Bacterial killing via a type IV secretion system

Abstract

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.

ARTICLE

Received 5 Sep 2014 | Accepted 29 Jan 2015 | Published 6 Mar 2015

DOI: 10.1038/ncomms7453

Bacterial killing via a type IV secretion system

Diorge P. Souza1, Gabriel U. Oka1, Cristina E. Alvarez-Martinez1,2, Alexandre W. Bisson-Filho1, German Dunger1, Lise Hobeika1, Nayara S. Cavalcante3, Marcos C. Alegria1,w, Leandro R.S. Barbosa4, Roberto K. Salinas1, Cristiane R. Guzzo1,5 & Chuck S. Farah1

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and proteinDNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the rst demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.

1 Departamento de Bioqumica, Instituto de Qumica, Universidade de Sao Paulo, Av. Prof. Lineu Prestes 748, Sao Paulo, SP 05508-000, Brazil.

2 Departamento de Gentica, Evoluao e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato 255, Campinas,SP 13083-862, Brazil. 3 Departamento de Fsica e Informtica, Instituto de Fsica de Sao Carlos, Universidade de Sao Paulo, Avenida Trabalhador Saocarlense 400, Sao Paulo, SP 13566-590, Brazil. 4 Departamento de Fsica Geral, Instituto de Fsica, Universidade de Sao Paulo, Caixa Postal 66318, Sao Paulo,SP 05315-970, Brazil. 5 Departamento de Microbiologia, Instituto de Cincias Biomdicas, Universidade de Sao Paulo, Av. Prof. Lineu Prestes 1374, Sao Paulo, SP 05508-900, Brazil. w Present address: Cristlia Produtos Qumicos Farmacuticos Ltda, Divisao de Biotecnologia, Rodovia Itapira-Lindia km 14, Itapira,

SP 13970-970, Brazil. Correspondence and requests for materials should be addressed to C.S.F. (email: mailto:[email protected]

Web End [email protected] ).

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Bacterial cells are continuously interacting with other bacterial and eukaryotic cells in a relentless battle for survival. These interactions have driven the evolution of

several mechanisms by which they quickly deploy proteinaceous and nucleic acid effectors that manipulate the behaviour of the target organism, often resulting in growth inhibition or death16. Distinct among these mechanisms are the type III, type IV and type VI secretion systems (T3SS, T4SS and T6SS, respectively) that are all capable of transferring proteins, and in the case of the T4SS, proteinDNA complexes, directly into neighbouring cells in a contact-dependent manner13. All three of these systems have been shown to be able to inject virulence factors into eukaryotic hosts, but only the T6SSs have been shown to deliver lethal toxins into bacterial cells7.

T4SSs are perhaps the most versatile of the macromolecular transport systems, being essential for host colonization by many medically important microbes813, the horizontal transfer of genetic material between bacteria and from bacteria to plants2 as well as the special cases of the Helicobacter pylori DNA uptake14 and Neisseria gonorrhoeae DNA release15 systems. The prototypical T4SS is that of the plant pathogen Agrobacterium tumefaciens that uses its Vir system to cause tumours in most dicotyledonous species16. T4SSs are generally made up of a core set of 12 proteins, named VirB1VirB11 plus VirD4 with a structural organization that is only now being elucidated: (i) a set of three cytoplasmic ATPases (VirB4, VirB11 and VirD4) that energize secretion; (ii) a periplasmic core complex made up of 14 repeats of a VirB7VirB9VirB10 trimer, in which VirB10 inserts into both inner and outer membranes and VirB7 is an outer membrane lipoprotein; (iii) an inner membrane complex that includes VirB3, VirB6 and VirB8; (iv) an extracellular pilus formed by VirB2 and VirB5; and (v) the periplasmic transglycosylase VirB1 (refs 2,1719).

Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs20,21. One of these distinguishing features is that the Xanthomonas citri (Xac) VirD4 protein (VirD4XAC2623), a

homologue of the coupling protein that recruits effectors to the T4SS for secretion2, interacts with a set of uncharacterized Xanthomonas VirD4-interacting proteins (XVIPs) that all contain a conserved C-terminal domain termed XVIPCD (XVIP-conserved domain)20. In this study, we show that these XVIPs are toxins secreted by the Xanthomonas T4SS, that secretion is dependent on the XVIPCD and that this special T4SS is used by Xanthomonas to kill other bacterial cells, thereby providing a competitive growth advantage in mixed bacterial communities.

ResultsA hypothesis for a new physiological role for a T4SS. The Xac XVIPs are highly diverse in size and architecture and lack a canonical signal sequence. Six Xac XVIPs (XAC0466, XAC0574, XAC1918, XAC2609, XAC2885 and XAC3634) are predicted to act within the periplasm as peptidoglycan (PG)-binding proteins, PG glycohydrolases, lytic transglycosylases, PG peptidases or lipases (Fig. 1a). Furthermore, the genes of these proteins, plus one other containing an HExxH metallopeptidase motif (XAC0096), are found downstream to, and potentially co-transcribed with, genes that code for proteins with unknown functions with predicted lipoprotein signal peptides that are expected to localize them within the bacterial periplasm (Fig. 1). One XVIP (XAC2609) has a complete PG-binding domain (residues 219280) and a region (residues 30120) with similarity to the catalytic region of proteins of glycoside hydrolase family 19 (GH19) that possess chitinase- or lysozyme-like activities22

(Fig. 1). XAC2609 has been shown to interact with the putative periplasmic lipoprotein XAC2610 and both are coded by genes within the Xac chromosomal T4SS vir locus20. These characteristics are very similar to those observed for certain T6SS effectors with hydrolytic activities against conserved PG and phospholipid components of the bacterial cell wall and their cognate immunity proteins23,24. On the basis of these observations, one hypothesis is that the Xac T4SS could secrete toxins participating in toxinimmunity protein systems.

XAC2609 lyses PG and XAC2610 is its inhibitor. To test the above hypothesis, we performed a set of in vitro experiments to determine whether XAC2609 has the predicted lysozyme-like activity. Puried recombinant XAC2609 is able to degrade PG from the Gram-positive Micrococcus luteus (Fig. 2a), reduces the optical density and introduces a marked lag in the growth curve of Bacillus subtilis cultures (Fig. 2b), and results in B. subtilis cell lysis (Fig. 2d). These activities are maintained in an XAC2609 fragment lacking the XVIPCD (XAC2609(1308); Fig. 2a) but are

not observed when using a mutant XAC2609(1308) in which

glutamic acid 48 within the predicted active site of the GH19 domain was changed to alanine (Fig. 2a,e). We observed similar lytic activity against PG (Fig. 2a) and B. subtilis (Fig. 2g) for a second XVIP, XAC0466, that has a domain architecture similar to XAC2609 plus a PG amidase domain (Fig. 1a). Furthermore, the XAC2609 lytic activity is inhibited by the addition of XAC2610 (Fig. 2a,b and f). XAC2610 is a specic inhibitor of XAC2609 since it was not able to inhibit the lytic activities of XAC0466 or hen egg white lysozyme (Fig. 2a). We also observed that XAC2609 and XAC2610 can be co-immunoprecipitated from Xac cells (Supplementary Fig. 1a) and that XAC2610 interacts with the catalytic XAC2609 N-terminal domain but not with the XVIPCD domain in vitro (Supplementary Fig. 1b). Due to these characteristics, we will therefore now refer to XAC2609 as X-TfeXAC2609 (for Xanthomonadaceae-T4SS effector) and to XAC2610 as X-TXAC2610 (for Xanthomonadaceae-T4SS immunity protein).

X-TXAC2610 structure is similar to PG hydrolase inhibitors. The X-TXAC2610 protein does not share sequence similarity with any proteins of known function. We therefore determined the crystal structure of X-TXAC2610(55267) (a fragment lacking the signal peptide, lipobox lipidation signal and a predicted unstructured region). Details of X-TXAC2610(55267) crystallization, data collection and renement are found in the Methods section and Supplementary Table 1. X-TXAC2610 has an N-terminal modied b-sandwich fold that includes a conserved integrin-like Ca2 -binding loop and an intramolecular disulde bond, connected via a single a-helix to a C-terminal domain made up of an uncommon single-layer six-stranded antiparallel b-sheet (Fig. 3a). A more detailed description of the X-TXAC2610 structure, including the Ca2 -binding loop and its semblance to a b-propeller fragment, is presented in Supplementary Fig. 2. Supplementary Figure 3 presents small-angle X-ray scattering data that is consistent with a monomeric structure in solution. What is most interesting about the X-TXAC2610 structure is that among its closest topological homologues are the periplasmic I-type lysozyme inhibitor PliI25,26 (Fig. 3b) and the P. aeruginosa immunity protein Tsi1 (Fig. 3c) that inhibits the lytic PG DL-endopeptidase activity of its cognate T6SS effector protein Tse1 (refs 7,27; see also Supplementary Fig. 4). This topological similarity includes the superposition of the ion-binding sites of the X-TXAC2610 and PliI proteins (Fig. 3d; Supplementary Fig. 4a). The above in vitro and structural results show that

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XVIP or X-Tfe

XAC3634 XAC2609 XAC0466 XAC0096 XAC0574 XAC2885 XAC1918 XAC3266 XAC4264 XAC0151 XAC0323 XAC1165 XAC3404

SLT

PGB PGB

XAC3633 XAC2610 XAC0467 XAC0097 XAC0573 XAC2884 XAC1917

513XVIPCD630

488XVIPCD584

16 XVIPCD136

Putative proximal immunity protein

(X-Tfi) with lipoprotein signal peptide

1 MLVRKISLVLALATIAAC....1 MLTRELARAVAGVIYLICVVSITAC ....31 MPPFRLLIPILAVALVAC....1 MRFPRAALLFLTALCAPALSAC....1 MAAHLPRRPALRQMLTPSLLALLLTAC ....

101 MKIKSVGCVALALFFITGC....

32 MQPRFPALLVLATSFFAVSC....

GH19

315XVIPCD431

120XVIPCD254

GH19 M23 metallopeptidase

Lipase 3

PGB

506XVIPCD646 317XVIPCD440

1 XVIPCD127

HExxH motif metallopeptidase

Lipase 3

DUF2974

DUF2974

271XVIPCD395

1 XVIPCD132

Lysozyme like

Ca2+

477XVIPCD606

AHH

735XVIPCD861

140XVIPCD279

Figure 1 | Xac X-Tfe proteins and their respective putative X-T immunity proteins. (a) Gene names and domain architectures of the 13 Xac X-Tfes. All these proteins contain a Xanthomonas VirD4-interacting protein conserved domain (XVIPCD)20. PGB, peptidoglycan-binding domain; GH19, glycohydrolase family 19; SLT, soluble lytic transglycosylase; Ca2 , Ca2 -binding domain; AHH, putative nuclease domain. (b) Gene names of putative lipoprotein signal peptide-containing Xac immunity proteins (X-Ts) and sequences of the putative lipoprotein signal peptides. All seven of these genes are upstream of, and potentially co-transcribed with, the neighbouring X-Tfe gene. Lipoprotein signal peptides are characterized by an N-terminal basic region (basic residues coloured blue), a central hydrophobic region (underlined) followed by an [LVI]-[ASTVI]-[AGS]-C lipobox motif (red)68. Also indicated are the positions of the putative translation initiation codons relative to those found in the original annotation of the Xac genome69.

OD (650 nm)

0.8

0.6

0.4

0.2

Buffer

XAC2609

XAC2609+ XAC2610

0.6

OD (600 nm)

0.4

XAC2610

0.2

0466Nt 2609NtE48A

2609Nt+2610

2609Nt

2609+2610

0

Buffer

0 2610

HEWL

0466Nt+2610

HEWL+2610

2609

0 60 120 180 240 300

Time (min)

Figure 2 | X-TfeXAC2609 is a peptidoglycan hydrolase and X-TXAC2610 is its specic inhibitor. (a) Analysis of M. luteus peptidoglycan degradation, as determined by decreases in optical density (OD) at 650 nm of cell wall suspensions treated with puried proteins X-TXAC261055267, 2610; full-length

X-TfeXAC2609, 2609; X-TfeXAC2609(1308) N-terminal fragment (residues 1308), 2609Nt; X-TfeXAC2609(1308_E48A) N-terminal fragment E48A mutant, 2609Nt-E48A; X-TfeXAC0466(1475) N-terminal fragment (residues 1475), 0466Nt; hen egg white lysozyme, HEWL. A suspension without the addition of any protein (buffer) is shown as a control. Error bars, s.d.; n 3. (b) Growth curves at 30 C of B. subtilis cells after incubation for 10 min without proteins

(blue), with X-TfeXAC2609 (red), X-TXAC2610 (purple) or a mixture of both proteins (green; error bars, s.d.; n 3). (cg) Fluorescence microscopy of

membrane-labelled B. subtilis cells after incubation for 5 min with c, buffer; d, X-TfeXAC2609; e, X-TfeXAC2609(1308_E48A); f, a X-TfeXAC2609 -X-TXAC2610 mixture; g, X-TfeXAC0466(1475). Scale bar, 5 mm.

X-TXAC2610 is in fact the cognate immunity protein of the X-TfeXAC2609 PG hydrolase toxin.

X-Tfe secretion is T4SS dependent and requires a XVIPCD. We next tested whether X-TfeXAC2609 is secreted in a manner dependent on the Xac T4SS. For this, we employed Xac DvirD4 and DvirB7 mutants, both of which are expected to abolish secretion of all T4SS substrates since VirD4 is the coupling protein required for recognition of T4SS substrates2 and

deletion of the Xac virB7 gene results in the disappearance of not just VirB7 but also VirB9 and VirB10 (ref 21), the principal components of the T4SS core complex28. Fig. 4a (top row) and Fig. 4b show that when Xac cells are spotted on nitrocellulose lters layered on Luria-Bertani (LB) agar plates, allowed to grow for 12 h and the cells washed away, we could not detect any secreted X-TfeXAC2609 bound to the membrane since the background immunouorescence signals of all Xac strains alone are the same as that observed for the Dxac2609 mutant. However,

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Wild type

virD4

virD4

+ virD4

virB7

virB7

+virB7

xac2609

xac2609

+ 2609

xac2609

+ 2609Nt

Xac only

20

Xac vs E.coli

100

80

60

40

XAC2609

XAC2609Nt

Figure 3 | X-TXAC2610 is structurally similar to peptidoglycan hydrolase inhibitors. (a) Ribbon representation of the X-TXAC2610 model (residues

55267). A Ca2 ion and Ca2 -coordinating residues are shown as a sphere and sticks, respectively. (b) Superposition of X-TXAC2610 (yellow)

and PliI (light blue, PDB code 3OD9, ref. 26). (c) Superposition of X-TXAC2610 (yellow) and Tsi1 (pink). Tse1, the hydrolase inhibited by Tsi1, is shown in green in the Tsi1Tse1 complex structure (PDB accession code 3VPJ, ref. 27). Stereo versions of b and c can be found in Supplementary Fig. 4. (d) Superposition of the X-TXAC2610 and PliI ion-binding sites.

Dashed lines show the pentagonal bipyramidal coordination of Ca2 in X-TXAC2610. The sticks colouring scheme for b and d are: C, yellow in

X-TXAC2610 and light blue in PliI; O, red; N, blue; Ca2 , yellow sphere; coordinating water, red sphere; Na , light blue sphere. Search for structural similarities were performed using the Dali server60. The r.m.s.d., fraction of the chain for the superposition and % sequence identity of X-TXAC2610

and Tsi1 are 3.4 , 82% and 7% respectively. For the superposition of X-TXAC2610 and PliI, these values are 3.7 , 78% and 12% respectively.

Normalized intensity

Figure 4 | X-TfeXAC2609 secretion requires a XVIPCD, a functional T4SS and is stimulated by co-culture with E. coli cells. (a) Xac cells only (top row) or 1:1 Xac:E. coli mixtures (bottom row) were spotted on nitrocellulose lters layered on LB agar plates, allowed to grow for 12 h, the cells washed away and bound X-TfeXAC2609 was immunodetected. The Xac wild-type (WT) strain (carrying pBBR-5GFP and empty pBRA vector) and the derived mutants (DvirD4, DvirB7 and Dxac2609) and complemented strains (DvirD4 virD4, DvirB7 virB7, Dxac2609 xac2609 and

Dxac2609 xac2609Nt (X-TfeXAC2609 N-terminal fragment)) used are

indicated above each lane. (b) Integrated infrared uorescence intensity of X-TfeXAC2609 immunodetection from seven independent experiments similar to those represented in a for Xac only (dark bars) and Xac:E. coli mixtures (light bars). Intensities were normalized to those measured for the WT Xac:E. coli mixture. Error bars, s.d. (c) Western blot detection of total cellular X-TfeXAC2609 produced by Xac strains used in a. The full blot is shown in Supplementary Fig. 9.

extracellular X-TfeXAC2609 is detected when wild-type (WT) Xac cells are mixed with an equal number of E. coli cells before spotting (Fig. 4a, bottom row and Fig. 4b). Moreover, E. coli-induced X-TfeXAC2609 secretion is abolished in the DvirD4 and DvirB7 strains and restored when plasmids for expression of VirD4 or VirB7 proteins are introduced in these strains (Fig. 4a,b). These results show that X-TfeXAC2609 is secreted by Xac on contact with E. coli cells in a T4SS-dependent manner. X-TfeXAC2609 secretion is dependent on its XVIPCD since extracellular X-TfeXAC2609 could only be detected in the experiments using a Xac Dxac2609 mutant carrying a plasmid expressing full-length X-TfeXAC2609 but not in mutant strains carrying empty plasmid or a plasmid that directs the expression of an X-TfeXAC2609 fragment lacking the XVIPCD (Fig. 4a,b). Detection of extracellular X-TfeXAC2609 in these experiments is not due to E. coli-induced Xac lysis since all Xac strains (except the uncomplemented Dxac2609 mutant), including both DvirD4 and DvirB7, produce intracellular X-TfeXAC2609 (Fig. 4c).

The Xac T4SS confers an advantage in growth competition experiments. We then asked whether the T4SS confers a competitive advantage to Xac in growth competition experiments against other bacterial species. While there are no signicant differences in the growth curves of the Xac WT and DvirD4 and

DvirB7 mutant strains in liquid culture or on semi-solid media (Supplementary Fig. 5), when Xac cells carrying a plasmid for the expression of cyan uorescent protein (CFP) and E. coli cells expressing yellow uorescent protein (YFP) were mixed and allowed to grow together as a co-culture on agar plates, we observed a signicant increase in both the number and the size of the yellow E. coli colonies/zones in the cyan background of the Xac DvirB7 strain when compared with that of the Xac WT strain (Fig. 5a,b). Xac competitiveness is recovered when the VirB7 protein is expressed in the DvirB7 strain (Fig. 5c). The same phenomenon was observed in competition experiments using Xac

DvirD4 and DvirB7 mutants expressing green uorescent protein (GFP) and non-uorescent E. coli (Supplementary Fig. 6a). Furthermore, after 38 h of co-culture growth, the Xac/E. coli ratio is two orders of magnitude lower when Xac DvirB7 strain is used, as compared with when the Xac WT strain or Xac DvirB7 strain carrying a plasmid for VirB7 expression are used (Fig. 5d). To further test whether T4SS mediates it effects in a contact-dependent manner, we placed a 0.2-mm pore lter between Xac and E. coli cells. Interestingly, the T4SS-dependent growth defect of Xac DvirB7 and DvirD4 mutant strains completely disappeared under this condition (Supplementary Fig. 6b), showing that direct contact is required for this T4SS function. Xac competitiveness against Chromobacterium violaceum, another Gram-negative species from a different bacterial class (Betaproteobacteria), was also observed to be T4SS dependent in the experiments similar to those shown in Fig. 5 (Supplementary Fig. 7).

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3

Log CFU ratio (Xac/E.coli)

2

1

0

1

2

Wild type

virB7

virB7

+virB7

t=0 h t=38 h

Figure 5 | Xac kills E. coli cells in a T4SS-dependent manner. (ac) Xac cells carrying a plasmid for the expression of CFP (cyan) and E. coli DH5a cells expressing YFP (yellow) were mixed at a 80:1 ratio, spotted on agar plates and allowed to grow together as a co-culture. The images show uorescence emitted from the resultant colonies. Xac strains used were: a wild-type (WT); b DvirB7; c DvirB7 strain carrying a plasmid for VirB7 expression. Scale bar for ac, 1 mm. (d) Ratio of the number of viable Xac and E. coli cells in experiments similar to those shown in ac after 0 and 38 h of co-culture growth. Xac WT,

DvirB7 strain and the DvirB7 strain with ectopic VirB7 expression (DvirB7 virB7) were used. Error bars represent the s.d. of three experiments.

(eg) Superposition of brighteld and uorescence microscopy images of single elds obtained after adding, to a thin agarose surface, mixtures containing equal amounts of E. coli and (e) Xac WT, (f) DvirB7 strain carrying a plasmid for VirB7 expression and (g) DvirD4 strain carrying a plasmid for VirD4 expression. In these experiments, Xac cells carry a plasmid for the expression of CFP (cyan, ef) or GFP (green, g), while E. coli cells carry a plasmid that expresses YFP (yellow, ef) or are non-uorescent (g). Arrows indicate E. coli cells that underwent lysis during the 5-min intervals between images. Similar results were observed in dozens of experiments of this kind. In comparison, death of Xac cells was very rarely observed. Scale bar for eg, 3 mm.

The Xac T4SS mediates bacterial killing. The competitive increment conferred by the Xac T4SS could be the result of secretion of factors that inhibit the growth of competing bacterial species or that promote direct killing of targeted adjacent cells. To distinguish between these two hypotheses, we used time-lapse microscopy to observe individual cells in Xac/E. coli co-cultures. Figure 5eg shows micrographs of single elds obtained shortly after mixing equal amounts of Xac and E. coli cells on a thin agarose surface. While we frequently observed the rapid lysis ofE. coli cells that were in direct physical contact with Xac WT cells (Fig. 5e), we did not observe these events when the DvirB7 and DvirD4 strains were used (Supplementary Fig. 8). Xac killing ofE. coli was restored using mutant strains carrying plasmids for VirB7 or VirD4 expression (Fig. 5f,g). Supplementary Movie 1 presents a series of images separated by 300-ms intervals that show that E. coli cell lysis is extremely rapid, with cells often bursting in a seemingly explosive manner in o1 s to a few seconds. By comparison, Xac cell lysis was very rarely observed in these experiments. The rapid and violent cell death that we observe contrasts with that which has been reported for T6SS-mediated killing by Pseudomonas aeruginosa, in which target cells change their morphology (cell rounding, blebbing and plasmolysis) over a period of many minutes before lysis29,30.

DiscussionWe have provided evidence that the Xac T4SS, and by extension its homologues in other Xanthomonadaceae species, target toxins (X-Tfes or XVIPs) to bacterial cells, thereby providing Xanthomonas an advantage to survive in an environment populated by a number of different bacterial species in a manner similar to that recently described for several T6SSs2933. Using the presence of a XVIPCD as search criteria, we have identied 500 potential X-Tfes in the public databases, of which all but two are derived from members of the Xanthomonadaceae family (Table 1). Most species that code for a Xanthomonadaceae-like T4SS code for

several X-Tfe proteins and, in most cases analysed so far, one X-Tfe gene is found next to the gene of a T4SS component with the remaining X-Tfe genes found spread throughout the genome. Many of these proteins carry domains that may work to kill or inhibit growth of other organisms. Especially abundant are lipase domains34 and domains involved in binding or modication of PG, found exclusively in bacterial cell walls35: for example, PG-binding domain 1 (ref. 36), transglycosylase SLT37, peptidase M23 (ref. 38), GH19 (refs 22,39) and NlpC/P60 (refs 38,40) domains (Table 1). Other examples are the Zeta toxin domain that produces an inhibitor of bacterial PG biosynthesis41, the AHH domain likely involved in killing bacteria via its putative nuclease activity42 and DUF2235 (ref. 43), a putative hydrolase recently found to be present in T6SS effectors44 (Table 1). Both Zeta toxin and AHH domains are found in toxinantitoxin systems where they are inhibited by specic immunity proteins41,42.

It is important to note that of the 500 X-Tfes (XVIPs) analysed in Table 1, a large majority has no recognizable domain other than the C-terminal XVIPCD. Therefore, by studying the XVIP repertoire, we can reasonably expect the number of activities of this newly characterized T4SS and its effectors to expand. The proposed general bacterial killing function for a large number of Xanthomonadaceae-T4SS effectors may have interesting biotechnological applications that lead to new drug targets and ways of ghting other pathogenic bacteria that cause disease in humans and plants. Furthermore, we speculate that some X-Tfes could provide defence against eukaryotic microbes that prey on Xanthomonads.

Finally, Ho et al.45 recently showed that P. aeruginosa uses its T6SS to attack neighbouring cells carrying a mating pair formation T4SS encoded by a conjugative plasmid. The initial speculation was that this T4SS-induced T6SS counterattack potentially blocks acquisition of parasitic foreign DNA. However, since we have now demonstrated that some T4SSs pose a more immediate lethal threat to neighbouring bacteria,

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Table 1 | Domains found in 500 proteins containing XVIPCDs.

Domain* Description Xac X-Tfes that contain this domain

Other examples found in other organisms GenBank accession codeOrganism

No. in 500 proteins analysed*

PG-binding domain 1 Binding to PG XAC2609 XAC0466 XAC3634

EIL93106R. fulvus Jip2EKQ64038X. axonopodis. pv. malvacearum CAJ25482X. campestris pv. vesicatoria

107

Lipase 3/DUF2974 Hydrolysis of ester linkages in triglycerides XAC0574 XAC2885

CCG35840X. citri pv. mangiferaeindicae EGD17149X. gardneri ATCC 19865

61

GH19 Hydrolysis of glycosidic bonds in chitin and PG XAC2609 XAC0466

EMF61220S. maltophilia EPM1 CAP51046X. campestris. pv. campestris

15

Peptidase M23 Hydrolysis of peptide bonds in PG XAC0466 EMI48206S. maltophilia AU12-09 AEL09382X. campestris pv. raphani

7

DUF2235 Uncharacterized alpha/beta hydrolase domain Not found CBA16126X. albilineans GPE PC73 CBA16292X. albilineans GPE PC73

6

Zeta toxin Phosphorylation of the PG precursor UNAG; inhibition of PG synthesis

Not found AEM51053S. maltophilia JV3EMI51042S. maltophilia AU12-09

3

AHHw Nuclease participating in toxinantitoxin system XAC3266 AGH78749X. axonopodis Xac29-1AGI06866X. citri subsp. citri Aw12879

3

SLTz Hydrolysis of glycosidic bonds in PG XAC3634|| EMI48206S. maltophilia AU12-09WP_016901345X. arboricola pv. juglandis

2

NlpC/P60 Hydrolysis of peptide bonds in PG Not found WP_017913944Xanthomonas sp. SHU308 WP_017910175Xanthomonas sp. SHU199

2

GH19, glycoside hydrolase family 19; PG, peptidoglycan; SLT, soluble lytic transglycosylase; Xac, Xanthomonas citri; XVIP, Xanthomonas VirD4-interacting protein; XVIPCD, XVIP-conserved domain *Analysis of the PFAM-A domains found in 500 proteins containing the XVIPCD. The proteins were identied by PSI-BLAST using the X-TfeXAC2609 XVIPCD sequence as the initial query. Of these 500 proteins, all but two are found in species of the Xanthomonadaceae family: Xanthomonas spp.: 377 proteins; Stenotrophomonas spp.: 101 proteins; Pseudomonas geniculata: 8 proteins; Dyella ginsengisoli: 5 proteins; Pseudoxanthomonas spadix: 4 proteins; Rhodanobacter fulvus: 2 proteins; Lysobacter spp.: 1 protein. The remaining two proteins were found in Pseudomonas syringae and Catenibacterium mitsuokai.

wSeven other XVIPs possibly have an AHH domain, although the matches were considered insignicant by PFAM (e-value: 0.0120.0027).

z15 other XVIPs possibly have a SLT domain, although the matches were considered insignicant by PFAM (e-value: 0.110.0009). All 17 XVIPs with a putative SLT domain also have a PG-binding domain 1.||While not identied by PFAM, XAC3634 has a predicted N-terminal SLT according to the Conserved Domain Database70 (see Fig. 1).

many bacterial species may have been under much stronger evolutionary pressure to have evolved an automatic T6SS counterattack response. This immediately suggests the hypothesis that, since very few T4SS have been characterized to date, T4SS-mediated bacterial killing may not be restricted to the Xanthomonadaceae family, and may in fact be a more widespread phenomenon.

Methods

Bacterial strains, growth conditions and cloning. Bacterial strains, oligonucleotides and plasmids used in this study are described in Supplementary Table 2. All strains were grown on LB or 2 TY (2 yeast extract and tryptone) media.

Concentrations of the antibiotics used: 100 mg ml 1 ampicilin, 20 mg ml 1 gentamicin, 100 mg ml 1 spectinomycin, 15 mg ml 1 tetracycline, 100 mg ml 1 chloramphenicol and 30 mg ml 1 kanamycin. For generation of the Xac Dxac2609 strain, B1 kb of the upstream and downstream regions of the xac2609 gene were amplied by PCR from Xanthomonas citri subsp. citri strain 306 genomic DNA and the two fragments were ligated to produce an in-frame deletion, leaving only the region coding for the rst 15 and last 12 codons. This sequence was then cloned into the BamHI restriction site of the pNPTS138 suicide vector (M. R. Alley, unpublished; described in Supplementary Table 2), thereby generating the pNPTS138-2609 plasmid. For generation of the Xac DvirD4 strain, a DNA fragment including the virD4 gene plus 0.55 kb upstream and downstream sequences were amplied by PCR and cloned into the NdeI/HindIII sites of the pUC19 vector. The resulting pUC19-virD4 vector was digested with the SacI restriction enzyme; as the virD4 gene has three of these sites, the digestion and subsequent re-ligation of the plasmid removed an internal fragment of 0.75 kb of virD4, generating the pUC19-DvirD4. This strategy removed 251 codons of the central region of the gene, leaving the rst 256 and the last 50 codons of virD4. This plasmid was digested with SpeI/SalI and the resulting DNA containing the virD4 gene fragment plus 0.55 kb upstream and downstream sequences was cloned into the pNPTS138, thereby generating the pNPTS138-virD4 plasmid. In-frame deletions in the virD4 and xac2609 genes were obtained by a two-step allelic exchange procedure as previously described for the virB7 gene21 using the pNPTS138-2609 and pNPTS138-virD4 vectors. For complementation of Xac mutant strains, genes virB7XAC2622, virD4XAC2623 and xac2609 (coding the full-length protein (residues

1431) or its N-terminal domain (residues 1306)) were cloned in the pBAD24-derived vector pBRA (M. Marroquin, unpublished; described in Supplementary Table 2), which contains an arabinose-induced promoter that is constitutively active in Xac. Genes virB7, virD4, xac2609 and xac2609Nt were amplied by PCR from Xac306 genomic DNA and PCR products were cloned directly into the

NcoI/SalI sites of pBRA. A recombinant XAC2610 (GenBank accessioncode AAM37459) fragment consisting of residues 22267 fused to an N-terminal His-tag (X-TXAC2610(His-22267)) and full-length XAC2609 (residues

1431; X-TfeXAC2609(1431); GenBank accession code AAM37458) were expressed as described previously20. In brief, DNA coding for residues 55267 of XAC2610 (X-TXAC2610(55267)) and N terminus (residues 1308; X-TfeXAC2609(1308)) and C

terminus (residues 311431; X-TfeXAC2609(311431)) fragments of XAC2609 were

amplied by PCR from Xac genomic DNA and cloned into the pET11a plasmid (Novagen) using the NdeI and BamHI cloning sites. The pET11a-XAC26091308

vector was used as a DNA template for the generation of the pET11a-XAC2609-E48A plasmid that codes for X-TfeXAC2609(1308_E48A) (the X-TfeXAC2609(1308)

protein with a Glu 48 to Ala mutation), using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). DNA coding for the N-terminal fragment of XAC0466 (residues 1475; X-TfeXAC0466(His-1475); GenBank accession code

AAM35357) was cloned into the pET28a vector using the NdeI and BamHI sites, expressing a protein fused to an N-terminal His-tag. All plasmids were conrmed by DNA sequence analysis.

Protein expression and purication. E. coli BL21(DE3)RP strain (Novagen) was used for the expression of the recombinant proteins. Cells were grown in 2 TY at

37 C and recombinant proteins were induced at mid-log phase for 34 h by the addition of 0.5 mM isopropyl-b-D-thiogalactoside. Cells were then harvested and lysed by sonication employing a Vibra-Cell VCX-500 instrument (Sonics & Materials Inc.). The rst chromatographic purication step was performed with a Q-sepharose anion exchange column (GE Healthcare) for all X-TfeXAC2609 and

X-TXAC2610 constructions. The second chromatographic step was an afnity purication using a nickel chelate column for X-TXAC2610(His-22267), a size

exclusion purication with a 26/600 Superdex 75 column for X-TXAC2610(55267),

X-TfeXAC2609(1308) and X-TfeXAC2609(311431) or a 26/600 Superdex 200 column

for X-TfeXAC2609(1431) and X-TfeXAC2609(1308_E48A). X-TfeXAC0466(His-1475) was

puried using a nickel chelate column.

PG hydrolase activity assay. Micrococcus luteus cell wall suspensions (Sigma) with an optical density at 650 nm (OD650nm) of 0.7 (0.8 mg ml 1) in 50 mM

sodium acetate (pH 5.0) and 2 mM CaCl2 were incubated in triplicate for 20 min at 30 C with the following treatments: (i) without proteins (buffer),(ii) with X-TXAC2610(55267), (iii) X-TfeXAC2609(1431), (iv) X-TfeXAC2609(1308),

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(v) X-TfeXAC2609(1308_E48A), (vi) X-TfeXAC0466(His-1475), (vii) HEWL,

(viii) X-TfeXAC2609(1431) plus X-TXAC2610(55267), (ix) X-TfeXAC2609(1308)

plus X-TXAC2610(55267), (x) X-TfeXAC0466(His-1475) plus X-TXAC2610(55267)

or (xi) HEWL plus X-TXAC2610(55267). X-TfeXAC2609-derived proteins,

X-TfeXAC0466(His-1475) and HEWL, were used at 1 mM concentration, while

X-TXAC2610(55267) was present at 2 mM. The reactions were stopped with the

addition of 500 mM sodium carbonate. Cell wall degradation was monitored as a decrease in OD650nm.

Bacterial growth inhibition assay. Bacillus subtilis PY79 cells were grown in2 TY to log phase (OD600nm 0.5). Cells were harvested by centrifugation and

resuspended to an OD600nm of 0.8 in 10 mM sodium acetate pH 5.0 and 1 mM

MgCl2. Incubations were performed in triplicate for 10 min at 30 C with only buffer or buffer containing 1 mM X-TfeXAC2609(1431) or 2 mM X-TXAC2610(55267)

or 1 mM X-TfeXAC2609(1431) plus 2 mM X-TXAC2610(55267). The bacteria were then

diluted with nine volumes fresh 2 TY medium and subsequent growth at 30 C

was monitored by measuring OD600nm.

Bacillus subtilis lysis by X-Tfes. Bacillus subtilis PY79 cells were grown in LB to OD600nm 0.5, harvested by centrifugation and resuspended to an OD600nm

of 0.8 in 10 mM sodium acetate (pH 5.0), 1 mM MgCl2. Incubations were

performed with only buffer or buffer containing (i) 1 mM X-TfeXAC2609(1431),

(ii) 1 mM X-TfeXAC2609(1308), (iii) 1 mM X-TfeXAC2609(1308_E48A), (iv) 2 mM

X-TXAC2610(55267), (v) 1 mM X-TfeXAC2609(1431) plus 2 mM X-TXAC2610(55267),

(vi) 1 mM X-TfeXAC2609(1308) plus 2 mM X-TXAC2610(55267), (vii) 1 mM

X-TfeXAC0466(His-1475) or (viii) 1 mM HEWL. After 5 min of treatment at 23 C,

cells were centrifuged, washed, resuspended in 2 TY and mounted on slides

covered with a layer of 25% LB plus 1.5% agarose. Membranes were stained with FM1-43 or FM5-95 (Molecular Probes) at a nal concentration of 1 mg ml 1 and the stained cells were analysed by uorescence microscopy.

Fluorescence microscopy. Microscopy was carried out using a Nikon Eclipse Ti microscope equipped with lters for GFP (GFP-3035B-000-ZERO, Semrock), CFP (CFP-2432A-000-ZERO, Semrock), YFP (YFP-2427-000-ZERO, Semrock) and membrane visualization (Texas Red BrightLine set; TXRED4040-B, Semrock), a Plan APO VC Nikon 100 objective (numerical aperture 1.4), a CFI Plan Apo

Lambda Nikon 2 objective and an Andor EMCCD i-Xon camera. The exposure

times varied from 0.2 to 1 s. Images were processed and analysed using the programs Nis Elements (version 3.07; Nikon) and ImageJ (http://rsbweb.nih.gov/ij/

Web End =http://rsbweb.nih.gov/ij/).

Antibody production, immunoprecipitation and immunoblot. Rabbit polyclonal antibodies were produced against X-TfeXAC2609(1431) and X-TXAC2610(His-22267).

Xac total cell lysates from WT and DvirB7XAC2622 (DB7) strains were used for

immunoprecipitation experiments with pre-immune serum or anti-X-TXAC2610 serum (1:1,000 dilution), followed by immunoblots with anti-X-TfeXAC2609and anti-X-TXAC2610 antibodies (1:1,000 dilution), performed as previously described21. Pre-immune serum, from the same animal later immunized with X-TXAC2610, was used as a negative control.

Fluorescence spectroscopy. Fluorescence assays were measured employing AVIV ATF-105 and Hitachi F-4500 uorometers. Qualitative proteinprotein interaction assays were carried out using 1 mM protein alone (X-TXAC2610(55267),

X-TfeXAC2609(1431), X-TfeXAC2609(1308) and X-TfeXAC2609(311431)) or combina

tions of X-TXAC2610(55267) and one of the three X-TfeXAC2609 fragments in

20 mM Tris-HCl pH 7.5, 50 mM NaCl, 3 mM EGTA and 4 mM CaCl2. Samples were pre-equilibrated at 25 C for 2 min, excited at 295 nm (bandwidth of 2.5 nm) and uorescence emission was recorded from 300 to 400 nm (bandwidth of 2.5 nm) at 0.2-nm intervals. The results of three experiments were averaged.

Crystallization, data collection and processing. Vapour diffusion sitting drop crystallization screens at 18 C were performed for X-TXAC2610(His-22267), but no

crystals were obtained. Two weeks incubation of this protein at room temperature produced an B23 kDa proteolytic fragment that formed thin needle-like crystals.

The N-terminal sequence of this fragment was determined by Edman degradation using a Sequenator model PPSQ-23 (Shimadzu) and MALDI-ToF measurements employing an Ettan MALDI-ToF mass spectrometer (GE Healthcare). The sequence was found to begin at residue 56 and mass spectroscopy analysis was consistent with a fragment consisting of X-TXAC2610 residues 55267. Recombinant X-TXAC2610(55267) protein (400 mM dissolved in 10 mM Tris-HCl pH 7.0,

20 mM NaCl) was submitted to crystallization trials and crystals were observed after 2 weeks over a reservoir solution containing 100 mM Bis-Tris-HCl pH 5.5, 200 mM (NH4)2SO4 and 25% (w/v) PEG3350. Crystals were transferred to

reservoir solution supplemented with 20% (v/v) glycerol and ash frozen at 100 K. X-ray diffraction data were collected using an in-house Rigaku MicroMax-007 HF microfocus rotating Cu anode generator (Institute of Chemistry, University of Sao Paulo) and the beamline W01B-MX2 of the Brazilian Synchrotron Light Laboratory (LNLS)46. The diffraction data were indexed, integrated and scaled using d*TREK47 and HKL2000 (ref. 48). Data collection statistics are shown in Supplementary Table 1.

X-ray structure determination and renement. The crystal structure of X-TXAC2610(55267) was determined by SIRAS (single isomorphous replacement

with anomalous scattering) using crystals soaked for 5 min with reservoir solution supplemented with 20% (v/v) glycerol and a saturated solution of mercury acetate. The initial phase estimation was carried out using SHELXD49 and autoSHARP50, followed by automatic model building by ARP/wARP51. The model was subsequently rened with the program Coot52 with rounds of TLS53 and restrained renement in Refmac554 from the CCP4 package55. Model quality was checked by Coot, Refmac5, MolProbity56, Rampage57 and PROCHECK58. Clear electron density was visible for all residues except for the rst four amino acids (residues 5558) that were not, therefore, included in the model. The electron density of some specic exposed loops (residues 5960, 139143, 218220 and 245249) had a relatively lower signal-to-noise level but was sufcient to model their backbones with condence. A total of 180 water molecules and a calcium ion were also modelled. The nal model has Rfactor and Rfree of 0.200 and 0.241, respectively

(Supplementary Table 1). Figures were prepared using PYMOL (http://www.pymol.org

Web End =http:// http://www.pymol.org

Web End =www.pymol.org ) and protein topology schemes with TopDraw 59. The coordinates of the X-TXAC2610(55267) X-ray crystal structure were deposited in the PDB under

accession code 4QTQ. Search for structural similarities were performed using the Dali server60. The Z-score, root mean squared deviation, number of residues superposed and fraction of the chain for the superposition of X-TXAC2610 and Tsi1 are 6.5, 3.4 , 120 and 82%, respectively, and for the superposition of X-TXAC2610 and PliI are 4.9, 3.7 , 95 and 78%.

Small-angle X-ray scattering (SAXS). SAXS experiments were collected at the D11A-SAXS1 beamline at the LNLS in Brazil. The radiation wavelength was 1.55 and a Pilatus detector at 936 mm distance was used to give a scattering vector(q 4p/lsin(y), where 2y is the scattering angle) ranging from 0.14 to 4.1 nm 1.

X-TXAC2610(55267) at 3.34, 1.67 and 0.84 mg ml 1 dissolved in 20 mM Tris-HCl pH 7.5 and 100 mM NaCl was exposed in frames of 3 and 10 s, and only normalized data without signicant radiation damage in the small q range were averaged to give the nal scattering curve. Buffer scattering curves were collected and subtracted from the samples scattering curve, taking into account its attenuation. The scattering curves were normalized by the protein concentration to check for interference effects61 or aggregation62 over the SAXS curves. Guinier analysis was used to calculate the protein radius of gyration, Rg, and to evaluate the monodispersity of the system. SAXS data analyses were performed using the ATSAS package63. The determination of the pair-distance distribution function, p(r), was conducted using the GNOM software64. After the p(r) calculation, 15 low resolution ab initio envelopes for X-TXAC2610(55267) were generated with

DAMMIN65 and aligned and averaged using the program suite DAMAVER66. The normalized spatial discrepancy between the models was 0.5620.011 and none were rejected. Comparisons of the scattering experimental data and X-ray protein models were performed using CRYSOL67.

X-TfeXAC2609 secretion analysis. GFP-labelled Xac strains (Xac WT pBRA GFP, Xac DvirD4 pBRA GFP, Xac DvirD4 pBRA-virD4 GFP, Xac DvirB7 pBRA GFP, Xac DvirB7 pBRA-virB7 GFP, Xac D2609 pBRA GFP, Xac D2609 pBRA-2609FL GFP and Xac D2609 pBRA-2609Nt GFP; Supplementary Table 2) and E. coli BL21(DE3)RIL ArcticExpress were grown separately in 2 TY to log phase

(OD600nm of 1.0), centrifuged, washed and resuspended to OD600nm 2.0. Xac

strains and E. coli were mixed at a 1:1 ratio. Xac alone or the mixtures (5 ml) were spotted onto a nitrocellulose transfer membrane (GE Healthcare). The lter was placed on LB agar media containing gentamicin and spectinomycin and maintained at 30 C for 12 h. Cells were washed away with running water for 2 min, the lter was blocked in 10% non-fat milk and then incubated with anti-X-TfeXAC2609 rabbit serum at 1:1,000 dilution. The primary antibody was detected using IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences) at 1:25,000 dilution. Membranes were scanned using an Odyssey CLx infrared imaging system (LI-COR Biosciences). Integrated uorescence intensities with median background subtracted were quantied using the Image Studio Lite (version 4.0) software package, normalized to the values measured for the WT Xac:E. coli mixture, and the results from seven independent experiments averaged. Western blots of total cellular X-TfeXAC2609 produced by the Xac strains were carried out with the same antibodies and documented using an Odyssey detection system as described above.

Xanthomonas growth in liquid and solid media. For analysis of Xac cell growth in liquid medium, the Xac WT, DvirB7 and DvirD4 strains were inoculated in triplicate in 2 TY medium with a starting OD600nm of 0.05, grown for 2 days at

30 C with agitation at 200 r.p.m. and the cell growth was monitored by measuring OD600nm and colony-forming unit (CFU) per ml at different time intervals.

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For analysis of cell growth on solid medium, 2.5 ml of Xac WT, DvirB7 and DvirD4 strains at OD600nm of 0.05 were plated onto LB agar medium. Colonies were resuspended in 1 ml of 2 TY after 32, 53 and 74 h of growth and serial dilutions

were plated on LB agar for CFU count.

Interbacterial growth competition experiments. Assays were performed in a manner similar to that described previously7,33. In brief, CFP-labelled Xac strains (Xac WT pUFR CFP, Xac DvirB7 pUFR CFP and Xac DvirB7 pUFR-virB7 CFP)

and E. coli DH5a YFP (Supplementary Table 2) were grown in 2 TY to log phase

(OD600nm 1.0), centrifuged, washed and resuspended to OD600nm 0.5. These

Xac cells were then mixed with E. coli diluted at an 80:1 Xac:E. coli ratio. The mixtures were spotted on LB agar plates containing gentamicin and allowed to grow together as a co-culture at 30 C. Fluorescence micrographs were collected after 48 h of incubation. Competition experiments of Xac cells (Xac WT GFP, Xac

DvirB7 GFP and Xac DvirD4 GFP; Supplementary Table 2) against C. violaceum were performed as above without antibiotic selection and analysed after 24 h of incubation. The results of competition experiments were quantied by determining the CFU of Xac and E. coli per colony of a co-culture initiated with a 180:1 Xac:E. coli mixture in which the initial Xac OD600nm 0.05. Xac strains used were Xac

WT pUFR, Xac DvirB7 pUFR and Xac DvirB7 pUFR-virB7 and the E. coli strain was E. coli BL21(DE3)RIL ArcticExpress. Final Xac:E. coli ratios were calculated after 38 h of competition by resuspending the colony, performing serial dilution and plating the cells in LB agar media containing tetracycline for selection of E. coli and ampicillin for selection of Xac.

Contact-dependent bacterial competition assays. GFP-labelled Xac strains (Xac WT pBRA GFP, Xac DvirD4 pBRA GFP, Xac DvirD4 pBRA-virD4 GFP, Xac DvirB7 pBRA GFP and Xac DvirB7 pBRA-virB7 GFP; Supplementary Table 2) andE. coli BL21(DE3)RIL ArcticExpress were grown in 2 TY to OD600nm 1.0,

collected by centrifugation, washed and resuspended to OD600nm 2.0. Xac strains

and E. coli were mixed at a 1:1 ratio. Xac alone or the mixtures were spotted onto a0.22 mm polycarbonate membrane (Whatman) placed on LB agar plates containing gentamicin and spectinomycin and the gfp uorescence emission from the Xac cells was documented after 24 h growth at 30 C. In parallel, no-contact experiments were carried out by growing Xac cells on an intervening lter placed on top of an E. coli BL21(DE3)RIL ArcticExpress lawn grown on LB agar plates containing gentamicin and spectinomycin. The intervening lter prevents direct contact between Xac and E. coli cells.

Microscopy analysis of Xac vs E. coli competition assays. Xac strains (Xac WT pUFR CFP, Xac DvirB7 pUFR CFP and Xac DvirB7 pUFR-virB7 CFP or Xac WT pBRA GFP, Xac DvirD4 pBRA GFP and Xac DvirD4 pBRA-virD4 GFP; see Supplementary Table 2) and E. coli cells (DH5a YFP or BL21(DE3)RIL ArcticExpress) were grown in 2 TY with appropriate antibiotics to OD600nm 1.0,

centrifuged, washed, resuspended to OD600nm 2.0, mixed at a Xac:E. coli 1:1 ratio

and 2 ml spotted onto an LB agarose pad (1.5% agarose in 25% LB medium). Interspecies competition was followed by uorescence and brighteld microscopy. Brighteld time-lapse data were acquired every 0.3 s. Image analysis and movie edition were performed using ImageJ software, and Supplementary Movie 1 was processed at 20 frames per second (that is, each second of the movie is equivalent to 6 s of the experiment).

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Acknowledgements

We thank Dr Frederico Gueiros-Filho for support in the microscopy studies and the

Brazilian Synchrotron Light Laboratory (LNLS) and the Central Analtica (Instituto de

Qumica, USP) for providing access to the X-ray beamlines. We thank Germn Sgro and

Miryam Marroquin for the kind gift of plasmids pBBR-5YFP and pBRA, respectively and

thank Cassia Docena for help in producing the virD4 knockout. This work was supported

by grants from the Fundaao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP) to

C.S.F. (# 2007/59425-0, 2005/59243-3, 2009/54009-3 and 2011/07777-5), FAPESP fel

lowships to D.P.S., A.W.B-F., C.E.A-M., G.D. and C.R.G., and CAPES fellowships to

D.P.S., A.W.B-F. and G.U.O. C.S.F and L.R.S.B thank CNPq for the nancial support.

Author contributions

D.P.S., G.U.O., C.E.A.-M., L.H., N.S.C. and M.C.A. performed the cloning, protein

purication and uorescence analysis. D.P.S. and G.U.O. performed the bacterial

competition and protein secretion assays. D.P.S., A.W.B.-F. and G.D. performed the

microscopy analysis. D.P.S., G.U.O., L.R.S.B., R.K.S., C.R.G. and C.S.F. performed the

structural studies. D.P.S., G.U.O. and C.S.F. analysed the data. D.P.S. and C.S.F. designed

the study, performed the bioinformatics analyses and wrote the paper.

Additional information

Accession codes X-ray crystallographic coordinates and structure factors for

X-TXAC2610 have been deposited in the Protein Data Bank with accession code 4QTQ.

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How to cite this article: Souza, D. P. et al. Bacterial killing via a Type IV secretion

system. Nat. Commun. 6:6453 doi: 10.1038/ncomms7453 (2015).

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