We initiated our investigation into the function of LXG proteins by characterizing the diversity and distribution of genes encoding these proteins across all sequenced genomes from Firmicutes. As noted previously, the C-terminal domains in the LXG family members we identified are highly divergent, exhibiting a wide range of predicted activities (Figure 1a) (Zhang et al., 2012). LXG protein-encoding genes are prevalent and broadly distributed in the classes Clostridiales, Bacillales and Lactobacillales (Figure 1A). Notably, a significant proportion of organisms in these taxa are specifically adapted to the mammalian gut environment. Indeed, we find that LXG genes derived from reference genomes of many of these gut-adapted bacteria are abundant in metagenomic datasets from human gut microbiome samples (Figure 1A and Figure 1—figure supplement 1). An LXG toxin that is predicted to possess ADP-ribosyltransferase activity – previously linked to interbacterial antagonism in Gram-negative organisms – was particularly abundant in a subset of human gut metagenomes (Zhang et al., 2012). Close homologs of this gene are found in Ruminococcus, a dominant taxa in the human gut microbiome, potentially explaining the frequency of this gene (Wu et al., 2011).10.7554/eLife.26938.003Figure 1.

(A) Dendogram depicts LXG-containing genera within Firmicutes, clustered by class and order. Circle size indicates the number of sequenced genomes searched within each genus and circle color represents percentage of those found to contain at least one LXG protein. For classes or orders in which no LXG domain-containing proteins were found, the number of genera evaluated is indicated in parentheses; those consisting of Gram-negative organisms are boxed with dashed lines. Grey boxes contain predicted domain structures for representative divergent LXG proteins. Depicted are LXG-domains (pink), spacer regions (light grey) and C-terminal polymorphic toxin domains (NADase, purple; non-specific nuclease, orange; AHH family nuclease, green; ADP-ribosyltransferase, blue; lipid II phosphatase based on orthology to TelC (defined biochemically herein), yellow; EndoU family nuclease, brown; unknown activity, dark grey). (B) Heatmap depicting the relative abundance (using logarithmic scale) of selected LXG genes detected in the Integrated Gene Catalog (IGC). A complete heatmap is provided in Figure 1—figure supplement 1. Columns represent individual human gut metagenomes from the IGC database and rows correspond to LXG genes. Grey lines link representative LXG toxins in (A) to their corresponding (≥95% identity) IGC group in (B).

DOI: http://dx.doi.org/10.7554/eLife.26938.003

The export of LXG proteins by the Esx pathway motivated us to investigate their capacity for mediating interbacterial antagonism. The C-terminal domains of TelA (TelA_tox) and TelC (TelC_tox) bear no homology to characterized proteins, so we first examined the ability of these domains to exhibit toxicity in bacteria. TelA_tox and TelB_tox* inhibited growth when expressed in the cytoplasm of E. coli, whereas TelC_tox did not exhibit toxicity in this cellular compartment (Figure 3A). Given the capacity of some interbacterial toxins to act on extracellular structures, we assessed the viability of Si cells expressing TelC_tox targeted to the sec translocon. In contrast to TelC_tox production, overexpression of a derivative bearing a signal peptide directing extracellular expression (ss-TelC_tox) exhibited significant toxicity (Figure 3B).10.7554/eLife.26938.008Figure 3.

(A) Viability of E. coli cells grown on solid media harboring inducible plasmids expressing the C-terminal toxin domains of the three identified Si^B196 LXG proteins or an empty vector control. (B) Si^B196 colonies recovered after transformation with equal concentrations of constitutive expression plasmids carrying genes encoding the indicated proteins. ss-TelC_tox is targeted to the sec translocon through the addition of the secretion signal sequence from S. pneumoniae LysM (SP_0107). Error bars represent ± SD (n = 3). Asterisk indicates a statistically significant difference in Si transformation efficiency relative to TelC_tox (p<0.05). (C) Viability of E. coli cells grown on solid media harboring inducible plasmids co-expressing the indicated proteins. Empty vector controls are indicated by a dash. Mean c.f.u. values ± SD (n = 3) are plotted. Asterisks indicate statistically significant differences in E. coli viability relative to vector control (p<0.05) (D) Intra-species growth competition experiments between the indicated bacterial strains. Competing strains were mixed and incubated in liquid medium or on solid medium for 30 hr and both initial and final populations of each strain were enumerated by plating on selective media. The competitive index was determined by comparing final and initial ratios of the two strains. Asterisks indicate outcomes statistically different between liquid and solid medium (n = 3, p<0.05). (E) Intra-species growth competition experiments performed as in (D) except for the presence of a filter that inhibits cell-cell contact. No contact, filter placed between indicated donor and susceptible recipient (∆telB ∆tipB) strains; Contact, donor and susceptible recipient strains mixed on same side of filter. Asterisks indicate statistically different outcomes (n = 3, p<0.05). Note that recipient cell populations have an Esx-independent fitness advantage in these experiments by virtue of their relative proximity to the growth substrate. (F) Inter-species growth competition experiments performed on solid or in liquid (E. faecalis) medium between Si wild-type and ∆essC donor strains and the indicated recipient organisms. Si²³⁷⁷⁵ lacks tipA and tipB and is therefore potentially susceptible to TelA and TelB delivered by Si^B196. Asterisks indicate outcomes where the competitive index of wild-type was significantly higher than an ∆essC donor strain (n = 3, p<0.05). Genetic complementation of the mutant phenotypes presented in this figure was confounded by inherent plasmid fitness costs irrespective of the inserted sequence. As an alternative, we performed whole genome sequencing on strains ∆essC, ∆telB, ∆telC, ∆telB ∆tipB, and ∆telC ∆tipC, which confirmed the respective desired mutation as the only genetic difference between these strains. Sequences of these strains have been deposited to the NCBI Sequence Read Archive (BioProject ID: PRJNA388094).

DOI: http://dx.doi.org/10.7554/eLife.26938.008

The Esx pathway is best known for its role in mediating pathogen-host cell interactions (Abdallah et al., 2007). Given this precedence, we considered the possibility that the antibacterial activity we observed may not be relevant physiologically. TelB degrades NAD⁺, a molecule essential for all cellular life, and therefore this toxin is not definitive in this regard. We next turned our attention to TelC, which elicits toxicity from outside of the bacterial cell (Figure 3B). This protein contains a conserved aspartate-rich motif that we hypothesized constitutes its enzymatic active site (Figure 4—figure supplement 1A). To gain further insight into TelC function, we determined the crystal structure of TelC_tox to 2.0 Å resolution (Table 2). The structure of TelC_tox represents a new fold; it is comprised of distinct and largely α-helical N- and C-terminal lobes (Figure 4A). The single β element of TelC_tox is a hairpin that protrudes from the N-terminal lobe. Although TelC_tox does not share significant similarity to previously determined structures, we located its putative active site within a shallow groove that separates the N- and C-terminal lobes. This region contains a calcium ion bound to several residues that comprise the conserved aspartate-rich motif. Site-specific mutagenesis of these residues abrogated TelC-based toxicity (Figure 4B,C, Figure 4—figure supplement 1B).10.7554/eLife.26938.011Figure 4.

(A) Space-filling representation of the 2.0 Å resolution TelC_tox X-ray crystal structure. Protein lobes (red and blue), active site cleft (white) and Ca²⁺ (green) are indicated. (B) TelC_tox structure rotated as indicated relative to (A) with transparent surface revealing secondary structure. (C) Magnification of the TelC active site showing Ca²⁺ coordination by conserved aspartate residues and water molecules. (D) Viability of S. aureus cells harboring inducible plasmids expressing the indicated proteins or a vector control. ss-TelC_tox is targeted for secretion through the addition of the signal sequence encoded by the 5’ end of the hla gene from S. aureus. Mean c.f.u. values ± SD (n = 3) are plotted. Asterisk indicates a statistically significant difference in S. aureus viability relative to vector control (p<0.05) (E) Representative micrographs of S. aureus expressing ss-TelC_tox or a vector control. Frames were acquired eight and 12 hr after spotting cells on inducing growth media. (F) Thin-layer chromotography (TLC) analysis of reaction products from incubation of synthetic Lys-type lipid II with buffer (Ctrl), TelC_tox, or TelC_tox and its cognate immunity protein TipC. (G) Partial HPLC chromatograms of radiolabeled peptidoglycan (PG) fragments released upon incubation of Lys-type lipid II with the indicated purified proteins. Schematics depict PG fragment structures (pentapeptide, orange; N-acetylmuramic acid, dark green; N-acetylglucosamine, light green; phosphate, black). Known fragment patterns generated by PBP1B + LpoB and colicin M serve as controls. (H) TLC analysis of reaction products generated from incubation of buffer (Ctrl), TelC_tox or TelC_tox and TipC with undecaprenyl phosphate (C55–P) (left) or undecaprenyl pyrophosphate (C55–PP) (right).

DOI: http://dx.doi.org/10.7554/eLife.26938.011

The majority of Esx substrates identified to-date belong to the WXG100 protein family. These proteins typically display secretion co-dependency and are essential for apparatus function. M. tuberculosis ESX-1 exports two WXG100 proteins, ESAT-6 and CFP10, and the removal of either inhibits the export of other substrates (Ates et al., 2016; Renshaw et al., 2002). LXG proteins do not belong to the WXG100 family; thus, we sought to determine how the Tel proteins influence Esx function in Si. Using Western blot analysis to measure TelC secretion and extracellular NADase activity as a proxy for TelB secretion, we found that telB- and telC-inactivated strains of Si retain the capacity to secrete TelC and TelB, respectively (Figure 5A–B). These data indicate that TelB and TelC are not required for core apparatus function and do not display secretion co-dependency.10.7554/eLife.26938.018Figure 5.

(A) NAD⁺ consumption assay of culture supernatants of the indicated Si^B196 strains. Mean densitometry values ± SD (n = 3) are plotted. Asterisk indicates statistically significant difference in NAD⁺ turnover compared to wild-type Si^B196 (p<0.05). (B) Western blot analysis of TelC secretion in supernatant (Sup) and cell fractions. (C) Western blot and coomassie stain analysis of CoIP assays of TelC-his₆ co-expressed with either WxgB-V or WxgC-V proteins. (D) Bacterial two-hybrid assay for interaction between Tel and WXG100-like proteins. Adenylate cyclase subunit T25 fusions (WXG100-like proteins) and T18 fusions (Tel proteins and fragments thereof) were co-expressed in the indicated combinations. Bait-prey interaction results in blue color production. (E) Model depicting Esx-dependent cell-cell delivery of LXG toxins between bacteria. The schematic shows an Si donor cell containing cognate TelA-C (light shades) and WxgA-C (dark shades) pairs intoxicating a susceptible recipient cell. Molecular targets of LXG toxins identified in this study are depicted in the recipient cell.

DOI: http://dx.doi.org/10.7554/eLife.26938.018

S. intermedius strains used in this study were derived from the sequenced strains ATCC 27335 and B196 (Supplementary file 1). S. intermedius strains were grown at 37°C in the presence of 5% CO₂ in Todd Hewitt broth (THYB) or agar (THYA) supplemented with 0.5% yeast extract. When needed, media contained spectinomycin (75 μg/mL) or kanamycin (250 μg/mL). S. aureus USA300 derived strains were grown at 37°C in tryptic soy broth (TSB) or agar (TSA) supplemented with chloramphenicol (10 μg/mL) and xylose (2% w/v) when needed. E. faecalis OG1RF and S. pyogenes 5005 were grown at 37°C on Brain Heart Infusion (BHI) media. P. aeruginosa PAO1 and B. thailandensis E264 were grown at 37°C on THYA. B. fragilis NCTC9343 was grown anaerobically at 37°C on Brain Heart Infusion-supplemented (BHIS) media. E. coli strains used in this study included DH5α for plasmid maintenance, BL21 for protein expression and toxicity assays and MG1655 for competition experiments. E. coli strains were grown on LB medium supplemented with 150 μg/mL carbenicillin, 50 μg/mL kanamycin, 200 μg/mL trimethoprim, 75 μg/mL spectinomycin, 200 μM IPTG or 0.1% (w/v) rhamnose as needed. For co-culture experiments with S. intermedius strains, E. coli, B. thailandensis, P. aeruginosa, S. aureus, E. faecalis, S. pyogenes were grown on THYA. BHIS agar supplemented with sheep’s blood was used when B. fragilis was grown in co-culture with S. intermedius. S. cerevisiae BY4742 was grown on Synthetic Complete -uracil (SC-ura) medium at 30°C.

S. intermedius mutants were generated by replacing the gene to be deleted with a cassette conferring resistance to spectinomycin (derived from pDL277) or kanamycin (derived from pBAV1K-T5), as previously described (Tomoyasu et al., 2010). Briefly, the antibiotic resistance cassette was cloned between ~800 bp of sequence homologous to the regions flanking the gene to be deleted. The DNA fragment containing the cassette and flanking sequences was then linearized by restriction digest, gel purified, and ~250 ng of the purified fragment was added to 2 mL of log-phase culture pre-treated for two hours with competence peptide (200 ng/ml) to stimulate natural transformation. Cultures were further grown for four hours before plating on the appropriate antibiotic. All deletions were confirmed by PCR.

All DNA manipulation procedures followed standard molecular biology protocols. Primers were synthesized and purified by Integrated DNA Technologies (IDT). Phusion polymerase, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB). DNA sequencing was performed by Genewiz Incorporated.

A comprehensive list of all clade names in the Firmicutes phylum was obtained from the List of Prokaryotic names with Standing in Nomenclature (http://www.bacterio.net/; updated 2017-02-02), a database that compiles comprehensive journal citations for every characterized prokaryotic species (Euzéby, 1997). This list was then compared with results obtained from a manually curated Jackhmmer search and LXG-containing Firmicutes were tabulated at the order, family, and genus levels (Finn et al., 2015; Mitchell et al., 2015). These results were binned into three categories based on the number of sequenced species and then further differentiated by the number of LXG-positive species within each genus. For species belonging to orders containing no predicted LXG encoding genes, the number of genera examined was tabulated and included in the dendogram.

The 240 nucleotide tags from the toxin domains were mapped using blastn to the Integrated Gene Catalog (Li et al., 2014) – a large dataset of previously identified microbiome genes and their abundances in several extensive microbiome studies (including HMP [Human Microbiome Project Consortium, 2012], MetaHiT [Qin et al., 2010], and a T2D Chinese cohort [Qin et al., 2012]). Genes to which at least one tag was mapped with >95% identity and >50% overlap were labeled as LXG genes. This set of LXG genes was further manually curated to filter out genes that lack the LXG targeting domain. In analyzing the relative abundance of the LXG genes across samples, relative abundances < 10⁻⁷ were assumed to represent noise and were set to 0. LXG genes that were not present above this threshold in any sample and samples with no LXG genes were excluded from the analysis.

Measurement of cellular NAD⁺ levels was performed as reported previously (Whitney et al., 2015). Briefly, E. coli strains harboring expression plasmids for Tse2, Tse6_tox, TelB_tox*, TelB_tox^R626A, TelB_tox*–TipB and a vector control were grown in LB media at 37°C to mid-log phase prior to induction of protein expression with 0.1% (w/v) rhamnose. 1 hr post-induction, cultures were diluted to OD₆₀₀ = 0.5 and 500 μL of cells were harvested by microcentrifugation. Cells were then lysed in 0.2 M NaOH, 1% (w/v) cetyltrimethylammonium bromide (CTAB) followed by treatment with 0.4 M HCl at 60°C for 15 min. After neutralization with 0.5 M Tris base, samples were then mixed with an equal volume of NAD/NADH-Glo Detection Reagent (Promega) prepared immediately before use as per the instructions of the manufacturer. Luciferin bioluminescence was measured continuously using a Synergy H1 plate reader. The slope of the luciferin signal from the linear range of the assay was used to determine relative NAD⁺ concentration compared to a vector control strain.

S. intermedius strains were grown to late-log phase before cells were removed by centrifugation at 3000 g for 15 min. Residual particulates were removed by vacuum filtration through a 0.2 um membrane and the resulting supernatants were concentrated 100-fold by spin filtration (30 kDa MWCO). NADase assays were carried out by mixing 50 μL of concentrated supernatant with 50 μL of PBS containing 2 mM NAD⁺ followed by incubation at room temperature for 2 hr. Reactions were terminated by the addition of 50 μL of 6M NaOH and incubated in the dark at room temperature for 15 min. Samples were analyzed by UV light at a wavelength of 254 nm and imaged using a FluorChemQ (ProteinSimple). Relative NAD⁺ consumption was determined using densitometry analysis of each of the indicated strain supernatants using the ImageJ software program (https://imagej.nih.gov/ij/).

To assess TelA and TelB toxicity in bacteria, stationary phase cultures of E. coli BL21 pLysS harboring the appropriate plasmids were diluted 10⁶ and each 10-fold dilution was spotted onto 3% LB agar plates containing the appropriate antibiotics. 0.1% (w/v) L-rhamnose and 100 μM IPTG were added to the media to induce expression of toxin and immunity genes, respectively. For TelB, plasmids containing the wild-type toxin domain (under non-inducing conditions) were not tolerated. To circumvent this, SOE pcr was used to assemble a variant (H661A) that was tolerated under non-induced conditions. Based on the similarity of TelB_tox to M. tuberculosis TNT toxin, this mutation likely reduces the binding affinity of TelB to NAD⁺ (Sun et al., 2015). To generate a TelB variant that exhibited significantly reduced toxicity under inducing conditions, a second mutation (R626A) was introduced in the toxin domain of TelB. For examination of TelC toxicity in S. intermedius, the gene fragment encoding TelC_tox was fused to the constitutive P96 promoter followed by a start codon and cloned into pDL277 (Lo Sapio et al., 2012). For extracellular targeting of TelC_tox in S. intermedius, the gene fragment encoding the sec-secretion signal (residues 1–30) of S. pneumoniae LysM (SP_0107) was fused to the 5’ end of telC_tox, each of the telC_tox site-specific variants and the telC_tox–tipC bicistron. 500 ng of each plasmid was transformed in S. intermedius B196 and toxicity was assessed by counting the number of transformants. For examination of TelC toxicity S. aureus, the gene fragment encoding TelC_tox was cloned into the xylose-inducible expression vector pEPSA5. For extracellular targeting, the gene fragment encoding the sec-secretion signal for hla was fused to the 5’ end of telC_tox and telC_tox^D401A. TelC-based toxicity was assessed in the same manner as was done for the above E. coli toxicity experiments except that xylose (2% w/v) was included in the media to induce protein expression. Detailed plasmid information can be found in Supplementary file 2.

S. aureus USA300 pEPSA5::ss-telC_202-CT and S. aureus USA300 pEPSA5 were resuspended in TSB and 1–2 μL of each suspension was spotted onto an 1% (w/v) agarose pad containing typtic soy medium supplemented with 2% (w/v) xylose and sealed.

Microscopy data were acquired using NIS Elements (Nikon) acquisition software on a Nikon Ti-E inverted microscope with a 60× oil objective, automated focusing (Perfect Focus System, Nikon), a xenon light source (Sutter Instruments), and a CCD camera (Clara series, Andor). Time-lapse sequences were acquired at 10 min intervals over 12 hr at room temperature. Movie files included are representative of three biological replicates for each experiment.

200 mL cultures of S. intermedius B196 wild-type and ΔessC strains were grown to stationary phase in THYB before being pelleted by centrifugation at 2500 × g for 20 min at 4°C. Supernatant fractions containing secreted proteins were collected and spun at 2500 × g for an additional 20 min at 4°C and subsequently filtered through a 0.2 μm pore size membrane to remove residual cells and cell debris. Protease inhibitors (1 mM AEBSF, 10 mM leupeptin, and 1 mM pepstatin) were added to the filtered supernatants prior to dialysis in 4L of PBS using 10 kDa molecular weight cut off tubing at 4°C. After four dialysis buffer changes, the retained proteins were TCA precipitated, pelleted, washed in acetone, dried and resuspended in 1 mL of 100 mM ammonium bicarbonate containing 8 M urea. The denatured protein mixture was then desalted over a PD10 column prior to reduction, alkylation and trypsin digestion as described previously (Eshraghi et al., 2016). The resulting tryptic peptides were desalted and purified using C18 spin columns (Pierce) following the protocol of the manufacturer before being vacuum dried and resuspended in 10 µL of acetonitrile/H₂O/formic acid (5/94.9/0.1, v/v/v) for LC-MS/MS analysis.

Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation nanoLC and a linear ion trap – Orbitrap hybrid mass spectrometer (ThermoFisher Scientific). Peptide samples were loaded onto the trap column, which was 150 µm x 3 cm in-house packed with 3 µm C18 beads, at flow rate of 5 µL/min for 5 min using a loading buffer of acetonitrile/H₂O/formic acid (5/94.9/0.1, v/v/v). The analytical column was a 75 µm x 10.5 cm PicoChip column packed with 1.9 µm C18 beads (New Objectives). The flow rate was kept at 300 nL/min. Solvent A was 0.1% formic acid in water and Solvent B was 0.1% formic acid in acetonitrile. The peptide was separated on a 90 min analytical gradient from 5% acetonitrile/0.1% formic acid to 40% acetonitrile/0.1% formic acid.

The mass spectrometer was operated in data-dependent mode. The source voltage was 2.10 kV and the capillary temperature was 275°C. MS¹ scans were acquired from 400 to 2000 m/z at 60,000 resolving power and automatic gain control (AGC) set to 1 × 10⁶. The top ten most abundant precursor ions in each MS¹ scan were selected for fragmentation. Precursors were selected with an isolation width of 1 Da and fragmented by collision-induced dissociation (CID) at 35% normalized collision energy in the ion trap. Previously selected ions were dynamically excluded from re-selection for 60 s. The MS² AGC was set to 3 × 10⁵.

Proteins were identified from the MS raw files using Mascot search engine (Matrix Science). MS/MS spectra were searched against the UniprotKB database of S. intermedius B196 (UniProt and UniProt Consortium, 2015). All searches included carbamidomethyl cysteine as a fixed modification and oxidized Met, deamidated Asn and Gln, acetylated N-terminus as variable modifications. Three missed tryptic cleavages were allowed. The MS¹ precursor mass tolerance was set to 10 ppm and the MS² tolerance was set to 0.6 Da. A 1% false discovery rate cutoff was applied at the peptide level. Only proteins with a minimum of two unique peptides above the cutoff were considered for further study. MS/MS spectral counts were extracted by Scaffold 4 (Proteome Software Inc.) and used for statistical analysis of differential expression. Three biological replicates were performed and proteins identified in all three wild-type replicates were included in further analysis. After replicate averaging, low abundance proteins (less than five spectral counts in wild-type) were excluded from the final dataset.

Overnight cultures of S. intermedius strains were used to inoculate 2 ml of THYB at a ratio of 1:200. Cultures were grown statically at 37°C, 5% CO₂ to mid-log phase, and cell and supernatant fractions were prepared as described previously (Hood et al., 2010).

Full-length TelC protein was expressed and purified as described below (see protein expression and purification) except that PBS buffer was used instead of Tris-HCl for all stages of purification. Ten milligrams of purified TelC protein was sent to GenScript for polyclonal antisera production.

Western blot analyses of protein samples were performed using rabbit α-TelC (diluted 1:2000) or rabbit α-VSV-G (diluted 1:5000, Sigma) and detected with α-rabbit horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000, Sigma). Western blots were developed using chemiluminescent substrate (SuperSignal West Pico Substrate, Thermo Scientific) and imaged with a FluorChemQ (ProteinSimple).

For intraspecific competition experiments donor and recipient strains were diluted in THYB to a starting OD₆₀₀ of 0.5 and 0.05, respectively. Cell suspensions were then mixed together in a 1:1 ratio and 10 μL of the mixture was spotted on THYA and grown at 37°C, 5% CO₂ for 30 hr. The starting ratio of each competition was determined by enumerating donor and recipient c.f.u. Competitions were harvested by excising the agar surrounding the spot of cell growth followed by resuspension of cells in 0.5 mL of THYB. The final donor and recipient ratio was determined by enumerating c.f.u. For all intraspecific experiments, counts of donor and recipient c.f.u. were obtained by dilution plating on THYA containing appropriate antibiotics. To facilitate c.f.u. enumeration of wild-type S. intermedius B196, a spectinomycin resistance cassette was inserted into the intergenic region between SIR_0114 and SIR_0115.

For interspecies competition experiments, donor and recipient strains were diluted in THYB to a starting OD₆₀₀ of 0.75 and 0.00075, respectively. Cell suspensions were then mixed together in a 1:1 ratio and 10 μL of the mixture was spotted on THYA and grown at 37°C, 5% CO₂ for 30 hr. The starting ratio of each competition was determined by enumerating donor and recipient c.f.u. Competitions were harvested by excising the agar surrounding the spot of cell growth followed by resuspension of cells in 0.5 mL of THYB. The final donor and recipient ratio was determined by enumerating c.f.u. Counts of donor and recipient c.f.u. were obtained by dilution plating on THYA containing appropriate antibiotics (S. intermedius), BHI under standard atmospheric conditions (E. coli, E. faecalis and S. pyogenes), LB under standard atmospheric conditions (P. aeruginosa and B. thailandensis) or BHIS supplemented with 60 μg/mL gentamicin under anaerobic conditions (B. fragilis).

Statistically significance was assessed for bacterial competition experiments through pairwise t-tests of competitive index values (n = 3 for each condition).

Stationary phase overnight cultures of E. coli BL21 pETDuet-1::telC, E. coli BL21 pETDuet-1::telC_202-CT (encoding TelC_tox) and E. coli BL21 pETDuet-1::tipC_Δss were used to inoculate 4L of 2 x YT broth and cultures were grown to mid-log phase in a shaking incubator at 37°C. Upon reaching an OD₆₀₀ of approximately 0.6, protein expression was induced by the addition of 1 mM IPTG followed by incubation at 18°C for 16 hr. Cells were harvested by centrifugation at 6000 g for 15 min, followed by resuspension in 35 mL of buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). Resuspended cells were then ruptured by sonication (3 pulses, 50 s each) and cellular debris was removed by centrifugation at 30,000 g for 45 min. Cleared cell lysates were then purified by nickel affinity chromatography using 2 mL of Ni-NTA agarose resin loaded onto a gravity flow column. Lysate was loaded onto the column and unbound proteins were removed using 50 mL of buffer A. Bound proteins were then eluted using 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 400 mM imidazole. The purity of each protein sample was assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining. All protein samples were dialyzed into 20 mM Tris-HCl, 150 mM NaCl.

Selenomethionine-incorporated TelC_202-CT was obtained by growing E. coli BL21 pETDuet-1::telC_202-CT in SelenoMethionine Medium Complete (Molecular Dimensions) using the expression conditions described above. Cell lysis and nickel affinity purification were also performed as described above except that all buffers contained 1 mM tris(2-carboxyethyl)phosphine.

Purified selenomethionine-incorporated TelC_202-CT was concentrated to 12 mg/mL by spin filtration (10 kDa cutoff, Millipore) and screened against commercially available crystallization screens (MCSG screens 1–4, Microlytic). Diffraction quality crystals appeared after 4 days in a solution containing 0.1 M Sodium Acetate pH 4.6, 0.1 M CaCl₂, 30% PEG400. X-ray diffraction data were collected using beamline 5.0.2 at the Advanced Light Source (ALS). A single dataset (720 images, 1.0° Δφ oscillation, 1.0 s exposure) was collected on an ADSC Q315r CCD detector with a 200 mm crystal-to-detector distance. Data were indexed and integrated using XDS (Kabsch, 2010) and scaled using AIMLESS (Evans and Murshudov, 2013) (table S2).

The structure of TelC_202-CT was solved by Se-SAD using the AutoSol wizard in the Phenix GUI (Adams et al., 2010). Model building was performed using the AutoBuild wizard in the Phenix GUI. The electron density allowed for near-complete building of the model except for N-terminal residues 202–211, two C-terminal residues and an internal segment spanning residues 417–434. Minor model adjustments were made manually in COOT between iterative rounds of refinement, which was carried out using Phenix.refine (Afonine et al., 2012; Emsley et al., 2010). The progress of the refinement was monitored by the reduction of R_work and R_free (Table 2).

Purified TelC_tox was dialyzed against 20 mM sodium acetate pH 4.6, 150 mM NaCl, 10 mM CaCl₂. Cross-linked peptidoglycan sacculi and lysostaphin endopeptidase pre-treated (non-cross-linked) sacculi from S. aureus were then incubated with 5 μM TelC_tox, 2.5 μg of cellosyl muramidase or buffer at 37°C for 18 hr. Digests were then boiled for 5 min at 100°C and precipitated protein was removed by centrifugation. The resulting muropeptides were reduced by the addition of sodium borohydride and analyzed by HPLC as described previously (de Jonge et al., 1992).

For the analysis of cell walls isolated from TelC-intoxicated cells, 1L of S. aureus USA300 pEPSA5::ss-telC_tox and S. aureus USA300 pEPSA5::ss-telC_tox^D401A cells were grown to mid-log phase prior to induction of protein expression by the addition of 2% (w/v) xylose. 90 min post-induction, cultures were rapidly cooled in an ice-water bath and cells were harvested by centrifugation. After removal of supernatants, cell pellets were resuspended in 40 mL of ice-cold 50 mM Tris-HCl pH 7.0 and subsequently added dropwise to 120 mL boiling solutions of 5% SDS. PG was isolated as described (de Jonge et al., 1992) and digested with either cellosyl muramidase or lysostaphin endopeptidase and cellosyl, reduced with sodium borohydride and analyzed by HPLC as described above.

Purified TelC_tox and TelC_tox–TipC_Δss complex were dialyzed against 20 mM sodium acetate pH 4.6, 150 mM NaCl, 10 mM CaCl₂. C¹⁴-labelled Lys-type lipid II was solubilized in 5 μL of Triton X-100 before being added to 95 μL of reaction buffer containing 15 mM HEPES pH 7.5, 0.4 mM CaCl₂ (excluded from the PBP1B-LpoB reaction), 150 mM NaCl, 0.023% Triton X-100 and either PBP1B–LpoB complex, TelC_tox, TelC_tox–TipC_Δss complex or Colicin M followed by incubation for 1 hr at 37°C. The reaction with PBP1B-LpoB was boiled and reduced with sodium borohydride. All the reactions were quenched by the addition of 1% (v/v) phosphoric acid and analyzed by HPLC as described (Bertsche et al., 2005). Three biological replicates were performed for each reaction. The lipid II degradation products of TelC_tox digestion were confirmed by mass spectrometry. Lipid II was kindly provided by Ute Bertsche and was generated as described previously (Bertsche et al., 2005).

For thin-layer chromatography (TLC) analysis of Lys-type lipid II degradation by TelC, TelC_tox or TelC_tox–TipC_Δss, 40 μM lipid II was solubilized in 30 mM HEPES/KOH pH 7.5, 150 mM KCl and 0.1% Triton X-100 before adding either 2 μM TelC_tox, 2 μM TelC_tox–TipC_Δss complex or protein buffer, followed by incubation for 90 min at 37°C. Samples were extracted with n-butanol/pyridine acetate (2:1) pH 4.2 and resolved on silica gel (HPTLC silica gel 60, Millipore) in chloroform/methanol/ammonia/water (88:48:1:10). For the undecaprenyl phosphate reactions 100 μM undecaprenyl phosphate (Larodan) was solubilized in 20 mM HEPES/KOH pH 7.5, 150 mM KCl, 1 mM CaCl₂ and 0.1% Triton X-100 before adding 2 μM TelC_tox (final concentration), 2 μM TelC_tox–TipC_Δss or protein buffer, followed by incubation for 5 hr at 25°C and 90 min at 37°C. Samples were extracted and separated by TLC as indicated above. For the undecaprenyl pyrophosphate synthesis reactions coupled to the degradation by TelC_tox0.04 mM Farnesyl pyrophosphate and 0.4 mM isopentenyl pyrophosphate were solubilized in 20 mM HEPES/KOH pH 7.5, 50 mM KCl, 0.5 mM MgCl₂, 1 mM CaCl₂, 0.1% Triton X-100 and incubated with 10 μM UppS and 2 μM TelC_tox (final concentrations) or protein buffer for 5 hr at 25°C and 90 min at 37°C. Samples were extracted and separated by TLC as indicated above.

To target TelC to the yeast secretory pathway, telC_tox was fused to the gene fragment encoding the leader peptide of Kluyveromyces lactis killer toxin (Baldari et al., 1987), generating ss-telC_tox. S. cerevisiae was transformed with pCM190 containing telC_tox, ss-telC_tox, a known toxin of yeast or empty vector and grown o/n SC-ura +1 ug/mL doxycycline. Cultures were resuspended to OD₆₀₀ = 1.5 with water and serially diluted 5-fold onto SC-ura agar. Plates were incubated at 30°C for 2 days before being imaged using a Pentax WG-3 digital camera. Images presented are representative of three independent replicate experiments. Proteolytic processing of the leader peptide of ss-TelC_tox was confirmed by western blot.

Solutions of 25 μM TelC_202-CT and 250 μM TipC_Δss were degassed prior to experimentation. ITC measurements were performed with a VP-ITC microcalorimeter (MicroCal Inc., Northampton, MA). Titrations consisted of 25 10 μL injections with 180 s intervals between each injection. The ITC data were analyzed using the Origin software package (version 5.0, MicroCal, Inc.) and fit using a single-site binding model.

E. coli BTH101 cells were co-transformed with plasmids encoding the T18 and T25 fragments of Bordetella pertussis adenylate cyclase fused to the proteins of interest. Stationary phase cells were then plated on LB agar containing 40 mg/mL X-gal, 0.5 mM IPTG, 50 mg/mL kanamycin and 150 mg/mL carbenicillin and grown for 24 hr at 30°C. Plates were imaged using a Pentax WG-3 digital camera. Images representative of at least three independent replicate experiments are presented.

E. coli BL21 (DE3) pLysS cells were co-transformed with plasmids encoding TelC-his₆ and WxgB-V or TelC-his₆ and WxgC-V. Cells were grown to an OD₆₀₀ of 0.6 prior to induction of protein expression with 0.5 mM IPTG for 6 hr at 30°C. Cultures were harvested by centrifugation and cell pellets were resuspended in Buffer A prior to lysis by sonication. Clarified lysates were then incubated with Ni-NTA resin and incubated at 4°C with rotation for 90 min. Ni-NTA resin was then washed four times with Buffer A followed by elution of bound proteins with Buffer B. After the addition of Laemmli sample buffer, proteins were separated by SDS-PAGE using an 8–16% gradient TGX Stain-Free gel (Bio-Rad). TelC-his₆ was visualized by UV activation the trihalo compound present in Stain-Free gels whereas WxgB-V and WxgC-V were detected by western blotting.