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Supplemental Information

Proteomic identification of novel secreted anti-bacterial toxins of the Serratia marcescens Type VI secretion system Maximilian J. Fritsch, Katharina Trunk, Juliana Alcoforado Diniz, Manman Guo, Matthias Trost, Sarah J. Coulthurst

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Supplemental Table S1: Bacterial strains and plasmids used in this study

Name Description Source or Reference

Bacterial strains

E. coli CC118(λpir) Cloning host and donor strain for pKNG101-derived marker exchange plasmids (λpir) (1)

E. coli DH5α Cloning host (2) E. coli MG1655 E. coli K-12 wild type laboratory strain (3)

E.coli HH26/pNJ5000 Mobilising strain for conjugal transfer of pKNG101-derived marker exchange plasmids (4)

P. fluorescens KT02 SmR derivative of P. fluorescens 55 (5) S. marcescens Db10 Wild type strain (6) JAD01 Db10, Δsip4, Δssp4 (ΔSMA3979, ΔSMA3980) this study JAD06 SmR derivative of JAD01 this study KK2 Db10, Δfha (ΔSMA2267) this study KT65 Db10, Δssp3 (ΔSMA1112) this study KT67 Db10, Δssp6 (ΔSMA4673) this study MJF4 Db10, ΔpppA, ΔppkA (ΔSMA2268, ΔSMA2275) this study MJF8 Db10, Δssp4 (ΔSMA3980) this study MJF9 Db10, Δssp5 (ΔSMA4628) this study MJF16 Db10 fha-HA (SMA2267-HA) this study SJC3 Db10, ΔclpV (ΔSMA2274) (5) SJC11 Db10, ΔtssE (ΔSMA2271) (5) SJC19 Db10, ΔpppA (ΔSMA2268) this study SJC25 Db10, ΔppkA (ΔSMA2275) this study SJC48 Db10 fha-HA, ΔppkA (SMA2267-HA, ΔSMA2275) this study SJC49 Db10 fha-HA, ΔpppA (SMA2267-HA, ΔSMA2268) this study Plasmids pBluescript KS+ High copy cloning vector (ApR) Stratagene

pSUPROM Vector for constitutive expression (KnR); gene of interest cloned under control of the E. coli PtatA promoter (7)

pSC812 ppkA (SMA2275) in pSUPROM this study pSC814 ppkA-HA in pSUPROM this study pSC863 ppkA-HA with D165N substitution in pSUPROM this study pSC815 pppA (SMA2268) in pSUPROM this study pSC830 ppkA (SMA2275) and pppA (SMA2268) and in pSUPROM this study

pBAD18-Kn Arabinose-inducible expression vector (KnR); gene of interest cloned downstream of ParaBAD promoter with its own ribosome binding site

(8)

pSC1231 ssp3 (SMA1112) in pBAD18-Kn this study pSC1232 ssp3 (SMA1112) and sip3 (SMA1111) in pBAD18-Kn this study pSC835 ssp4 (SMA3980) in pBAD18-Kn this study pSC836 ssp4 (SMA3980) and sip4 (SMA3979) in pBAD18-Kn this study pSC1233 ssp4 (SMA3980) and SMA3978 in pBAD18-Kn this study

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Name Description Source or Reference

pSC837 ssp4 (SMA3980), sip4 (SMA3979) and SMA3978 in pBAD18-Kn this study

pSC838 ssp5 (SMA4628) in pBAD18-Kn this study pSC839 ssp5 (SMA4628) and sip5a (SMA4629) in pBAD18-Kn this study pSC842 ssp5 (SMA4628) and sip5b (SMA4630) in pBAD18-Kn this study

pSC840 ssp5 (SMA4628),sip5a (SMA4629) and sip5b (SMA4630) in pBAD18-Kn this study

pSC1235 ssp6 (SMA4673) in pBAD18-Kn this study

pSC1237 N-terminal fusion of E. coli ompA signal peptide (codon 1-24) to ssp3 (SMA1112) in pBAD18-Kn this study

pSC1234 N-terminal fusion of E. coli ompA signal peptide (codon 1-24) to ssp4 (SMA3980) in pBAD18-Kn this study

pSC841 N-terminal fusion of E. coli ompA signal peptide (codon 1-24) to ssp5 (SMA4628) in pBAD18-Kn this study

pSC1236 N-terminal fusion of E. coli ompA signal peptide (codon 1-24) to ssp6 (SMA4673) in pBAD18-Kn this study

pKNG101 Suicide vector for allelic marker exchange (strAB (SmR), sacBR, mobRK2, oriR6K) (9)

pSC105 pKNG101-derived marker exchange vector for generation of ΔpppA (ΔSMA2268) chromosomal in-frame deletion this study

pSC111 pKNG101-derived marker exchange vector for generation of ΔppkA (ΔSMA2275) chromosomal in-frame deletion this study

pSC904 pKNG101-derived marker exchange vector for generation of Δfha (ΔSMA2267) chromosomal in-frame deletion this study

pSC1249 pKNG101-derived marker exchange vector for generation of Δssp3 (ΔSMA1112) chromosomal in-frame deletion this study

pSC828 pKNG101-derived marker exchange vector for generation of Δssp4 (ΔSMA3980) chromosomal in-frame deletion this study

pSC829 pKNG101-derived marker exchange vector for generation of Δssp5 (ΔSMA4628) chromosomal in-frame deletion this study

pSC1241 pKNG101-derived marker exchange vector for generation of Δssp6 (ΔSMA4673) chromosomal in-frame deletion this study

pSC618 pKNG101-derived marker exchange vector for generation of Δssp4 Δsip4 (ΔSMA3980 ΔSMA3979) chromosomal deletion this study

pSC822

pKNG101-derived marker exchange vector for generation of chromosomal C-terminal HA-tag fusion to fha (SMA2267); the codons of the HA-tag are followed by a HindIII site and the last ten codons of fha in order to maintain the polycistronic organisation of the locus.

this study

pSC161

pKNG101-derived marker exchange vector for generation of chromosomal C-terminal HA-tag fusion to fha (SMA2267) and ΔpppA (ΔSMA2268) chromosomal in-frame deletion; the codons of the HA-tag are followed by a HindIII site and the last six codons of pppA in order to maintain the polycistronic organisation of the locus.

this study

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Supplemental Fig. S1. Quality control of proteomics data. Technical reproducibility of label-free quantitation (LFQ). A, The reproducibility of four technical replicates of secretome samples of S. marcescens Db10 was analysed by plotting the LFQ intensity data of each replicate against the average. B, Comparison of LFQ intensities between S. marcescens wild type strain (WT) and mutant strains SJC3 (ΔclpV) or SJC19 (ΔpppA). LFQ intensities of WT proteins plotted against SJC3 are shown in blue and proteins with significant changes (WT/ ΔclpV ≥ 4; p < 0.05) are shown in green and labelled with gene identifiers. LFQ intensities of WT proteins plotted against SJC19 are shown in red. C, Distribution of peptide intensity changes between the wild type strain and the ΔclpV mutant. The ratios of LFQ intensities between the WT strain and the T6SS mutant strain ΔclpV were plotted against their frequency. Data shows that the majority of proteins do not change and centre around 0.

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Supplemental Fig. S2: Multiscatter plot of biological replicates. The LFQ intensities of each replicate and of each strain were plotted against each other. Linear regression values are shown for each chart showing a high level of reproducibility for all samples.

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Supplemental Fig. S3. Sip5a and Sip5b share sequence and secondary structure similarity. Pairwise, global sequence alignment of Sip5a (SMA4629) and Sip5b (SMA4630) using the EMBOSS stretcher alignment tool (http://www.ebi.ac.uk; (10)). Sip5a and Sip5b share 26.4% and 39.6% sequence identity and similarity, respectively. Residues were highlighted in Jalview 2.7 (11, 12) using a Clustalx colour scheme. Secondary structures were predicted using PSIPRED v3.0 (13) and helices are shown above and below the amino acid sequences.

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Supplemental Fig. S4. Additional analysis of newly-identified T6SS-secreted effectors Ssp3-Ssp6. A, Heterologous expression of T6SS-secreted proteins with Sec signal sequences in E. coli. Genes encoding Ssp5 and Ssp6 proteins, with or without the OmpA Sec signal sequence, were heterologously expressed in E. coli MG1655 from an inducible plasmid. Serial dilutions of E. coli strains were spotted on M9 minimal media plates and gene expression was repressed or induced with D-Glucose or L-arabinose, respectively. The expressed genes are indicated on the left and the density of the inoculum is given along the top of the images. B, Anti-bacterial killing activity of S. marcescens strains Db10 (WT), T6SS− mutant SJC3 (ΔclpV) and effector mutants KT65 (Δssp3), MJF8 (Δssp4), MJF9 (Δssp5) and KT67 (Δssp6) against P. fluorescens. Error bars show standard error of the mean (SEM) with n ≥ 4.

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Supplemental Fig. S5. The C-terminal domain of S. marcescens PpkA is not homologous to P. aeruginosa PpkA and is predicted to contain a pre-albumin fold. A, Pairwise global sequence alignment between S. marcescens PpkA (SMA2275) and P. aeruginosa PpkA (PA0074) using the EMBOSS stretcher alignment tool (http://www.ebi.ac.uk; (10)). The alignment is coloured by residue conservation (30%) in Jalview 2.8 (11, 12). The catalytic aspartate residue D165 of S. marcescens PpkA is indicated by a star above the sequence. Conserved residues in the kinase domain are highlighted in red and residues of the predicted transmembrane domains are highlighted in magenta. The proline-rich region and the periplasmic von Willebrand factor A domain of P. aeruginosa PpkA are highlighted in green and cyan, respectively. Residues comprising a predicted pre-albumin fold in the periplasmic C-terminal domain of S. marcescens PpkA are boxed in red. B, Protein structure prediction of the C-terminal domain of S. marcescens PpkA using Phyre2 (18). The predicted model shows a pre-albumin fold between residues 409 and 476 of S. marcescens PpkA. The structural model of the PpkA C-terminal domain (residues 367 to 480) was based on three structural templates: SCOP domains d1HL8A1 (Conf. 95%), d1UWYA1 (Conf. 91%) and d1Z0MB1 (Conf. 51%). The sequence coverage of the templates in the alignment and in the structural model is highlighted in red. White coloured regions in the structure, which were not covered by the templates, were modelled ab initio at low confidence. Arrows (beta sheets) and helices (alpha helices) above the S. marcescens PpkA sequence indicate the predicted secondary structure.

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Supplemental Fig. S6. The phospho-peptide binding and phosphorylation site of S. marcescens and P. aeruginosa Fha are conserved. A, Multiple, global sequence alignment of the phospho-peptide binding site of bacterial Fha homologues and the FHA1 domain of S. cerevisiae RAD53. Conserved residues involved in phospho-peptide binding are highlighted in red and the position of beta-sheets in the S. cerevisiae RAD53 structure is indicated by black arrows on top of the alignment (14). B, Multiple, global sequence alignment of Ser/Thr phosphorylation sites of bacterial Fha homologues. Threonine residues that align with the phosphorylated Threonine residues of S. marcescens and P. aeruginosa Fha homologues are highlighted in red. A and B, Sequences were aligned using Clustal Omega (15) and the alignment was coloured by percentage identity in Jalview 2.8 (11, 12). The sequences of those bacterial Fha homologues (COG3456) were selected which were encoded in the same T6SS gene cluster as homologues of the kinase PpkA (COG0515) and the phosphatase PppA (COG0631), based on the study by Boyer et al. (16). In addition, Fha homologues of P. ananatis LMG 20103 and Pantoea sp. aB valens were also included since their sequences showed close homology to S. marcescens Fha and were encoded within T6SS clusters with PpkA and PppA homologues (17). The UniProt accession numbers of

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the sequences are as follows: Azoarcus sp., A1KCE4; B. japonicum, Q89P85; P. aeruginosa (Fha1), Q9I751; P. aeruginosa (Fha2), Q9I360; P. ananatis, D4GCZ7;P. denitrificans, Q9I360; P. entomophila, Q1IFS5; P. fluorescens, Q4K3P0; P. profundum, Q6LUE6; P. syringae, Q87U85; Pantoea sp., E0LZ84; R. loti, Q98IL9; R. sphaeroides, Q3IWJ8; S. cerevisiae (RAD53), P22216; S. degradans, Q21KJ1; S. frigidimarina, Q080U3; V. fisheri, Q5E654; V. parahaemolyticus, Q87HC2; V. vulnificus, Q8D6T0; X. axonopodis, Q8PF63; X. campestris (Fha1), Q3BTP7; X. campestris (Fha2), Q3BMR8. The sequence of S. marcescens Fha (SMA2267) is available from the Sanger Institute (http://www.sanger.ac.uk/resources/downloads/bacteria/serratia-marcescens.html).

Supplemental Fig. S7 Detection of Fha Thr438 phosphorylation. A, Coomassie stained SDS-PAGE of representative immunoprecipitated samples from Db10 (WT), MJF16 (fha-HA), SJC48 (fha-HA, ΔppkA) and SJC49 (fha-HA, ΔpppA). Samples were immunoprecipitated from liquid grown cultures using anti-HA agarose. Protein bands corresponding to the Fha-HA fusion proteins are indicated on the right hand side of the gel. B, Representative extracted ion chromatogram of the phosphorylated peptide AEMpTMILDEANNPFK of HA-tagged Fha in Db10 (ctrl.), MJF16 (WT), SJC48 (ΔppkA) and SJC49 (ΔpppA). Note that the total abundance of Fha was greater in ΔpppA than in WT.

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