - efp S.o. -His6 pbbr1mcs-2-p earp - earp S.o - efp S.o. -His6- R32K pbbr1mcs-2-p earp - earp S.o - efp S.o. -His6- R32A E. coli into pbad33 P40-P45 / P41-P44 Overlap PCR fragment of C-terminal His 6 - Tag efp substitution variant R33A from S. oneidensis into pbad33 P40-P43 /P41-P42 Overlap PCR fragment of C-terminal His 6 -Tag efp substitution variant R33K from S. oneidensis into pbad33 P46/ P47 PCR fragment of C-terminal His 6 -Tag earp from S. oneidensis into pbad24 P15 / P41 PCR fragment of C-terminal His 6 -Tag efp from S. oneidensis into pbbr1mcs-2 including the P earp native operon promoter P15 / P47 PCR fragment of earp from S. oneidensis into pbbr1mcs-2 including the P earp native operon promoter P15 / P41 PCR fragment of of earp and C-terminal His 6 -Tag efp from S. oneidensis into pbbr1mcs-2 including the P earp native operon promoter P15-P45 / P44-P41 Overlap PCR fragment of earp and C- terminal His 6 -Tag efp from S. oneidensis into pbbr1mcs-2 including the P earp native operon promoter P15-P43 / P41-P44 Overlap PCR fragment of earp and C- terminal His 6 -Tag efp from S. oneidensis into pbbr1mcs-2 including the P earp native operon promoter

17 Supplementary Table 3. Strains used in Strain description Source Escherichia coli DH5αλpir reca1 enda1 gyra96 thi-1 hsdr17 supe44 7 WM3064 rela1 laczya-argf U169 80lacZ M15 λpir thrb1004 pro thi rpsl hsds lacz ΔM15 RP Δ(araBAD) 567ΔdapA 1341::[erm pir(wt)] LMG194 F - lacx74, gale thi rpsl phoa (Pvu II) ara714 leu::tn10 Strp r, Tet r MG1655 E. coli K-12 reference strain 8 MG-CR MG1655 ΔlacZ::tet rpsl150 ΔcadBA 9 P cadba ::lacz MG-CR-efp MG1655 ΔlacZ::tet rpsl150 efp::npt ΔcadBA This study P cadba ::lacz MG-CR-efp-fcl MG-CR-efp; fcl::cat This study MG-CR-efp-rmlD MG-CR-efp; rmld::cat This study BW25113 (arad-arab)567, lacz4787(::rrnb-3), lambda -, rph-1, (rhad-rhab)568, hsdr514 JW4107 BW25113 efp::npt 10 JW4116 BW25113 epma::npt 10 JW4106 BW25113 epmb::npt 10 BW-Δefp/epmA::npt BW25113 Δefp epma::npt This study BW-Δefp/epmB::cat BW25113 Δefp; epmb::cat This study Shewanella oneidensis MR-1 S79 Shewanella oneidensis MR-1 wildtype 11 efp S.o. SO_2328 This study earp S.o. SO_2329 This study efp S.o. / earp S.o. SO_2328/ SO_2329 This study rmlc S.o. SO_3160 This study Pseudomonas P. aeruginosa PAO1 wt PA4684 transposon mutant from the 12 control (PW8884) Washington Genome Center PA01 mutant library, Tc r PA2851 efp (PA2851) transposon mutant from the 12 Washington Genome Center PA01 mutant library, Tc r PA2852 earp (PA2852) transposon mutant from the 12 Washington Genome Center PA01 mutant library, Tc r Eukaryotic cell lines A549 hypotriploid human cell line with a modal chromosome number of 66, occurring in 24% of cells. Six markers are present in single copies in all cells: der(6)t(1;6) (q11;q27);?del(6) (p23); del(11) (q21), del(2) (q11), M4 and M5. Most cells had two X and two Y chromosomes. Chromosomes N2 and N6 had single copies per cell; and N12 and N17 usually had 4 copies. W. Metcalf, University of Illinois, Urbana-Champaign 4 10 ATCC CCL185

18 Supplementary Table 4. selected polyproline containing proteins from P. aeruginosa PAO1

19 Supplementary Dataset 1. Distribution of EF-P, EpmA, EpmB, EpmC, EarP, and RmlD homologs in the representative genome set. Taxonomy for all genomes is shown based on phyla or phyla and class for Proteobacteria. The genome indicates the species and strain in addition to the genome sequencing identifier (uid). The genome id indicates the short genome identifier composed of the species name and uid used in sequence analyses. The number of homologs of each particular protein of interest encoded in each genome are provided. Supplementary Dataset 2. EF-P, EpmA, EpmB, EpmC, EarP, and RmlD sequences identified in the representative genome set. Sequence identifiers correspond to the genome identifier in Table S1 and the locus tag of the sequence. 1. Ude, S. et al. Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches. Science 339, (2013). 2. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, (2000). 3. Lassak, J., Henche, A.L., Binnenkade, L. & Thormann, K.M. ArcS, the cognate sensor kinase in an atypical Arc system of Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 76, (2010). 4. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD promoter. J. Bacteriol. 177, (1995). 5. Kovach, M.E. et al. Four new derivatives of the broad-host-range cloning vector pbbr1mcs, carrying different antibiotic-resistance cassettes. Gene 166, (1995). 6. Fried, L., Lassak, J. & Jung, K. A comprehensive toolbox for the rapid construction of lacz fusion reporters. J. Microbiol. Meth. (2012). 7. Macinga, D.R., Parojcic, M.M. & Rather, P.N. Identification and analysis of aarp, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii. J. Bacteriol. 177, (1995). 8. Blattner, F.R. et al. The complete genome sequence of Escherichia coli K-12. Science 277, 1453-& (1997). 9. Ruiz, J., Haneburger, I. & Jung, K. Identification of ArgP and Lrp as transcriptional regulators of lysp, the gene encoding the specific lysine permease of Escherichia coli. J. Bacteriol. 193, (2011). 10. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2(2006). 11. Venkateswaran, K. et al. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. Int. J. Syst. Bacteriol. 49 Pt 2, (1999). 12. Jacobs, M.A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100, (2003).

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