BcsK_C Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsL_B

Abstract

The type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK_C is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsK_C is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pulldown assays demonstrated that BcsK_C interacts with BcsL_B, another conserved T6SS component. Internal deletions within BcsK_C revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL_B. Fractionation experiments showed that BcsK_C can be in the cytosol or tightly associated with the outer membrane and that BcsK_C and BcsL_B form a high molecular weight complex anchored to the outer membrane that requires BcsF_H (a ClpV homolog) to be assembled. Together, our data show that BcsK_C/BcsL_B interaction is essential for the T6SS activity in B. cenocepacia.

Introduction

The Burkholderia cepacia complex comprises closely related species of Gram-negative bacteria commonly found in the environment (1, 2). B. cepacia complex bacteria are opportunistic human pathogens that establish chronic lung infections in immunocompromised patients with chronic granulomatous disease or, most commonly, cystic fibrosis (3). In most cystic fibrosis centers worldwide and more particularly in Canada, Burkholderia cenocepacia is one of the most common B. cepacia complex species recovered from patients (4, 5) and is frequently associated with the most severe infections (6).

Novel factors for B. cenocepacia survival in vivo were discovered in our laboratory by signature-tagged mutagenesis using a rat model of chronic lung infection (7), and these included a type 6 secretion system (T6SS)³ (7, 8). The T6SS is a newly recognized secretion system widespread among Gram-negative pathogens and symbionts that are in close interaction with eukaryotic cells (9–11). Several proteins specifically secreted by the T6SS into culture supernatants have been identified. These proteins usually lack any conventional N-terminal hydrophobic signal peptide and include the hemolysin coregulated-like protein (Hcp) (12), the valine-glycine repeat family proteins (VgrG) (13), and in some cases non-Hcp, non-VgrG substrates (12, 14, 15). Hcp is secreted by most of the T6SSs studied to date, and its detection in culture supernatants is considered the hallmark of a functional T6SS (8, 16–19). Hcp and VgrG proteins might also be part of the T6SS structural scaffold displayed at the bacterial surface and might be released into culture supernatants by shearing forces (20, 21).

The T6SS contributes to the pathogenicity of many bacteria (12, 16, 17, 22, 23). In Vibrio cholerae, bacteria-host contact and endocytosis are required to trigger the translocation of VgrG-1 into the host cell cytosol. Cytosolic VgrG-1 mediates actin cross-linking impairing the phagocytic cell functions (24). Although in some bacteria the T6SS has evolved to target eukaryotic cells, the Pseudomonas aeruginosa T6SS has been recently shown to deliver toxins into other bacteria. This novel T6SS property would confer a fitness advantage to P. aeruginosa growing in bacterial communities (25).

Typically, the T6SS is poorly expressed under in vitro laboratory conditions, but it is quorum sensing regulated (19, 26) and induced in vivo during infection (9, 11, 27). We have recently identified AtsR, a sensor kinase-response regulator hybrid that negatively regulates the T6SS expression in B. cenocepacia K56-2 (8). Deletion of atsR causes increased T6SS activity, as exemplified by hypersecretion of Hcp into culture supernatant (8). Infection of macrophages with the atsR deletion strain induces cytoskeletal rearrangements, resulting in macrophages displaying actin-rich cellular projections (8). This phenotype relies on T6SS activity and has only been described for B. cenocepacia, but the B. cenocepacia effector molecules and the eukaryotic targets involved in this process are unknown and currently under investigation in our laboratory.

Similarity between bacteriophage proteins and the T6S-associated Hcp and VgrG proteins suggests that the T6SS is structurally and evolutionarily related to bacteriophages (21, 28). Several components highly conserved among T6SSs have been investigated, and some light has been shed on their localization, function, and interaction with other T6SS components. These include: (i) the integral inner membrane IcmF-like protein (ImpL_M) from Agrobacterium tumefaciens (29); (ii) the outer membrane lipoprotein SciN (30); (iii) the polytopic inner membrane protein SciZ anchoring the T6SS to the cell wall (31) in enteroaggregative Escherichia coli; (iv) the proteins VipA in V. cholerae (32) and its homolog IglA in Francisella novicida, which interact with VipB and IglB (11), respectively; and (v) the energizing component ClpV, which disintegrates cytosolic tubule like structures made of VipA and VipB (32).

This study focuses on BcsK_C, a T6SS component from B. cenocepacia homologous to IglB. We show here that BcsK_C is critical for the T6SS activity of B. cenocepacia K56-2. Experiments employing the bacterial two-hybrid system confirmed that BcsK_C also interacts with BcsL_B, a protein homologous to IglA, and support the notion that interactions between BcsL_B- and BcsK_C-like proteins are a common feature of T6SSs. Internal deletions of BcsK_C allowed us to characterize the region required for BcsL_B binding and demonstrated that the N terminus of BcsK_C is essential and sufficient for interaction with BcsL_B. Furthermore, cell fractionation experiments revealed that BcsK_C can be tightly associated with the outer membrane and that BcsK_C, in association with BcsL_B, forms a membrane associated high molecular weight complex whose assembly requires BcsF_H (ClpV homolog). Together, this work establishes BcsK_C as a critical component of the T6S apparatus.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Media

Bacterial strains and plasmids used in this study are listed in supplemental Table S1. Bacteria were routinely cultured in LB (Difco) at 37 °C. E. coli cultures were supplemented, as required, with the following antibiotics (final concentrations): 100 μg/ml ampicillin, 30 μg/ml tetracycline, 30 μg/ml kanamycin, 30 μg/ml chloramphenicol, 50 μg/ml trimethoprim, and 50 μg/ml gentamicin. B. cenocepacia cultures were supplemented, as required, with 100 μg/ml trimethoprim, 100 μg/ml tetracycline, and 50 μg/ml gentamicin. We used the unified nomenclature proposed by Cascales (33) and assigned the alphabetical character designated by Shalom et al. (34) to the T6SS genes from B. cenocepacia K56-2.

General Molecular Techniques

DNA manipulations were performed as described previously (35). T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. Transformation of E. coli DH5α, E. coli SY327, and E. coli KEM5 was done by the calcium chloride protocol (36). Mobilization of complementing plasmids and mutagenesis plasmids into B. cenocepacia K56-2 was performed by triparental mating using E. coli DH5α carrying the helper plasmid pRK2013 (37, 38). DNA amplification by PCR was performed using a PTC-221 DNA engine (MJ Research, Incline Village, Nevada) with Taq, Proof Start, or HotStar HiFidelity DNA polymerases (Qiagen). DNA sequences of all of the primers used in this study are described in supplemental Table S2. DNA sequencing was performed at the DNA sequencing facility of York University (Toronto, Canada). The computer program BLAST was used to analyze the sequenced genome of B. cenocepacia strain J2315.

Mutagenesis of B. cenocepacia K56-2 and Complementing Plasmids

Unmarked and nonpolar deletions were performed as described previously (39). Details about the construction of the deletion plasmids are available in the supplemental materials. Plasmid pDelatsR was used to delete atsR (BCAM0379) in B. cenocepacia K56-2. Deletions were subsequently performed in the atsR-deleted mutant, B. cenocepacia K56-2 ΔatsR. Plasmids pDelT6SS, pDelTU1, pDelTU2, and pDelTU3 were used to delete the entire T6SS gene cluster: T6SS transcriptional units 1, 2, and 3, respectively. Each gene within transcriptional unit 2 was individually deleted. Plasmids pDelbcsM, pDelbcsL_B, pDelbcsK_C, pDA45, pDelbcsI_E, pDelbcsH_F, pDelbcsG_G, pDelbcsF_H, and pDelbcsE_A were used to delete BCAL0340, 0341, 0342, 0343, 0344, 0345, 0346, 0347, and BCAL0348, respectively. Gene deletions were first analyzed by PCR and also confirmed by Southern blot hybridization.

The bcsK_C gene was PCR-amplified using primer pair 3401–3393. Amplicons were digested with the restriction enzymes KpnI-SacI and cloned into a similarly digested pME6000 plasmid, giving rise to pBcsK_C. We took advantage of two SphI restriction sites within the bcsK_C gene sequence to generate an in-frame deletion of 0.45 kb. The bcsK_C gene was PCR-amplified as described above and digested with KpnI, SacI, and SphI, generating three fragments (0.13, 0.45, and 0.93 kb). Only the 0.13- and 0.93-kb fragments were gel-purified using the QiaQuick gel extraction kit (Qiagen) and cloned as above into the KpnI-SacI-digested pME6000 plasmid, giving rise to pBcsK_CΔ40–189.

A FLAG epitope was fused to the N termini of BcsK_C and BcsK_CΔ40–189 as follows. The bcsK_C and bcsK_CΔ40–189 genes were PCR-amplified using primer pair 3700–3701. Amplicons were digested with the restriction enzymes HindIII-BamHI and cloned into a similarly digested pEL-1 plasmid giving rise to pBcsK_C-FLAG and pBcsK_{CΔ40–189-FLAG}.

Preparation of Culture Supernatant Proteins

Culture supernatant proteins were precipitated with trichloroacetic acid as described previously (8). Ten μg of protein were loaded on a 16% SDS-PAGE. Detection was performed with a Brilliant Blue G colloidal staining according to the manufacturer's recommendations (Sigma).

Pulldown Assay and MS

Protein G-agarose beads (Roche Applied Science; 20-μl packed volume/sample) were washed three times with 20 volumes of TBS. Five μg of FLAG M2 monoclonal antibody (Sigma) per sample were incubated with the bead suspensions with constant agitation for 2 h at 4 °C using a Barnstead Thermolyne LABQUAKE (Barnstead International, Dubuque, IA). The beads were washed three times with 20 volumes of TBS. Overnight cultures of B. cenocepacia K56-2 mutants ΔatsR ΔbcsK_C (pEL-1), ΔatsR ΔbcsK_C (pBcsK_C-FLAG), and ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}) were 10-fold diluted in 50 ml of prewarmed LB. After 3 h of incubation at 37 °C, the cultures were centrifuged for 10 min at 10,000 × g at 4 °C. Bacterial pellets were washed twice with 10 ml of TBS and resuspended in 4 ml of TBS containing a protease inhibitor mixture (Roche Applied Science). The cells were lysed by three passages in a French press (Thermo Scientific) using a miniature French pressure cell at 600 p.s.i. The cell lysates were centrifuged first at 5000 × g at 4 °C for 15 min, and the supernatants were transferred in fresh tubes, centrifuged at 21,000 × g for 15 min, and collected. The bead suspensions conjugated to the FLAG antibody were added to 1 ml of each supernatant and incubated for 3 h at 4 °C with constant rotation. The beads were washed three times with 10 volumes of TBS and resuspended in 100 μl of TBS. Twenty μl of bead suspension were boiled and loaded in duplicate on a 16% SDS-PAGE gel. After electrophoresis, detection was performed on half of the gel by silver staining adapted from Blum et al. (40). The other half of the gel was transferred to a nitrocellulose membrane for immunoblot analysis (see below).

Protein bands were excised from a silver-stained one-dimensional gel using an Ettan Spot-Picker (Amersham Biosciences). In-gel trypsin digestion was performed in the Functional Proteomics Facility of the University of Western Ontario using the Waters MassPREP automated station. Five μl of submitted sample was injected for LC/MS/MS analysis as described elsewhere (8). The data were processed using the Proteinlynx Global Server software, and a database search was performed using MASCOT search engine (Matrix Science, London, UK). This search also provides a score estimating the probability that the observed match between experimental data and each protein in the database is a random event. The higher the protein score, the higher the confidence that the protein in the sample corresponds to the match.

Bacterial Two-hybrid System, β-Galactosidase Assay, and Plasmid Constructions

In vivo interaction between BcsK_C and BcsL_B was investigated using a bacterial adenylate cyclase two-hybrid system kit (Euromedex). This system is based on the interaction-mediated reconstitution of the adenylate cyclase activity in an adenylate cyclase-deficient E. coli reporter strain (41). The catalytic domain of adenylate cyclase (CyaA) from Bordetella pertussis consists of two complementary fragments, T25 and T18, that are not active when physically separated. When these two fragments are fused to interacting polypeptides and coexpressed in E. coli, heterodimerization of the hybrid proteins restores the activity CyaA, leading to cyclic AMP synthesis and transcriptional activation of the lac operon. Therefore, interaction between two hybrid proteins in E. coli will generate high levels of β-galactosidase activity, which can be readily scored on X-gal-containing medium and quantified.

BcsL_B and BcsK_C were fused to T25 and T18, respectively (details are available in the supplemental materials). Plasmid pT25BcsL_B encodes a hybrid protein made of BcsL_B fused to the C terminus of the T25 fragment (T25-BcsL_B). Plasmids pT18BcsK_C and pT18BcsK_CΔ40–189 encode hybrid proteins made of BcsK_C or a truncated variant fused to the C-terminal end of T18. Plasmids pBcsK_CT18 and pBcsK_CΔ40–189T18 encode hybrid proteins made of BcsK_C or a truncated variant fused to the N-terminal end of T18.

Sequential 25-amino acid deletions were performed within BcsK_C (details are available in the supplemental materials). Deletion of amino acids 38–62, 63–87, 88–112, 113–137, 138–162, 163–187, 188–212, 213–237, and 213–496 were performed, giving rise to plasmids pBcsK_CΔ38–62T18, pBcsK_CΔ63–87T18, pBcsK_CΔ88–112T18, pBcsK_CΔ113–137T18, pBcsK_CΔ138–162T18, pBcsK_CΔ163–187T18, pBcsK_CΔ188–212T18, pBcsK_CΔ213–237T18, and pBcsK_CΔ213–496T18, respectively, expressing truncated BcsK_C fused to the N terminus of T18.

The E. coli KEM5 reporter strain (lacking any endogenous adenylate cyclase activity) harboring pT25BcsL_B was cotransformed with the recombinant plasmids expressing hybrid proteins fused to the T18 domain. Bacterial cultures were grown for 16 h in LB at 30 °C with antibiotics as required and isopropyl β-d-1-thiogalactopyranoside (0.5 mm). Samples were taken to measure the bacterial growth (A_{600 nm}) and the β-galactosidase activity (A_{420 nm} and A_{550 nm}) upon lysis and incubation with O-nitrophenyl-β-d-galactoside according to Miller (42). Enzyme activities (Miller units) were calculated using the equation [(A_{420 nm} − 1.75 A_{550 nm}) × 1000]/[reaction time (min) × culture volume (ml) × A_{600 nm}]. Each sample was analyzed in triplicate during three independent experiments. Four μl of each culture were also spotted on a LB agar plate containing X-gal (40 μg/ml), isopropyl β-d-1-thiogalactopyranoside (0.5 mm), and antibiotics as required. Incubation was performed for 24 and 48 h at 30 and 37 °C.

Cell Fractionation, Sucrose Density Gradient, and Western Blot Analysis

Overnight cultures were diluted in prewarmed LB. After 3 h of incubation at 37 °C, the cultures were centrifuged for 10 min at 10,000 × g at 4 °C. Bacterial pellets were washed with 50 mm Tris-HCl, pH 7.4, and resuspended in 5 ml of the same buffer containing a protease inhibitor mixture, PMSF (1 mm), DNase (50 μg/ml), and RNase (50 μg/ml). The cells were lysed by three passages in a French press at 600 p.s.i. Unbroken cells were removed by centrifugation twice at 20,000 × g for 15 min at 4 °C. An aliquot of the supernatant corresponding to the total cell lysate was stored at −20 °C, and the remainder was processed to separate the soluble fraction from the total membrane fraction as follows. The membranes were pelleted by ultracentrifugation at 150,000 × g for 1 h at 4 °C in a Beckman 70Ti rotor. An aliquot of the supernatant corresponding to the soluble fraction was stored at −20 °C. To increase the purity of the membrane preparation, the pellet was resuspended in 2 ml of 50 mm Tris-HCl, pH 7.4, containing 10% (w/w) sucrose, a protease inhibitor mixture, and 1 mm PMSF and loaded on top of a discontinuous sucrose gradient consisting of 4 ml of 15% (w/w) sucrose placed on top of 4 ml of 55% (w/w) sucrose made in 50 mm Tris-HCl, pH 7.4. Ultracentrifugation was performed at 37,000 rpm for 1 h at 4 °C in a Beckman SW41. The membranes were collected using a syringe by puncturing the side of the tube slightly below the membrane ring. An aliquot corresponding to the total membrane fraction was stored at −20 °C.

The protein concentration of each fraction was determined by Bradford assay (Bio-Rad), and 10 μg of protein were loaded on 12% SDS-PAGE gels. Unless indicated, the samples were not boiled prior to loading. After electrophoresis, the gels were transferred to nitrocellulose membranes for immunoblot analysis. The membranes were incubated with the 4RA2 monoclonal antibody (Neoclone) cross-reacting with the B. cenocepacia RNA polymerase subunit 1 mm (cytosolic control) and the FLAG M2 monoclonal antibody (Sigma). The Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes) was used as a secondary antibody. Detection was performed using the Odyssey Infrared Imager (LI-COR Biosciences).

Protein solubilization experiments were performed based on the work of Ma et al. (29). Briefly, the membrane fractions were incubated with either 50 mm Tris-HCl, pH 7.4 (control), 1 m NaCl, 6 m urea, 3% Triton X-100, 2% N-lauroylsarcosine, or 1% SDS in ice for 1 h and centrifuged at 150,000 × g for 1 h at 4 °C in a Beckman 100Ti rotor to separate soluble and insoluble fractions. The pellet was resuspended in 50 mm Tris-HCl, pH 7.4, in the same volume as the soluble fraction and analyzed by Western blot.

To increase the separation of the inner and outer membranes, the cells were incubated at room temperature for 15 min in 20% (w/w) sucrose made in 50 mm Tris-HCl, pH 7.4, with protease inhibitor. DNase (50 μg/ml), RNase (50 μg/ml), EDTA (0.5 μm), and lysozyme (0.1 mg/ml) were added prior to cell lysis. The soluble lysate was loaded on top of a discontinuous sucrose gradient consisting of 4 ml of 25% (w/w) sucrose placed on top of 2 ml of 60% (w/w) sucrose made in 50 mm Tris-HCl, pH 7.4, and centrifuged as indicated previously. The sucrose concentration of the purified total membranes was lowered to 30% (w/w) sucrose and loaded on top of a second discontinuous sucrose gradient consisting of 0.6 ml of 60% and 1.16-ml layers of 56, 53, 50, 47, 44, 41, 38, 35, and 32% (w/w) sucrose. The membrane fractionation was performed by ultracentrifugation at 37,000 rpm for 40 h at 4 °C in a Beckman SW41 rotor. The gradient was collected from top to bottom using a syringe by puncturing holes in the tube every 0.3 cm. Protein content was assessed by measuring the absorption at 280 nm, and the quality of the fractionation was assessed by the detection of the outer membrane porin BCAM1931, by measuring the enzymatic activity of the NADH oxidase (inner membrane marker) (43) and the refractive index of each fraction. Ten-μl aliquots of the collected fractions were loaded on a 12% SDS-PAGE gel. After electrophoresis, the gel was transferred to a nitrocellulose membrane for immunoblot analysis or silver-stained. The samples need to be boiled to detect BCAM1931.

Electroelution

Purified total membranes from B. cenocepacia K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) and B. cenocepacia K56-2 ΔatsR ΔT6SS (pBcsK_C-FLAG) were prepared as above. One mg of total membrane proteins was loaded in several wells and separated by electrophoresis on 12% SDS-PAGE gels. After electrophoresis, a 4-mm-wide band from the top of the separating gel (containing the high molecular weight proteins) was excised from each gel and minced. The proteins were electroeluted in volatile buffer (50 mm NH₄HCO₃, 0.1% SDS) using a model 422 Electro-Eluter (Bio-Rad) and Green Membrane caps (molecular mass cutoff of 3,500 Da) for 6 h at 20 mA. After the elution the volatile buffer was removed using a spin vacuum, leaving concentrated proteins. The pellets were resuspended with 80 μl of volatile buffer, and protein concentration was estimated by measuring absorbance at 280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Seventy μg of protein were boiled for 10 min and loaded on a 16% SDS-PAGE gel. Protein was detected by silver staining, and selected protein bands were excised and analyzed by mass spectrometry as indicated above.

Macrophage Infection Assay

The C57BL/6 murine bone marrow-derived macrophage cell line ANA-1 (44) was maintained in DMEM with 10% (v/v) FBS and grown at 37 °C in a humidified atmosphere of 95 and 5% CO₂. Bacteria were added to ANA-1 cells at a multiplicity of infection of 50:1 as described previously (8) and incubated for 4 h at 37 °C. The images were digitally processed using the Northern Eclipse version 6.0 imaging analysis software (Empix Imaging, Mississauga, Canada).

RESULTS

BcsK_C Is Essential for T6SS Activity

The bcsK_C gene (BCAL0342) encodes a 496-amino acid protein (∼55 kDa). BLASTP analysis revealed that BcsK_C contains a domain of unknown function, DUF877, and belongs to a family of uncharacterized bacterial proteins that are associated with the T6S locus of many Gram-negative bacteria including Burkholderia mallei (TssB), P. aeruginosa (PA0084), V. cholerae (VipB), Salmonella enterica serovar Typhimurium (SciI), Edwardsiella tarda (EvpB), and F. novicida (IglB).

We have previously demonstrated that inactivation of atsR in B. cenocepacia K56-2 leads to increased T6SS activity, as exemplified by (i) hypersecretion of an Hcp-like protein and (ii) cytoskeletal reorganization with the formation of cellular projections upon infection of ANA-1 murine macrophages (8). Following these two phenotypes we investigated the importance of BcsK_C in the T6SS activity of B. cenocepacia K56-2. Analysis of culture supernatants (Fig. 1A) revealed that in contrast to K56-2 ΔatsR, the K56-2 ΔatsR ΔbcsK_C double mutant was unable to release Hcp, consistent with a secretion defect. Hcp secretion was restored upon introduction of pBcsK_C, which constitutively expresses BcsK_C into K56-2 ΔatsR ΔbcsK_C (Fig. 1A). Furthermore, more than 60% of ANA-1 macrophages displayed cellular projections upon infection with K56-2 ΔatsR (Fig. 1B, upper left panel), whereas this phenotype was absent in macrophages infected with K56-2 ΔatsR ΔbcsK_C. Introduction of pBcsK_C into K56-2 ΔatsR ΔbcsK_C (Fig. 1B, lower left panel) restored the ability of these bacteria to mediate cytoskeletal rearrangements in macrophages. Identical results were obtained with pBcsK_C-FLAG (supplemental Fig. S1), indicating that fusion of a FLAG epitope to the N terminus of BcsK_C does not alter the protein function. Together, these results demonstrate that BcsK_C is essential for T6SS function in B. cenocepacia K56-2.

FIGURE 1.

BcsK_C is required for T6SS activity. A, secretion assay. SDS-polyacrylamide gel electrophoresis analysis of concentrated culture supernatants recovered from B. cenocepacia K56-2 ΔatsR containing the control plasmid pME6000 (ΔatsR pME6000), K56-2 ΔatsR ΔbcsK_C (pME6000), K56-2 ΔatsR ΔbcsK_C (pBcsK_C), K56-2 ΔatsR ΔbcsK_C (pBcsK_CΔ40–189), and K56-2 ΔatsR Δhcp (pHcp). Shown at left is the molecular mass marker with the indicated sizes (kDa). The black arrow highlights the position of secreted Hcp. B, phase contrast microscopy of infected ANA-1 macrophages. The infections were performed at a multiplicity of infection of 50:1 for 4 h with the following strains: B. cenocepacia K56-2 ΔatsR (pME6000), K56-2 ΔatsR ΔbcsK_C (pME6000), K56-2 ΔatsR ΔbcsK_C (pBcsK_C), and K56-2 ΔatsR ΔbcsK_C (pBcsK_CΔ40–189). The white arrows indicate cellular projections.

The N Terminus of BcsK_C Is Required for T6SS Activity

We took advantage of two conveniently located SphI restriction sites in bcsK_C to generate a 0.45-kb truncation of the gene, causing an in frame deletion of 150 amino acids within the BcsK_C N terminus (BcsK_CΔ40–189). Plasmid pBcsK_CΔ40–189 could not restore Hcp secretion in K56-2 ΔatsR ΔbcsK_C (Fig. 1A). Moreover, ANA-1 macrophages infected with K56-2 ΔatsR ΔbcsK_C (pBcsK_CΔ40–189) or K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}) did not display the cellular projections typical of T6SS activity (Fig. 1B, lower right panel, and supplemental Fig. S1). Western blot analysis of bacterial cell lysates prepared from K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) and K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}) demonstrated that BcsK_C-FLAG and BcsK_{CΔ40–189-FLAG} were similarly expressed (see below). Thus, a lack of complementation by pBcsK_{CΔ40–189-FLAG} was not due to a defect in protein expression. Together, these data indicate that the N-terminal portion of BcsK_C (amino acids 40–189) is critical for the T6SS activity, suggesting that this region may be required for protein-protein interaction essential for T6SS assembly/stability, correct localization of the protein within the cell, or the intrinsic function of the protein.

BcsK_C Interacts with BcsL_B, and the N Terminus of BcsK_C Is Required for the Interaction

Studies in V. cholerae and F. novicida show that BcsK_C homologs interact with a small T6SS protein homologous to BcsL_B from B. cenocepacia (32, 45). To investigate whether BcsK_C also interacts with BcsL_B or with other proteins, we performed pulldown assays using cell lysates from B. cenocepacia K56-2 ΔatsR ΔbcsK_C (pEL-1), K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG), and K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}). As shown in Fig. 2A, an additional protein with an apparent mass of ∼20 kDa was copurified with BcsK_C-FLAG but not with BcsK_{CΔ40–189-FLAG}, suggesting that the N-terminal portion of BcsK_C is required to bind the 20-kDa protein. Mass spectrometry was performed on the 20-kDa polypeptide and on the corresponding region in lane K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}) as control. Mass spectra were searched against the NCBI database with the MASCOT search engine. Identified peptides from the 20-kDa protein matched with good confidence BcsL_B (BCAL0341; 19.2 kDa) with a total protein score of 467 and were absent in the control lane K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}).

FIGURE 2.

BcsK_C interacts with BcsL_B. Shown is an immunoprecipitation assay using anti-FLAG coupled protein G-agarose beads and cell lysates from B. cenocepacia K56-2 ΔatsR ΔbcsK_C harboring pEL-1 (control), pBcsK_C-FLAG, or pBcsK_{CΔ40–189-FLAG}. A, silver staining. B, anti-FLAG Western blot analysis. The diagonal arrows highlight the positions of BcsK_C-FLAG and BcsK_{CΔ40–189-FLAG}, whereas the asterisk indicates the position of the copurified 20-kDa protein, BcsL_B. Protein bands of ∼25 and 53 kDa (indicated LC and HC, respectively) in all samples correspond to the light and heavy chains of the anti-FLAG antibody.

Bacterial two-hybrid experiments confirmed the BcsK_C/BcsL_B interaction. BcsL_B was used as bait and fused to the T25 domain of the adenylate cyclase from B. pertussis (pT25BcsL_B). BcsK_C and BcsK_CΔ40–189 were used as prey and fused either to the N terminus of the adenylate cyclase T18 domain using the pUT18 plasmid (pBcsK_CT18 and pBcsK_CΔ40–189T18) or to the C terminus of the T18 domain using the pUT18C plasmid (pT18BcsK_C and pT18BcsK_CΔ40–189). E. coli KEM5 (adenylate cyclase-deficient) harboring pT25BcsL_B was cotransformed with the recombinant plasmids expressing BcsK_C hybrid proteins. Bacterial cultures were spotted on a X-gal containing agar plate, and transcriptional activation of the lac operon indicative of protein interaction was visualized by the formation of blue colonies (Fig. 3A). The extent of interaction was also estimated by measuring the β-galactosidase activity (Fig. 3B). E. coli KEM5 colonies harboring pT25BcsL_B together with the vectors pUT18 or pUT18C remained white, indicating that BcsL_B fused to the T25 domain does not interact with the T18 domain alone expressed from pUT18 or pUT18C plasmids. In contrast, E. coli KEM5 (pT25BcsL_B + pBcsK_CT18) and E. coli KEM5 (pT25BcsL_B + pT18BcsK_C) formed blue colonies, denoting interacting hybrid proteins and thus confirming that BcsK_C interacts with BcsL_B. However, E. coli KEM5 (pT25BcsL_B + pBcsK_CΔ40–189T18) and E. coli KEM5 (pT25BcsL_B + pT18BcsK_CΔ40–189) formed white colonies, clearly demonstrating that BcsK_CΔ40–189 does not interact with BcsL_B and indicating that the N terminus of BcsK_C is critical for interaction with BcsL_B. These results were validated with β-galactosidase assays, thus confirming the interaction between full-length BcsK_C and BcsL_B and the critical role of the BcsK_C N terminus in the interaction (Fig. 3B).

FIGURE 3.

The N terminus of BcsK_C is required for interaction with BcsL_B. A, protein interaction using the bacterial two-hybrid system. E. coli harboring pT25BcsL_B was cotransformed with pUT18 and pUT18C control vectors or with the recombinant plasmids expressing BcsK_C or BcsK_CΔ40–189 hybrid proteins fused to the T18 domain in pUT18 (pT18BcsK_C and pT18BcsK_CΔ40–189) and in pUT18C (pBcsK_CT18 and pBcsK_CΔ40–189T18). Bacterial cultures were spotted on a X-gal containing agar plate and transcriptional activation of the lac operon indicative of protein interaction was visualized by the formation of blue colonies. B, quantification of the β-galactosidase activity of E. coli harboring pT25BcsL_B cotransformed with pUT18, pUT18C, pBcsK_CT18, pT18BcsK_C, pBcsK_CΔ40–189T18, and pT18BcsK_CΔ40–189. The enzyme activities are expressed in Miller units. The data shown are the means of three independent experiments done in triplicate. The error bars represent the standard deviation.

To determine the minimal amino acid sequence of BcsK_C required for BcsL_B binding, we constructed a panel of sequential deletions of bcsK_C (Fig. 4). Truncated BcsK_C proteins were fused to the adenylate cyclase T18 domain and tested for interaction with T25BcsL_B. Physical interactions between the hybrid proteins were detected on X-gal-containing plates. Among the deletions tested, only BcsK_CΔ38–62T18, BcsK_CΔ163–187T18, BcsK_CΔ188–212T18, BcsK_CΔ213–237T18, and BcsK_CΔ213–496T18 could bind T25BcsL_B and thus activate the lacZ reporter gene (Fig. 4). Interactions between BcsK_CT18, BcsK_CΔ38–62T18, BcsK_CΔ213–237T18, and BcsK_CΔ213–496T18 and T25BcsL_B were detected on the plate after only 24 h of incubation at 30 °C (data not shown). By contrast, interactions between either BcsK_CΔ163–187T18-T25BcsL_B or BcsK_CΔ188–212T18-T25BcsL_B required 48 h of incubation, indicating that BcsK_CΔ163–187T18 and BcsK_CΔ188–212T18 interact weakly with T25BcsL_B. After 1 week of incubation, BcsK_CΔ113–137T18 and BcsK_CΔ138–162T18 eventually turned blue, whereas BcsK_CΔ63–87T18 and BcsK_CΔ88–112T18 remained unable to activate lacZ (data not shown). Together, these results suggest that the N terminus of BcsK_C (amino acids 2–212) is sufficient for the interaction with BcsL_B. Within the BcsK_C N-terminal domain, amino acids 88–162 are important for BcsK_C/BcsL_B interaction, and more precisely the region encompassing amino acid 63–112 is essential for interaction with BcsL_B.

FIGURE 4.

Identification of BcsK_C domains required for interaction with BcsL_B. Protein interaction using the bacterial two-hybrid system. E. coli harboring pT25BcsL_B was cotransformed with recombinant plasmids expressing sequentially truncated BcsK_C hybrid proteins fused to the T18 domain in pUT18. Bacterial cultures were spotted on a X-gal containing agar plate, and transcriptional activation of the lac operon indicative of protein interaction was visualized by the formation of blue colonies.

BcsK_C Is Tightly Associated with the Outer Membrane

BcsK_C lacks a signal peptide sequence as predicted by the SignalP 3.0 program (54, 55), and in silico analysis of the BcsK_C protein using different transmembrane helices prediction programs available at the ExPASY Proteomics and PSIPRED servers (46, 47) was not conclusive. BcsK_C is predicted to have either no transmembrane domain (DAS, SOSUI, and TMHMM) or one transmembrane domain (HMMTOP, TMpred, TopPred, MEMSAT) whose position and orientation differ depending on the algorithm used (supplemental Fig. S2).

Cell fractionations were performed to determine the subcellular localization of BcsK_C-FLAG and its truncated derivative BcsK_{CΔ40–189-FLAG}. As shown in Fig. 5A, BcsK_C-FLAG and BcsK_{CΔ40–189-FLAG} proteins localized both in the soluble fraction and the membrane fraction, suggesting that deletion of the N terminus of BcsK_C does not affect the protein localization. The membrane fraction containing BcsK_C-FLAG was incubated with various reagents (Fig. 5B). BcsK_C-FLAG was not released with high salt (1 m NaCl) but was partially solubilized by 6 m urea and nonionic detergent (3% Triton X-100). BcsK_C-FLAG was completely solubilized by strong ionic detergents (1% SDS) and 2% N-lauroylsarcosine, which solubilizes both inner and outer membrane under the conditions tested (supplemental Fig. S3). Together, these data suggest that BcsK_C is a tightly associated to the bacterial membrane. The membrane localization of BcsK_C was further investigated by using sucrose density gradients to separate the inner from the outer membrane. As shown in Fig. 5C, BcsK_C-FLAG did not cofractionate with the NADH oxidase activity but with BCAM1931, an outer membrane porin from B. cenocepacia, indicating that BcsK_C-FLAG is associated with the outer membrane.

FIGURE 5.

BcsK_C is tightly associated with the outer membrane. A, Western blot analysis of total cell lysate (T), soluble (S), and total membrane (Mb) fractions recovered from B. cenocepacia K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG), K56-2 ΔatsR ΔbcsK_C (pBcsK_{CΔ40–189-FLAG}), and B. cenocepacia K56-2 ΔatsR ΔT6SS (pBcsK_C-FLAG) using anti-FLAG and anti-RNAP α subunit antibodies (cytosolic control). Ten μg of protein were loaded on a 12% SDS-PAGE gel. Molecular mass marker sizes (kDa) are shown on the left. The arrow highlights the position of the BcsK_C-FLAG-containing complex. The faint bands seen in the membrane fractions (lines 3, 6, and 9) at the RNAP α subunit location (∼37 kDa) correspond to an unspecific protein cross-reacting with the anti-FLAG antibody (see Fig. 6B). B, protein solubilization. Total membranes were incubated with 50 mm Tris-HCl, pH 7.4 (Buffer), 1 m NaCl, 3% Triton X-100 (TX100), 2% N-lauroylsarcosine (LS), or 1% SDS. Insoluble (pellet, P) and soluble (supernatant, S) fractions were loaded on a 12% SDS-PAGE gel and analyzed by Western blot using an anti-FLAG antibody. C, sucrose density gradient. Inner and outer membranes were separated on a discontinuous sucrose gradient. The protein content was calculated by measuring the A_{280 nm}, and the quality of the fractionation was assessed by measuring the enzymatic activity of the NADH oxidase (inner membrane marker) and by detecting the porin BCAM1931 (outer membrane marker). Fractions 1–24 were loaded on 12% SDS-PAGE gels and silver-stained or analyzed using an anti-FLAG antibody. The position of BCAM1931, BcsK_C-FLAG and the high molecular mass complex (HMWC) are indicated. The positions of the inner membrane-containing (IM) and outer membrane-containing (OM) fractions are indicated.

BcsK_C Forms a High Molecular Weight Complex Containing BcsL_B and Also Requiring BcsF_H for Its Formation

Expression of BcsK_C-FLAG in B. cenocepacia K56-2 ΔatsR ΔbcsK_C leads to the formation of a high molecular weight complex detected with the anti-FLAG antibody at the top of the separating gel (Fig. 5A, lane 3), suggesting multimerization of BcsK_C-FLAG. This complex localizes almost exclusively in the membrane fraction (Fig. 5A, lanes 2 and 3) and is anchored to the outer membrane (Fig. 5C). The complex was absent when BcsK_{CΔ40–189-FLAG} was expressed in K56-2 ΔatsR ΔbcsK_C, suggesting that the N terminus of BcsK_C is required for the complex formation (Fig. 5A, lanes 3 and 6). Moreover, although a complex is formed when BcsK_C-FLAG is expressed in B. cenocepacia K56-2 ΔatsR ΔbcsK_C, expression of BcsK_C-FLAG in a strain where all the T6SS encoding genes have been deleted (K56-2 ΔatsR ΔT6SS) did not allow the complex formation, indicating that other T6SS components are required (Fig. 5A, lanes 3, 6, and 9).

We investigated which T6SS components were required for the formation of the BcsK_C-FLAG-containing complex. In B. cenocepacia K56-2 the T6SS gene cluster is organized in three putative transcriptional units (Fig. 6A). To identify the components involved in the BcsK_C-containing complex formation, each of the three transcriptional units were individually deleted in the K56-2 ΔatsR (K56-2 ΔatsR ΔTU1, K56-2 ΔatsR ΔTU2, and K56-2 ΔatsR ΔTU3). The cell lysates were prepared from the different mutant strains expressing BcsK_C-FLAG and analyzed by Western blot. As shown Fig. 6B, strains K56-2 ΔatsR ΔTU1 (pBcsK_C-FLAG) (lane 3) and K56-2 ΔatsR ΔTU3 (pBcsK_C-FLAG) (lane 5) but not K56-2 ΔatsR ΔTU2 (pBcsK_C-FLAG) (lane 4) could produce the BcsK_C-containing complex, suggesting that the TU2 contains the critical components for complex formation. Each gene of the TU2 was systematically deleted in K56-2 ΔatsR (Fig. 6A). Cell lysate analysis of individual gene mutants within the TU2 revealed that BcsL_B and BcsF_H were required for the formation of the BcsK_C-FLAG-containing complex (Fig. 6B, lanes 7 and 13).

FIGURE 6.

BcsL_B and BcsF_H are required for the formation of the BcsK_C-containing high molecular mass complex. A, genetic map of the T6SS gene cluster. This cluster has been designated bcs cluster for B. cenocepacia survival. The arrows represent the location and direction of gene transcription. BCAL gene designations according to the annotation of the B. cenocepacia J2315 genome (53) are shown above, and the bcs annotation of the genes is shown below. The genes icmF-like (BCAL0351, bcsB_M), clpV-like (BCAL0347, bcsF_H), and hcp_Bc (BCAL0343, bcsJ_D), which are hallmarks of T6SSs in other bacteria, are shown. The black arrow represents bcsK_C. The putative transcriptional units TU1, TU2, and TU3 are boxed. B, anti-FLAG Western blot analysis of total cell lysates recovered from B. cenocepacia K56-2 mutant strains harboring pBcsK_C-FLAG. Lane 1, K56-2 ΔatsR; lane 2, K56-2 ΔatsR ΔT6SS; lane 3, K56-2 ΔatsR ΔTU1; lane 4, K56-2 ΔatsR ΔTU2; lane 5, K56-2 ΔatsR ΔTU3; lane 6, K56-2 ΔatsR ΔbcsM; lane 7, K56-2 ΔatsR ΔbcsL_B; lane 8, K56-2 ΔatsR ΔbcsK_C; lane 9, K56-2 ΔatsR Δhcp; lane 10, K56-2 ΔatsR ΔbcsI_E; lane 11, K56-2 ΔatsR ΔbcsH_F; lane 12, K56-2 ΔatsR ΔbcsG_G; lane 13, K56-2 ΔatsR ΔbcsF_H; lane 14, K56-2 ΔatsR ΔbcsE_A; lane M, molecular mass markers with the indicated sizes (kDa). The asterisk indicates that the sample has been boiled prior to loading. Black arrows indicate BcsK_C or the high molecular mass complex (HMWC).

The complex is heat-sensitive because it was not detectable in samples that were heated prior to loading (Fig. 6B, lane 1*). To investigate the composition of the complex unheated membrane, samples prepared from K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) and K56-2 ΔatsR ΔT6SS (pBcsK_C-FLAG) were individually run on a SDS-PAGE gel. A slice from the top of the separating gels containing high molecular weight proteins (and the BcsK_C-containing complex in the case of K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG)) was excised. The proteins were electroeluted and concentrated, and the samples were heated prior to loading on a SDS-PAGE gel to disassociate the components of the BcsK_C-containing complex. Comparison of the protein profiles of K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) and K56-2 ΔatsR ΔT6SS (pBcsK_C-FLAG) revealed two extra bands in the electroeluted sample from K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) with apparent masses of ∼55 and ∼20 kDa (Fig. 7). Mass spectrometry was performed on those two proteins. Identified peptides matched with a score of 507 and 643 to BCAL0342 (BcsK_C) and BCAL0341 (BcsL_B), respectively, indicating that BcsL_B is also part of the complex containing BcsK_C and again confirms the interaction between BcsK_C and BcsL_B.

FIGURE 7.

The high molecular mass complex contains BcsK_C and BcsL_B. Seventy μg of electroeluted high molecular mass proteins recovered from B. cenocepacia K56-2 ΔatsR ΔbcsK_C (pBcsK_C-FLAG) (left lane) and K56-2 ΔatsR ΔT6SS (pBcsK_C-FLAG) (right lane) were boiled and loaded on a 16% SDS-PAGE gel. The asterisks highlight the positions of the 55- and 20-kDa proteins subjected to mass spectrometry analysis and corresponding to BcsK_C and BcsL_B, respectively. Shown at left are the molecular weight markers with the indicated sizes (kDa).

DISCUSSION

In this study, we show that deletion of bcsK_C abolishes T6SS-associated phenotypes in B. cenocepacia (Hcp secretion and induction of the cytoskeletal rearrangements in macrophages after infection), demonstrating that BcsK_C is a key component of the B. cenocepacia T6SS. Most T6SSs identified to date encode a BcsK_C homolog, and the importance of BcsK_C-like proteins in T6SS activity has already been underlined in other Gram-negative bacteria such as enteroaggregative E. coli, E. tarda, and F. novicida (11, 12, 14). In B. mallei, the BcsK_C homolog TssB (BMAA0743) is not required for Hcp secretion, but it is necessary for virulence in hamsters (22). B. mallei contains three paralogs sharing more than 30% homology with TssB amino acid sequence. Redundancy might explain why Hcp is still secreted but does not explain the lack of virulence when TssB is mutated. Schell et al. (22) speculated that TssB is a secreted effector instead of a component of the T6S apparatus, but this remains to be demonstrated.

We also demonstrated that BcsK_C could interact with BcsL_B as shown by our immunoprecipitation and bacterial two-hybrid assays. This was an expected result because interactions between proteins homologous to BcsK_C and BcsL_B have already been reported in other T6S systems (11, 32, 45). For example, in F. novicida, the central region of IglA (BcsL_B homolog) is required for interaction with IglB (BcsK_C homolog) (45). Using a similar approach, our study reveals that the region required to bind BcsL_B is limited to the N terminus of BcsK_C, in particular to a segment encompassing amino acids 63–112. The N terminus of BcsK_C, likely because of its ability to bind BcsL_B, is essential for T6SS activity. PSIPRED analysis predicted this region to form two α helices preceding a β strand. Ab initio structural modeling of BcsK_C, VipB, IglB, and PA0084 using the I-Tasser server (48) revealed a similar fold in the regions corresponding to amino acids 63–112 of BcsK_C, which consists of paired α-helices (supplemental Fig. S4). Therefore, we propose that this is a conserved domain among BcsK_C-like proteins for the interaction with BcsL_B-like proteins.

Using a purified protein fusion containing the N terminus of ClpV and soluble cell lysate from V. cholerae Bönemann et al. (32) demonstrated that the N terminus of ClpV interacts with the VipA/VipB complex (BcsL_B/BcsK_C homolog). Interaction between BcsF_H (ClpV homolog) and BcsK_C/BcsL_B has not been detected in our pulldown assays or our electroelution/resolution of the BcsK_C-containing complex under the conditions tested. In V. cholerae ClpV is eight times less abundant than VipA and VipB (32). Thus, if the same ratio is true for B. cenocepacia, ClpV and BcsL_B/BcsK_C interactions might be more difficult to detect when BcsK_C is used as bait in immunoprecipitation assays. Moreover, because the energizing component ClpV has been shown in V. cholerae to degrade BcsL_B/BcsK_C-like cytosolic complexes, it is possible that ClpV interacts with BcsL_B/BcsK_C transiently. Interaction between IglA/IglB in F. novicida has been shown to be required for the stability of each protein (45). In our experimental conditions BcsK_C and derivatives are constitutively expressed on a multi-copy plasmid at higher levels than it would be in the wild type strain. Constitutive expression of BcsK_{CΔ40–189-FLAG} (unable to bind BcsL_B) in B. cenocepacia K56-2 ΔatsR or of BcsK_C-FLAG in a B. cenocepacia K56-2 ΔatsR ΔbcsL_B background likely bypassed the protein stability issue because no reduction of the protein expression levels or degradation products were noticed in the different cell lysates.

BcsL_B and BcsK_C homologs have been proposed to have different cellular localizations. In V. cholerae, the BcsL_B and BcsK_C homologs VipA and VipB have been described as nonsecreted cytosolic proteins (32). In F. novicida, the BcsL_B and BcsK_C homologs IglA and IglB have been described as strictly cytosolic proteins (11) or as surface-exposed proteins (49, 50). In E. tarda, EvpA (BcsL_B) interacts with the periplasmic domain of IcmF (12), suggesting that BcsL_B homologs might have a periplasmic location. By using a different approach and constitutively expressing BcsK_C on a plasmid in B. cenocepacia K56-2 ΔatsR (overexpressing the other T6SS components), we show that BcsK_C can be found in the cytosol but also in strong association with the outer membrane. In our fractionation analysis we noticed that BcsK_C forms a high molecular weight complex anchored to the outer membrane. The formation of this complex depends on two other T6SS encoded proteins, BcsL_B and BcsF_H (ClpV homolog). Purification and analysis of the complex composition showed that BcsL_B is also part of the complex (Fig. 7). The high molecular weight complex containing BcsK_C and BcsL_B may be indicative of the Type VI secretion structure; however, no other proteins could be detected as part of the complex under the conditions tested. If any other proteins are part of the complex, their amount is likely below the level of detection of our technique. In V. cholerae, BcsL_B and BcsK_C homologs VipA and VipB form cytosolic tubules disassembled by ClpV (32). However, in contrast to VipA/VipB tubules, the B. cenocepacia BcsL_B/BcsK_C complex was consistently detected in the membrane fraction, but not in the soluble fraction containing the cytosolic proteins. Furthermore, the BcsL_B/BcsK_C complex requires ClpV for its formation, whereas in V. cholerae the VipA/VipB tubule-like structures are severed by ClpV (32). Together, these data suggest that even though the BcsL_B/BcsK_C complex and the VipA/VipB tubules are made of BcsK_C/BcsL_B-like proteins, they have different localizations and likely reflect different stages in the biogenesis of the T6S apparatus. Leiman et al. (21) found that the characteristics of this VipA/B structure resemble those of the contracted T4 phage tail sheath (51) and speculated that VipA/B may provide energy to the T6SS-mediated secretion and membrane insertion process through conformational changes that mimic those that occur during phage tail contraction. Because of the similarities between the architectures of the VipA/VipB tubules and the bacteriophage T4 tail sheath, Bönemann et al. (52) proposed recently that VipA/VipB tubules might represent structural core components of the T6SS and that biogenesis of T6SSs might require ClpV to export VipA and VipB subunits into the periplasm, followed by VipA/VipB tubule reformation. We propose that the BcsL_B/BcsK_C complex that we describe in this study reflects precisely the tubule formation in the periplasmic space, which would be anchored to the outer membrane. Further characterization of BcsK_C/BcsL_B-like complexes is needed to understand their role in the functioning of the T6SS.