Several bacterial species possess more than one T6SS gene clusters in their genome, but only one main cluster was found in the sequenced A. tumefaciens strains so far (Boyer et al., 2009; Lin et al., 2013; Huang et al., 2015; Cho et al., 2018). A.
tumefaciens T6SS was first noticed by a secretome analysis that has identified the
secretion of Hcp (Wu et al., 2008), which is named based on its co-regulation of hemolysin protein (Williams et al., 1996). As Hcp is considered as a hallmark of a functional T6SS, each of the genes in the T6SS gene cluster of A. tumefaciens C58 were
subjected to in-frame deletion analysis to determine the genes essential for Hcp secretion (Lin et al., 2013). A. tumefaciens strain C58 encodes one T6SS main cluster containing 2 operons, imp operon and hcp operon, and another orphan vgrG-associated operon named as vgrG2 operon located elsewhere (Figure 1A). It is noteworthy that the T6SS outer membrane protein TssJ found in other bacterial species is not encoded in A.
tumefaciens genome though it is considered as an essential component of T6SS
membrane complex (Durand et al., 2015). TssL and TssM are two inner membrane proteins that form a complex in A. tumefaciens, and the ATPase domain of TssM is required for Hcp secretion and therefore proposed to energize T6SS machine assembly (Ma et al., 2009; Ma et al., 2012).
A. tumefaciens T6SS is transcriptionally induced by acid signal via ChvG/ChvI
two-component system (Wu et al., 2012). When grown in neutral minimal medium, transcription of imp operon is suppressed by a mature periplasmic ExoR protein that binds to ChvG to inhibit the signal transduction of ChvG/ChvI two-component system.
While periplasmic ExoR is not stable under acidic environment; the inhibition caused by ExoR is lost and transcription of imp operon is induced by ChvG/ChvI. The hcp operon is expressed at basal level at neutral pH but also upregulated by acid signal at
transcriptional level. The acid-induced T6SS transcription is also found in transcriptome analysis (Yuan et al., 2008; Heckel et al., 2014).
Other than transcriptional regulation, T6SS is also post-translationally regulated. In P.
aeruginosa, TagQRST proteins on the membrane are able to sense the perturbation of
the membrane triggering the self-phosphorylation of PpkA followed by phosphorylation of Fha, a forkhead associated domain-containing protein, by PpkA (Hsu et al., 2009;
Basler et al., 2013; Casabona et al., 2013). It is proposed that this regulation is
associated with an interesting phenotype, so called “tit-for-tat”; P. aeruginosa T6SS is efficiently firing only when it is attacked by other T6SS from the target cell (Basler et al., 2013). While PpkA is responsible for phosphorylating Fha and activate T6SS, PppA dephosphorylates Fha and represses T6SS, and this regulation pathway is called
“threonine phosphorylation pathway (TPP)” (Mougous et al., 2007). While Fha is the phosphorylation substrate by PpkA in P. aeruginosa and Serratia marcescens (Fritsch et al., 2013), TssL instead of Fha is phosphorylated by PpkA first found in A. tumefaciens, and recently in Vibrio alginolyticus (Yang et al., 2018). In A. tumefaciens, TssL
phosphorylation is required for recruitment of Fha for activation of type VI secretion (Lin et al., 2014). T6SS is also negatively regulated at post-translational level by TagF
protein in a TPP-independent manner (Silverman et al., 2011; Lin et al., 2018). In P.
aeruginosa, deletion of tagF gene increases Hcp secretion, and it is independent from
TPP since Hcp secretion of strains with Fha phosphorylation site substitution mutant is still de-repressed in the tagF deletion mutant (Silverman et al., 2011). In A. tumefaciens, TagF is fused with PppA (Atu4331). Overexpression of TagF-PppA or TagF domain only in A. tumefaciens abolished Hcp secretion. Same as P. aeruginosa, it is
independent from TPP since TssL is still phosphorylated when T6SS is repressed by TagF overexpression (Lin et al., 2018). Interaction between TagF and Fha is shown to be essential for TagF-dependent repression in both A. tumefaciens and P. aeruginosa (Lin et al., 2018).
In A. tumefaciens strain C58, three toxin-immunity pairs were identified, namely type VI DNase effector and immunity 1 and 2 (tde1-tdi1, tde2-tdi2) and type VI
peptidoglycan amidase effector and immunity, tae-tai (Ma et al., 2014) (Figure 1A).
Tde1 and Tde2 have been shown to exhibit DNase activity while biochemical activity of Tae as a peptidoglycan amidase has not been reported. All of these toxins have toxicity when expressed by an inducible promoter in bacterial cells while Tde but not Tae toxins exhibits T6SS-dependent interbacterial toxicity (Ma et al., 2014). Previous study
indicates that Tde1 and Tde2 are translocated via the cognate VgrG proteins, and both of them require adaptor/chaperone, Tap-1for Tde1 and Atu3641 for Tde2, for secretion and interbacterial toxicity (Bondage et al., 2016). Also, the interaction relationship among Tap-1, Tde1 and VgrG1 have been determined; Tap-1 and Tde1 can form a complex independent of VgrG1 but they require each other to be loaded onto VgrG1.
Interestingly, though PAAR protein is believed to be critical for T6SS assembly (Shneider et al., 2013; Cianfanelli et al., 2016a), deletion of paar gene (atu4352) only reduced but not abolished type VI secretion in A. tumefaciens (Bondage et al., 2016).
Tae was found to be co-precipitated by Hcp in A. tumefaciens although no direct interaction was identified when co-expressed in E. coli (Lin et al., 2013). Based on the evidence that Tae secretion is always correlated with Hcp secretion, Tae may be loaded in the lumen of Hcp tube for secretion, like Tse2 in P. aeruginosa (Silverman et al., 2013). In this study, I found that Tde effector not only requires cognate VgrG for delivery, Tde effector loading onto VgrG is also required for VgrG secretion. The protein-protein interaction data indicates that A. tumefaciens VgrG1 and VgrG2 can interact with TssA, TssF, TssG and TssK. Tde loading onto VgrG enhances the interaction between VgrG and T6SS baseplate protein TssK. I also found that TssK interacts with TssM and this interaction is lost in the absence of Tde. Thus, we proposed
that TssK is able to sense the conformational change of VgrG once loaded with its cognate Tde effector and trigger T6SS machine assembly via interaction with TssM. In summary, our study reveals a novel mechanism found in A. tumefaciens and such mechanism may be also conserved in other bacterial species as a strategy to save energy by limiting T6SS machine firing when there is no effector in the cell.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Bacterial strains used in this study are listed in Table 3. A. tumefaciens was grown in 523 medium at 28 ℃ and E. coli was cultured in LB medium at 37 ℃ unless indicated otherwise (Lin et al., 2013; Ma et al., 2014; Bondage et al., 2016). The antibiotics were used as the concentrations below. Gentamycin (Gm): 50 μg/mL for A. tumefaciens and 30 μg/mL for E. coli. Spectinomycin: 200 μg/mL. Kanamycin: 50 μg/mL. Amipicilin:
100 μg/mL. Chloramphenicol: 150 μg/mL.
Medium preparation
LB medium was prepared with dissolving 25 g of the prepared powder (Becton,
Dickinson and Company, Sparks, USA) containing 10 g tryptone, 5 g yeast extract and 10 g sodium chloride and adjusted pH to 7.0 per liter followed by autoclave
sterilization.
523 medium was prepared by dissolving 10 g sucrose, 8 g casein enzymatic
hydrolysate, 4 g yeast extract, 3 g K2HPO4, 0.3 g MgSO4. H2O in 1 liter water and the
pH was adjusted to 7.0 followed by autoclave sterilization (Kado and Heskett, 1970).
I-medium was prepared by dissolving 3 g K2HPO4, 1 g NaH2PO4, 1 g NH4Cl, 0.15 g KCl and 9.76 g MES (C6H13NO4S. H2O) in 900 mL water (Lai and Kado, 1998). For T6SS induction, the pH was adjusted to 5.5 (Wu et al., 2012). After autoclaving, 100 mL of sterile 20 % glucose, 1 mL of sterile FeSO4. 7H2O (250 mg/ 100 mL), 1.25 mL of sterile 1 M MgSO4. 7H2O and 1 mL of sterile 0.1 M CaCl2 were added.
For agar plates, 15 g agar was added to 1 L medium above followed by autoclave sterilization.
DNA preparation
Plasmid DNA was extracted using Presto Mini Plasmids Kit (Geneaid, Taiwan).
Polymerase chain reaction (PCR) for colony PCR and plasmid construction involves 2X Ready Mix A (Zymeset) by following manufacturer’s protocol. A. tumefaciens tssK gene and its upstream ribosome binding site was amplified from pRL-TssK (EML1301) (Table 3) with primers tssK_BamHI_F and tssK_SalI_R (Table 4) containing restriction sites for BanHI and SalI. The PCR product and pTrc200 vector were double-digested
T4 DNA ligase (New England BioLabs, Ipswich, USA). The plasmid construct was confirmed with colony PCR, enzyme digestion, sequencing and western blot of A.
tumefaciens cells harboring the plasmid.
Mutant construction
In-frame deletion of A. tumefaciens mutants were generated with pJQ200KS suicide plasmid (Quandt and Hynes, 1993) via a double crossover process (Ma et al., 2009). In brief, after transformation by electroporation, transformants were selected with 523 agar plate containing gentamycin without sucrose. The Gm resistant colonies were further cultured in LB broth without Gm overnight followed by serial dilutions and spreading onto 523 agar plates containing 5% sucrose without Gm. Bacterial cells undergoing second crossover are able to survive on plates containing 5% sucrose. The deletion mutants were confirmed by colony PCR and western blot.
Type VI secretion assay
Type VI secretion assay was performed as described (Bondage et al., 2016). In brief, A.
tumefaciens strains were cultured in 523 medium overnight and sub-cultured with
OD600nm 0.2 as the initial cell density in I medium (pH 5.5) or 523 medium, depending on the purpose of the experiments, for 6 hours at 25 ℃. After subculture, the
supernatant and the bacterial cells were separated by centrifugation with 10,000 g for 10 min. Total cell pellets were adjusted to OD600nm 5 and supernatant was filtered with low protein-binding 0.22 μm sterilized filter units (Milipore, Tullagreen, Ireland). Proteins in the supernatants were precipitated by incubation of 1 mL supernatant with 150 μL of 100% trichloroacetic acid (TCA) and 30 μL of 10% deoxycholic acid (DOC) at 4 ℃ overnight followed by centrifugation at 17,000 g, 15 min at 4 ℃. The resulting protein pellets were solubilized with 10 μL of 1 M tris with original pH. All the samples were added with 4X sodium dodecyl sulfate (SDS) sample loading buffer (SSB) and 1/10 volume of 1 M dithiothreitol (DTT) to result in protein samples in 1X SSB and 0.1 M DTT and boiled for 10 min. The samples were then centrifuged with 10,000 g, 10 mins at 4℃ and the resulting supernatant containing solubilized proteins were subjected to SDS-PAGE and western blot analysis.
Western blot analysis
Proteins were usually analyzed with 12 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) except protein whose sizes are under 20 kDa and over 80 kDa are
analyzed by 15 % and 7 % SDS-PAGE, respectively. Appropriate amounts of
solubilized protein samples were loaded into SDS-PAGE for electrophoresis followed by transferring onto a polyvinylidene difluoride (PVDF) membrane. The PVDF
membrane containing protein samples was first blocked with TBST buffer (2.42 g Tris-base, 8 g NaCl, 2 g KCl, and 0.5 mL Tween 20 in liter ddH2O) containing 5 % skim milk overnight in the cold room, and then changed to TBST containing 5 % skim milk and primary antibody with optimal titer (1:2,500 for Hcp; 1:4,000 for RpoA; 1:4,000 for Tde1; 1:2,000 for Tae; 1:1,000 for VgrG; 1:1,000 for VgrG1; 1:4,000 for TssA; 1:1,000 for TssE; 1:1,000 for TssK; 1:4,000 for TssB; 1:2,000 for ClpV; 1:5,000 for Tap-1;
1:3,000 for HA tag; 1:10,000 for His tag) at room temperature for at least 1 hr (Lin et al., 2013; Bondage et al., 2016). The PVDF membranes were washed with TBST buffer three times, 5 min each, followed by hybridizing with horseradish
peroxidase-conjugated anti-rabbit secondary antibody (1:20000). Finally, membranes were washed with TBST four times, 5 min each, and Western Lightening ECL Pro Enhanced
Chemiluminescence Substrate (PerkinElmer, Watham, USA) was added to produce the chemical luminescence following the user manual. The chemiluminescent was detected
and visualized with X-ray films.
Co-purification assay in E. coli
E. coli co-purification was performed essentially as described previously (Lin et al.,
2013). In brief, 5 ml overnight culture of E. coli strain BL21(DE3) containing protein expression plasmids was subcultured into 25 ml LB containing appropriate antibiotics for 3 hours and induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) with final concentration of 0.5 mM after subculture for 1 hour. Bacterial cells were collected by centrifugation at 10,000 g, 10 mins and the cell pellet was washed and resuspended with 5 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, 1 mM
phenylmethylsulfonyl fluoride (PMSF), pH8.0). Cells were broken with Constant Cell Disruption System (Constant System, Northants, UK) with 40,000 psi. Part of the cell lysate was saved as an input sample. Approximately 1700 μL cell lysate was incubated with 100 μL Ni-NTA resin pre-equilibriumed with lysis buffer for 30 mins at 4 ℃. After binding with the protein, Ni-NTA column containing the specific proteins was washed with 1 ml of wash buffer (50 mM NaH2PO4, 0.3 M NaCl, 30 mM imidazole, pH 8.0)
300 mM NaCl, 250 mM imidazole, pH 8.0). Input and elute samples were further analyzed with SDS-PAGE followed by western blot analysis.
Biochemical fractionation of A. tumefaciens proteins
Fractionation of A. tumefaciens cytoplasmic proteins, periplasmic proteins and
membrane proteins was described previously (Ma et al., 2009). In general, 20 ml of overnight cultured A. tumefaciens cells in 523 medium was subcultured into 200 ml I medium (pH 5.5) for 6 hours in 25 ℃. Cells were collected and resuspended in 5 mL buffer containing 50 mM Tris-Cl, pH 7.5, 20 % sucrose 2 mM EDTA and 1 mM (PMSF) with addition of lysozyme powder to a final concentration of 0.5 mg/mL and incubated at room temperature with gentle rocking for 1 hour followed by centrifugation at 10,000 g for 10 mins at 4 ℃. The supernatant was saved as periplasmic proteins.
Pellets were resuspended in 5 ml of buffer containing 50 mM Tris-Cl (pH 7.5), 0.2 M KCl and 1 mM PMSF. Cells were broken by French Pressure Cell Press (Thermo, Needham Heights, USA) with 40,000 psi and the cytosol and membrane fractions were separated by centrifugation at 150,000 g for 1 hour in micro-centrifuge tube with thicker walls. After centrifugation, the supernatant was collected as cytosol fraction and the
pellet was solubilized in 50 mM Tris-Cl (pH 7.5) buffer containing 0.2 M KCl and 1 mM PMSF by adding n-dodecyl-β-D-maltoside (DDM) to the final concentration is 1
%. Protein fractions were analyzed by SDS-PAGE followed by western blot analysis.
Co-purification assay in A. tumefaciens
A. tumefaciens strain producing His-tagged specific protein expressed on pRL662
was cultured in 10 ml 523 medium overnight and subcultured into 100 ml I medium (pH 5.5) for 6 hours. Same with co-purification experiment in E. coli, cells were broken with Constant Cell Disruption System and the proteins were co-purified with a Ni-NTA column. The input and elute samples were analyzed with SDS-PAGE followed by western blot analysis.
Bacterial two hybrid
The bacterial two hybrid assay was conducted following the protocol (Battesti and Bouveret, 2012). E. coli strain DHM1 was transformed by heat shock with pT18 and pT25 plasmids expressing adenylate cyclase T18 or T25 domain fused protein. After
selection of the colonies harboring the two plasmids on LB plates containing
chloramphenicol and ampicillin, single colony transformants were cultured in 3 mL LB containing antibiotics and 0.5 mM IPTG. After 30℃ overnight culture, 2 μL of the bacterial culture was dropped onto LB agar plates containing chloramphenicol,
ampicillin, 0.5 mM IPTG and 40 μg/mL X-Gal followed by incubation at 30℃ for two days for colorimetric signal development.
RESULTS
VgrG cargo effector loading is required for cognate VgrG secretion
A. tumefaciens strain C58 encodes two vgrG genes and three T6SS toxin-immunity
pairs, namely, Tde1-Tdi1, Tde2-Tdi2 and Tae-Tai (Figure 1A) (Lin et al., 2013; Ma et al., 2014). Among the T6SS toxin effectors, Tde1 and Tde2 are DNases and Tae is predicted to be a peptidoglycan amidase. Two VgrG proteins, VgrG1 and VgrG2, are mainly different at the C-terminus, which confers the effector specificity toward Tde1 and Tde2 respectively (Bondage et al., 2016). To determine whether effector loading onto VgrG spike plays any role on the T6SS assembly or firing, type VI secretion assay (Figure 2) to detect the secretion of Hcp and VgrG proteins, a hallmark of a functional T6SS, was conducted in wild type C58 and each of the effector-immunity pair deletion mutants (Figure 1B). The VgrG antibody used in this study is able to detect both VgrG1 and VgrG2, and they can be differentiated with their molecular weight; the upper band is VgrG1 and the lower band is VgrG2 (Lin et al., 2013). As controls, Hcp, VgrG1/2, Tde1, and Tae are secreted from wild type C58 but not from ΔtssL. In single, double, and triple toxin-immunity pair mutants, it is interesting to note that VgrG1 secretion is coincided with the secretion of its cognate Tde1 effector (C58 and Δtde2-tdi2).
Similarly, VgrG2 secretion is only detected from strains capable of Tde2 delivery, i. e.
C58, Δtde1-tdi1, Δtae1-tai1, Δtae1-tai1Δtde1-tdi1. Because of low abundance of endogenous cellular Tde2, we are unable to detect Tde2 secretion but Tde2-mediated antibacterial activity has been demonstrated previously (Bondage et al., 2016). While Hcp secretion level is similar to wild type in each of single toxin-immunity pair deletion mutants; surprisingly, no Hcp secretion could be detected in tde-tdi double deletion mutant (Δtdei). In other words, Hcp is secreted only when either of the VgrG proteins is secreted. We also found that Δtae-tai mutant has a polar effect as downstream VgrG1, Tap-1, and Tde1 proteins were not detected. As a result, no VgrG1 and Tde1 secretion could be detected whenever tae-tai gene pair is deleted. To confirm whether the loss of Hcp and VgrG secretion is indeed caused by the absence of Tde1 and Tde2,
complementation test was carried out. Indeed, expression of Tde1 but not Tde2 in Δtdei restored VgrG1 secretion whereas Tde2 but not Tde1 restored VgrG2 secretion (Figure 1C). Hcp secretion is restored whenever VgrG1 or VgrG2 is secreted.
Previous study showed that secretion and/or antibacterial activity of Tde1 and Tde2 require specific adaptor/chaperone proteins for loading onto their cognate VgrG spike for delivery (Bondage et al., 2016). Taken together with the secretion assay results from
Figure 1B and 1C, we hypothesize that cargo effector loading (such Tde1 onto VgrG1) is not only required for effector delivery but such effector-VgrG interaction may also affect cognate VgrG’s function as a T6SS machine component and therefore impact VgrG and Hcp secretion. While the secretion results in various toxin-immunity pair mutants is consistent with our hypothesis, it remains possible that it is the protein accumulation of Tde in the cell affects T6SS machine function rather than the
interaction of Tde-VgrG. To rule out this possibility, we conducted secretion assay using T6SS effector adaptor/chaperone deletion mutant, that is Δtap-1 and Δatu3641 (Figure 3). Tap-1 is a DUF4123-containing protein which is a T6SS adaptor/chaperone protein in both V. cholerae and A. tumefaciens (Liang et al., 2015; Unterweger et al., 2015;
Bondage et al., 2016). In A. tumefaciens, Tap-1-Tde1-VgrG1 interaction was
demonstrated in our previous study (Bondage et al., 2016); Tde1 and Tap-1 require each other to interact with VgrG1 and Tap-1-Tde1 complex may form first followed by loaded onto VgrG1while the actual interaction interface in the complex is not determined yet. Atu3641, a DUF2169-containing protein, was proposed to be an adaptor/chaperone of Tde2 since there is no Tde2-mediated antibacterial activity when atu3641 is deleted. Also, co-IP data showed that VgrG2 but not VgrG1 interacts with
loaded onto VgrG2 (Devanand Bondage, unpublished result). As shown in Figure 3, VgrG1 is secreted only in the presence of Tap-1 and vice versa for VgrG2 requiring Atu3641 for its secretion. This result excluded the possibility that Tde protein accumulation in the cell may affect T6SS function since Tde effector in these two mutants still accumulates significant amounts in the cell. Taken together, we suggested that cargo effector loading onto cognate VgrG protein is important for VgrG secretion, and this may be a mechanism that A. tumefaciens uses to save energy when Tde effector is not loaded onto VgrG.
VgrG proteins interact with baseplate components TssAFGK
To further elucidate the mechanism underlying the requirement of Tde effector loading
for VgrG secretion, we went on studying the subcellular localization and the protein interaction network of VgrG. We first co-expressed His-tagged VgrG1 with each of T6SS baseplate components in E. coli for co-purification assay (Figure 4 and 5). The results showed that TssA, TssF, TssG and TssK are co-purified with VgrG1 but not TssE, suggesting the direct interaction between VgrG1 with TssA, TssF, TssG and TssK in the absence of other Agrobacterium T6SS proteins. TssA-VgrG interaction has been
demonstrated in both P. aeruginosa and EAEC using B2H (Planamente et al., 2016;
Zoued et al., 2016). Furthermore, TssF-VgrG and TssG-VgrG interaction is also
demonstrated in EAEC with B2H and co-IP (Brunet et al., 2015). However, TssK-VgrG B2H result was negative in EAEC (Zoued et al., 2013).
To confirm the interaction relationship between VgrG and baseplate components, we then used B2H and pulldown assay in A. tumefaciens to confirm the interactions.
B2H is a quick and sensitive assay for identifying interactions between two proteins
B2H is a quick and sensitive assay for identifying interactions between two proteins