Killing and lysis of prey bacteria differs for each of the five effectors

Bacteria use type VI secretion systems (T6SSs) to manipulate host cells during pathogenesis or to kill competing bacteria, which, in some cases, increases horizontal gene transfer. These functions largely depend on T6SS regulation, dynamics, and the set of effectors that the system delivers into the target cells. Here, we show that Acinetobacter baylyi ADP1 assembles a highly dynamic T6SS capable of killing and lysing bacterial cells. T6SS function depends on conserved T6SS components as well as Acinetobacter-specific genes of unknown function. Five different effectors, encoded next to VgrG or PAAR proteins and their cognate immunity proteins, cause distinct changes in the prey cells, resulting in various degrees of their lysis. Prey lysis correlates with the rate of DNA transfer from prey to predator, suggesting that lytic effectors are required for efficient T6SS-dependent horizontal gene transfer in naturally competent bacteria.

Here, we characterized the dynamics of the T6SS of A. baylyi ADP1 using live-cell fluorescence microscopy and identified and characterized five T6SS effectors and their cognate immunity proteins. We could demonstrate that none of the effectors are required for T6SS assembly and that each kills the target cells by a distinct mechanism. Moreover, we demonstrate that the efficiency of horizontal gene transfer, promoted by the T6SS-mediated lysis of sensitive bacteria, depends on the mechanism of target cell killing.

Interestingly, the T6SS of Vibrio cholerae is part of the competence regulon, and therefore, killing of target cells may contribute to horizontal gene transfer (). Acinetobacter baylyi ADP1 is naturally competent throughout most of its growth () and encodes a single constitutively active antibacterial T6SS (). Recently, the combination of natural competence and T6SS-mediated bacterial killing in A. baylyi was shown to contribute to the transfer of a plasmid from target cells to the predator, suggesting that this may play a role in the spread of antibiotic resistance in the related A. baumannii strains ().

T6SS effectors may constitute extensions of any of the secreted components Hcp, VgrG, or PAAR (), or bind non-covalently to these, then termed “cargo” effectors (). Some cargo effectors require an adaptor/chaperone protein for secretion, which are not secreted themselves (). To prevent self-intoxication, anti-bacterial effectors are accompanied by cognate immunity proteins, often encoded in close proximity to the corresponding effector ().

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

The T6SS is composed of three distinct substructures: the membrane complex, the baseplate, and the sheath-tube complex (). The envelope-spanning membrane complex is usually composed of TssJ, TssL, and TssM and anchors the T6SS to the cell envelope (). The baseplate is composed of TssE, TssF, TssG, TssK (), and, in some organisms, a TssA variant (). The baseplate serves as a platform for the polymerization of the contractile sheath-tube complex and connects it to the membrane complex. The contractile sheath, consisting of VipA (TssB) and VipB (TssC), forms around the inner tube, which is composed of Hcp (), by adding the sheath subunits at the end that is distal from the baseplate (). The initiation of the assembly and the polymerization may require TssA (). Furthermore, a spike complex is situated at the tip of the Hcp tube, which is composed of a VgrG trimer () and a PAAR protein (). The contraction of the sheath is thought to propel the Hcp tube with its associated spike complex into the extracellular medium or the target cell (). The contracted sheath is recycled in an ATP-dependent manner by ClpV or ClpB ().

The type VI secretion TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via multiple contacts and serves as assembly platform for tail tube/sheath polymerization.

Bacteria secrete various substrates by specialized secretion systems to manipulate their environment (). The type VI secretion system (T6SS) () gene clusters are found in more than 25% of all sequenced Gram-negative bacteria, but mostly in the proteobacteria (). Systems similar to the proteobacterial T6SS have been discovered in Francisella (), Bacteroidetes (), and more recently in Amoebophilus asiaticus (), overall constituting four phylogenetically distinct subgroups.

When we incubated the T6SSstrain with the Tle1- and Tse2-sensitive strains, we observed reduced recoveries of the sensitive strains comparable with those obtained during our previous assays (compare Figures 5 D and 5E with Figure 6 B). In addition, similar to the observations made for the competition with E. coli, the lipase effector Tle1 induced lysis of the non-immune A. baylyi strain (vipA-sfGFP clpV-mCherry2 Δtli1-tle1) as documented by the leakage of DNA out of cells, the decrease in contrast of the bacterial cytosol, and the rapid accumulation of SYTOX Blue signal ( Figures 6 C). On the other hand, the Tse2-effector-mediated killing resulted in a high level of inhibition of the non-immune A. baylyi strain (vipA-sfGFP clpV-mCherry2 Δtse2 Δtsi2a-tsi2b; Figure 6 B), however, no clear cell lysis was observed, and the cells accumulated SYTOX Blue rather slowly ( Figure 6 D). Importantly, the competition of the T6SSstrain with the Tle1-sensitive strain produced significantly more double-resistant mutants than the competition of the T6SSstrain with the Tse2-sensitive strain or the T6SS-independent transfer ( Figure 6 A). Overall, these data suggest that the mechanisms of killing and lysis of target cells have major implications for DNA release and thus efficiency of horizontal gene transfer.

To account for DNA transfer independent of T6SS-mediated killing, a T6SS-deficient, spectinomycin-resistant strain (T6SS) was used as a control strain. To exclude possible differences in uptake and integration of the counter-selectable cassettes from the Tle1- and Tse2-sensitive strains, we transformed the T6SSstrain with equal amounts of the genomic DNA of both sensitive strains and enumerated the resulting double-resistant mutants. The number of transformants obtained with the genomic DNA was not significantly different, indicating that both cassettes incorporate with similar efficiency ( Figure 6 A).

(C and D) Time-lapse microscopy illustrating the distinct lysis phenotypes of the Δtli1-tle1 (C) and the Δtse2 Δtsi2a-2b (D) sensitive strains (vipA-sfGFP clpV-mcherry2 background) incubated with unlabeled wild-type A. baylyi ADP1. The top rows show a merge of phase contrast, GFP (green), mCherry (magenta), and SYTOX (cyan) channels. The bottom rows show the increase in the fluorescence of the cell-impermeable DNA stain SYTOX Blue upon the loss of cell membrane integrity. The mixtures were imaged every 30 s for 1 hr. The scale bars represent 1 μm.

(B) Quantitative competition assay measuring the recovery of the indicated strains coincubated as in (A). The dashed lines indicate the means and the error bars indicate the SD. n. s. = not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

(A) The level of DNA transfer between the indicated strains was tested by enumerating the clones having acquired a resistance cassette after 4 hr of coincubation. The control transformations of the T6SS + strain with genomic DNA are labeled as Δtli1-tle1::rpsL’-kan R and Δtse2 Δtsi2a-2b::rpsL’-kan R .

T6SS-mediated killing of prey cells by the naturally competent A. baylyi ADP1 can liberate the DNA of the prey and thereby promote horizontal gene transfer (). We speculated that effectors causing the release of cellular content, like Tae1 and Tle1, should cause a higher transformation rate than those not directly leading to lysis, like Tse2. To test this hypothesis, we competed a spectinomycin-resistant, T6SS-active A. baylyi ADP1 derivative (T6SS) against the Tle1- and the Tse2-sensitive strains ( Figures 6 A and 6B). The sensitive strains carry the kanamycin resistance cassette, disrupting the immunity protein-encoding genes. Successful transfer of DNA can thus be monitored by selecting for spectinomycin and kanamycin double-resistant strains.

The putative immunity protein is co-encoded in the same operon upstream of tle1, which we termed Tli1 (ACIAD3426). Tli1 carries a predicted cleavable N-terminal signal sequence as is common for the cognate phospholipase immunity proteins (). The recovery of the sensitive strain was significantly reduced when competed against the parental or single effector strain. The recovery was restored when competed against the ΔtssM or the Δtle1 strains ( Figure 5 E). These results confirm that Tle1 is a T6SS effector and that Tli1 is its cognate immunity protein.

Tle1 (ACIAD3425) was predicted to be a phospholipase belonging to family 4 of T6SS-associated phospholipases (). It matches an alpha/beta-hydrolase fold (Gene3D 3.40.50.1820) and the abhydrolase_5 domain (PF12695.5; Figure S1 C). The Tle1 single effector strain significantly reduced the recovery of E. coli and led to intermediate lysis of E. coli in the CPRG conversion assay ( Figures 3 A and 3C). Surprisingly, when the Tle1 single effector strain was co-incubated with E. coli, the E. coli cells first shrank without an increase of SYTOX Blue signal and then reinflated, coinciding with their permeabilization ( Figure 3 H; Movie S3 ).

A manual inspection of gene ortholog neighborhoods of tse2 revealed the presence of Tse2 homologs mostly in γ-proteobacteria, but also in α- and β-proteobacteria. Multiple copies of the immunity proteins, up to five consecutive ones in Klebsiella pneumonia W14 and Photorhabdus luminescens subsp. luminescens DSM 3368, were a common feature in γ-proteobacteria, but only two duplications were observed for β-proteobacteria, and none were observed for α-proteobacteria ( Figure S3 B). Immunity protein duplications seem to be common, and all may contribute to immunity (). These evolved paralogs were speculated to provide immunity against diverged corresponding effectors arising in the population ().

The two putative immunity proteins Tsi2a (ACIAD3112) and Tsi2b (ACIAD3113), encoded in the opposite direction downstream of the effector ( Figure S1 B), are predicted to contain a cleavable N-terminal signal sequence, indicating that they are periplasmically localized and suggesting that the subcellular target of Tse2 is accessible from the periplasm. The recovery of the Δtsi2a/Δtsi2b/Δtse2-sensitive strain was significantly reduced after incubation with the parental or the single effector strain, but unchanged when incubated with the ΔtssM or Δtse2 strains ( Figure 5 D). These data indicate that Tse2 is a T6SS effector and that Tsi2a, Tsi2b, or both confer immunity toward Tse2.

Tse2 (ACIAD3114) is a homolog of the recently described Tse3(ACX60_11695) in A. baumannii ATCC 17978, which was found to be an antibacterial effector, but its mechanism of action remained unknown (). The single effector strain significantly reduced the recovery of E. coli, however, lysis, indicated by the CPRG conversion assay, was delayed compared with the other single effector strains ( Figure 3 A). Interestingly, E. coli only slowly gained SYTOX Blue signal during its interaction with the Tse2 single effector strain, and the signal remained low. This is in contrast to what we observed when E. coli was lysed by other effectors ( Figure 3 G; Movie S3 ). The slow increase in SYTOX Blue signal suggests that Tse2 leads to a low-level permeabilization of E. coli, which is consistent with the delayed conversion of CPRG by LacZ and suggests that the cell envelope remains largely impermeable to CPRG and LacZ.

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis.

Two putative immunity proteins sharing 82% sequence identity are encoded downstream of Tse1, which we termed Tsi1a (ACIAD1795) and Tsi1b (ACIAD1796; Figure S1 B). A Tse1 ortholog was only found in Burkholderia cenocepacia (excluding Acinetobacter), and immunity protein duplications were restricted to Acinetobacter, ranging from one to three copies ( Figure S3 A). Both Tsi1a and Tsi1b are predicted to contain four transmembrane helices. The sensitive strain (lacking both Tsi1a and Tsi1b) was inhibited by the single effector strain carrying the restored Tse1 ( Figure 5 C). However, no inhibition was observed when competed against the ΔtssM strain or the Δtse1’ strain as well as the parental strain containing the IS element. When tssM was deleted in the single Tse1 effector strain, no reduction in recovery of the sensitive strain could be detected ( Figure 5 C). This confirms that Tse1 is secreted in a T6SS-dependent manner and that Tsi1a, Tsi1b, or both are the cognate immunity proteins.

We removed the insertion sequence (IS) element and restored the full-length Tse1 based on the multiple sequence alignment with the homologous effectors (ACIAD1790–1794 fusion; Figures S1 B and S2 ). The full-length Tse1 is predicted to carry four C-terminal transmembrane helices and had a low-quality match for the short-chain dehydrogenase/reductase active site (PS00061; Figure S1 C). The single effector strain significantly reduced the recovery of E. coli and led to intermediate lysis of E. coli in the CPRG conversion assay ( Figures 3 A and 3C). The competition microscopy showed that, in some cases, lysis proceeded similar to what had been observed for Tae1, where E. coli rounded up and then burst, whereas in other cases, E. coli shrinks slightly and lyses ( Figure 3 F; Movie S3 ). Both processes take longer compared with the lysis induced by the other effectors.

Downstream of VgrG2 (ACIAD1788), a protein we termed Tap1 (ACIAD1789) is encoded that shows weak homology to the DUF4123 domain found in T6SS effector chaperones (TECs), also referred to as adaptor proteins (). The downstream gene terminates at a copy of the insertion element IS1236, suggesting that the original gene was disrupted by IS1236 ( Figure S1 B). Indeed, a BLAST search of the N-terminal fragment Tse1’ (ACIAD1790) in the UniParc () database yielded longer proteins in various Acinetobacter strains, which were in the genomic neighborhood of VgrG and Tap1 homologs and whose N-terminal regions were similar to Tse1’.

The gene downstream of tae1 encodes a protein we termed Tai1 (ACIAD0169), which carries a predicted cleavable N-terminal signal sequence, suggesting that Tai1 is the cognate immunity protein of Tae1. When the Δtae1/Δtai1 strain was competed with the parental strain or the Tae1 single effector strain, the recovery of the Δtae1/Δtai1 strain was significantly reduced. This was fully dependent on T6SS activity and presence of tae1, since the recovery was restored when competed against the Δtae1 and the ΔtssM strains ( Figure 5 B). This indicates that Tae1 is a peptidoglycan-targeting T6SS effector and that Tai1 is the cognate immunity protein.

The remaining putative T6SS effectors are encoded downstream of VgrGs. Bioinformatic analysis of the sequence of Tae1 (ACIAD0168) suggested that it is a peptidoglycan-hydrolyzing amidase, which has no clear homology to any of the four currently known families (). Tae1 contains two predicted peptidoglycan-binding domains (LysM, PF01476.19, and IPR002477), a D-alanyl-D-alanine carboxypeptidase zinc-binding domain (IPR009045) and a peptidoglycan-hydrolyzing domain (hydrolase_2, PF07486.11; Figure S1 C), suggesting that Tae1 cleaves the peptide crosslinks of peptidoglycan. The single effector strain significantly reduced the recovery of E. coli and induced its lysis to a level comparable to that of the parental strain ( Figures 3 A and 3C). Imaging the competition with E. coli revealed that lysing E. coli often round up and burst ( Figure 3 E; Movie S3 ), which is consistent with the prediction that Tae1 encodes a peptidoglycan-targeting effector.

The protein encoded downstream of Tpe1, which we termed Tpi1 (ACIAD0054), contains a predicted N-terminal transmembrane helix, and we hypothesized it to constitute the cognate immunity protein to Tpe1 ( Figure S1 B). The competition of the sensitive strain (lacking both Tpe1 and Tpi1) against the parental and the single effector strains led to a significantly reduced recovery of the sensitive strain, whereas there was no such reduction when competed against the ΔtssM and the Δtpe1 strains ( Figure 5 A). This indicates that Tpe1 is a T6SS effector and Tpi1 is its corresponding immunity protein. The fact that no E. coli inhibition or lysis was detected suggests that either E. coli is resistant to the action of Tpe1 or that E. coli can outgrow its effects without lysis.

The smallest of the putative effectors, Tpe1 (ACIAD0053), is encoded in an operon with two PAAR proteins ( Figure S1 B). It is predicted to contain a zinc metallopeptidase active site, PS00142 ( Figure S1 C). The single effector strain was unable to significantly reduce the recovery of E. coli or induce its lysis ( Figures 3 A and 3C). Additionally, the imaging of the competition with E. coli showed no increase in signal from the DNA-binding dye SYTOX Blue, suggesting that no E. coli cell permeabilization was occurring ( Movie S3 ).

To investigate the role of the individual effectors, strains lacking all but one of the effectors (single effector strains) were constructed. All five single effector strains secreted Hcp and displayed sheath dynamics similar to the parental strain ( Figures 3 C–3H; Movie S3 ). The strains were tested for their ability to lyse or inhibit growth of E. coli ( Figure 3 A). To dissect the mode of action of the individual effectors, we also incubated the strains with E. coli and imaged the competition for 30 min to 1 h at 30°C on a Luria-Bertani (LB) agarose pad containing SYTOX Blue as an indicator for cell permeability ( Figures 3 D–3H; Movie S3 ). Since putative immunity proteins were identified in the vicinity of the effectors, we constructed strains lacking the immunity-effector pairs and tested their growth inhibition due to interactions with the corresponding single effector, the parental, the single effector deletion, and the ΔtssM strains ( Figures 5 A–5E).

(A–E) Quantitative competition assays measuring recovery of the sensitive strains and the specified mutants after 4 hr of coincubation. The error bars indicate the SD, the long dashed lines indicate the mean recovery of the sensitive strains and the short dashed lines indicate the mean recovery of the aggressors. n. s. = not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

There Is No Crosstalk between the Five T6SS Effectors and Their Cognate Immunity Proteins

Figure 5 There Is No Crosstalk between the Five T6SS Effectors and Their Cognate Immunity Proteins

To test if the T6SS in the ΔE strain is capable of inflicting damage, we co-incubated the strain with both P. aeruginosa PAO1 and its ΔretS variant. Interestingly, both the wild-type and the ΔretS strain inhibited the A. baylyi ΔE strain to the same level as its parental strain. However, the inhibition of A. baylyi was significantly reduced when the ΔtssM strain was co-incubated with the P. aeruginosa wild-type or ΔretS strains ( Figure 4 ). This is consistent with previous observations () and suggests that the ΔE strain is likely damaging at least the outer membrane of target cells and thus induces retaliation by P. aeruginosa.

Quantitative competition assays measuring recovery of the indicated A. baylyi and P. aeruginosa strains upon 4 hr of coincubation. The dashed lines indicate the means and the error bars indicate the SD. n. s. = not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

The fact that T6SS effectors are often found encoded in an operon with a secreted structural component and the cognate immunity protein allowed us to identify five putative effectors and their cognate immunity proteins in A. baylyi ADP1 ( Figure S1 B). An effector-deficient strain (ΔE), lacking all five identified effectors, was still able to secrete Hcp, and its T6SS activity and dynamics, observed by fluorescence microscopy, were unaffected ( Figures 3 B and 3C ; Movie S1 ). However, we were unable to detect a growth inhibition of E. coli or its lysis when competed against the ΔE strain ( Figure 3 A). Moreover, no E. coli permeabilization was detected by fluorescence microscopy using SYTOX Blue as a cell permeability reporter ( Movie S3 ). This suggests that there is no remaining antibacterial effector secreted by the ΔE strain, and that none of the effectors are structural or functional components of the secretion system itself.

(D–H) Time-lapse microscopy of the competitions of the parental strain (D) and the tae1 (E), tse1 (F), tse2 (G), and tle1 (H) single effector A. baylyi ADP1 strains with E. coli. The representative frames were chosen to illustrate the distinct lysis phenotypes elicited by the indicated effectors. The top rows show a merge of phase contrast, GFP (for VipA-sfGFP in green), mCherry (for ClpV-mCherry2 in magenta), and SYTOX (in cyan) channels. The bottom rows show the increase in the fluorescence of the cell-impermeable DNA stain SYTOX Blue on the loss of cell membrane integrity. The scale bars represent 1 μm. The arrows indicate the cells that lose membrane integrity throughout the time lapse. Except for (F), the competitions were imaged every 30 s for 30 min. For (F), the competitions were imaged every 1 min for 1 hr.

(C) Hcp detected in the culture supernatant of the indicated strains after TCA precipitation, separation by PAGE, and subsequent staining with Coomassie. For comparison, representative images of the endpoints of the lysis assay from (A) are shown.

(B) Representative image of effector deficient strain (ΔE vipA-sfGFP clpV-mCherry2) shows the merge of phase contrast, GFP (in green) and mCherry (in magenta) channels on the left; the GFP channel in the middle; and mCherry channel on the right. The scale bar is equivalent to 1 μm.

(A) Quantitative competition assay measuring recovery of the indicated strains after 4 h of coincubation of E. coli with the indicated aggressor strains is shown on the left. The error bars indicate the standard deviation, the long dashed lines indicate the means of the E. coli recovery and the short dashed lines indicate the means of the aggressor recovery. Lysis assays measuring CPRG conversion upon release of LacZ from E. coli cells incubated with the indicated A. baylyi strains for the indicated time is shown on the right. The lysis assays were performed in biological triplicate and in at least technical tetraplicate. n. s. = not significant; ∗ = p < 0.05; ∗∗∗ = p < 0.01.

Bioinformatic analysis of ACIAD2685 suggested the presence of two N-terminal transmembrane helices and that the N- and C-termini are localized in the cytoplasm. The T6SS activity of the ΔACIAD2685 strain was severely attenuated. There was no detectable Hcp secretion and no inhibition of E. coli ( Figures 2 B and 2C). However, the more sensitive CPRG conversion assay indicated that E. coli lysis was still occurring, albeit to a severely reduced extent ( Figure 2 B). These results are in agreement with the strongly reduced frequency of T6SS assembly observed by fluorescence microscopy, similar to what had been observed for the ΔtagX strain ( Figure 2 A; Movie S1 ). TssM is encoded right downstream of ACIAD2685, therefore, we cannot exclude a potential polar effect of the in-frame deletion.

The recently characterized L,D-endopeptidase TagX is thought to be involved in forming a hole in the peptidoglycan, allowing the assembly of the T6SS (). Accordingly, we were unable to detect Hcp in the supernatant of the ΔtagX strain, and the E. coli inhibition was similar to that caused by the ΔtssM strain ( Figures 2 B and 2C). Interestingly, the more sensitive CPRG conversion assay indicated that the ΔtagX strain is still capable of lysing E. coli, although to a much lesser extent than the parental strain ( Figure 2 B), suggesting that the T6SS is still partially active in the absence of TagX. This was confirmed by fluorescence microscopy, which revealed a strongly reduced frequency of T6SS sheath assembly initiation ( Figure 2 A; Movie S1 ), indicating that TagX is dispensable for the T6SS mode of action after the assembly is initiated by a TagX-independent mechanism.

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis.

TagF was reported to act as a posttranslational repressor of the H1-T6SS in Pseudomonas aeruginosa PAO1 (). However, we observed no change in E. coli inhibition or frequency of T6SS sheath assembly in the ΔtagF strain. Furthermore, both the lysis of E. coli and the Hcp secretion were unaffected ( Figures 2 A–2C; Movie S1 ). This suggests that TagF has a different function in A. baylyi ADP1 or that it does not act as a repressor under the tested conditions.

The ΔACIAD2698 strain was phenotypically indistinguishable from the parental strain ( Figures 2 A–2C; Movie S1 ). On the other hand, the ΔACIAD2693 strain secreted Hcp, but displayed an intermediate phenotype in the quantitative E. coli competition assay ( Figures 2 B and 2C). Even though the E. coli inhibition was significantly decreased in the absence of ACIAD2693, the lysis of E. coli was indistinguishable from that induced by the parental strain ( Figure 2 B). The decreased inhibition of E. coli is in agreement with the reduction in the number of sheath assemblies per cell ( Figure 2 A). However, the dynamics of the individual T6SS structures were unaltered ( Movie S1 ). Even though ACIAD2693 overlaps with the essential vipA, a polar effect is unlikely the reason for the decreased T6SS activity since the VipA-sfGFP fluorescence was comparable to that of the parental strain ( Figure 2 A).

Very little is known about the Acinetobacter-specific T6SS components ACIAD2693 and ACIAD2698. ACIAD2698 contains a single predicted N-terminal transmembrane helix with the C terminus being disordered and residing in the periplasm. A similar analysis suggested that ACIAD2693 carries a cleavable N-terminal signal sequence and an intrinsically unstructured C-terminal region.

TagN was proposed to be required for anchoring the T6SS to the peptidoglycan (). In ADP1, the TagN homolog (ACIAD2682) is the only protein encoded in the core cluster bearing a predicted peptidoglycan binding domain and a cleavable N-terminal signal sequence. Surprisingly, the ΔtagN strain secreted Hcp and displayed only an intermediate phenotype both in the quantitative competition assay and the lysis assay ( Figures 2 B and 2C). Furthermore, it had fewer active T6SS structures ( Figure 2 A; Movie S1 ). Peptidoglycan was shown to be dispensable for the T6SS activity in V. cholerae (). However, V. cholerae seems to lack T6SS-associated peptidoglycan anchoring proteins ().

No Hcp could be detected in the supernatant of the ΔtssM strain ( Figure 2 C) and neither the Δhcp nor the ΔtssM strains inhibited the growth of E. coli or induced its lysis ( Figure 2 B). Moreover, no dynamic sheath structures were detected in the ΔtssM or Δhcp strains ( Figure 2 A), however, some static VipA-sfGFP foci were observed in the ΔtssM strain. This was in contrast to the ΔtssE strain, in which we found dynamic VipA-sfGFP foci associated with the cell periphery ( Movie S1 ). Nonetheless, those are unlikely to be functional assemblies, because we were unable to detect Hcp in the supernatant of the ΔtssE strain, and the recovery and lysis of E. coli were indistinguishable from that of the ΔtssM strain ( Figures 2 B and 2C). We cannot exclude a potential polar effect of the tssE deletion on the downstream-encoded TssF and TssG, which were shown to be essential components of T6SS (). Although TssE homology to gp25 of the T4 phage suggests its critical role in the assembly and function of T6SS (), it was shown for V. cholerae that a ΔtssE strain retains detectable T6SS activity ().

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis.

The type VI secretion TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via multiple contacts and serves as assembly platform for tail tube/sheath polymerization.

To describe the dynamics of the T6SS assembly in ADP1, we first constructed a vipA-sfGFP and clpV-mCherry2 strain, which then served as a parental strain for in-frame deletion mutants unless indicated otherwise ( Figure S1 A). Live-cell fluorescence microscopy showed that T6SS sheath structures assembled in approximately 15.0 ± 4.2 s (average ± SD, n = 60) and contracted shortly thereafter. Usually, only a single assembling sheath could be observed per cell at any given time. Occasionally, T6SS sheaths polymerized across the whole cell and bent, presumably due to colliding with the cell envelope. On contraction, ClpV-mCherry2 co-localized with the contracted sheath and disassembled it within approximately 40.1 ± 13.4 s (n = 60; Figures 1 B and 1C ; Movie S2 ). Importantly, the T6SS activity of the vipA-sfGFP/clpV-mCherry2 strain was indistinguishable from that of the wild-type strain in its ability to lyse or inhibit growth of Escherichia coli as well as secrete Hcp ( Figures 2 B and 2C ), indicating that the fluorescent protein tags have no influence on the T6SS function.

(C) Hcp detected in the culture supernatant of the indicated strains after trichloroacetic acid (TCA) precipitation, separation by PAGE and subsequent staining with Coomassie. Representative pictures of the endpoints of the lysis assays from (B) are shown for comparison.

(B) The quantitative competition assay measuring recovery of the indicated strains after 4 hr of coincubation of E. coli with the indicated aggressor strains is shown on the left. The error bars indicate the SD, the long dashed lines indicate the mean value of the E. coli recovery, and the short dashed lines indicate the mean value of the aggressor recovery. n. s. = not significant; ∗∗∗ p < 0.001. Lysis assays measuring CPRG conversion upon release of LacZ from E. coli cells incubated with the indicated A. baylyi strains for the indicated time are shown on the right. The lysis assays were performed in biological triplicate and technical hexaplicate for all competitions except for the parental and the ΔtssM strains for which biological and technical hexaplicates were performed.

(A) Large fields of view of the indicated mutants of A. baylyi ADP1 showing the merge of phase contrast, GFP (for VipA-sfGFP in green), and mCherry (for ClpV-mCherry2 in magenta) channels. A close up of the GFP channel of a selected region of interest is shown as an inset. The scale bars of the large fields of view represent 2 μm and those of the insets represent 0.5 μm.

(C) Kymographs depicting the three examples of assembly, contraction, and subsequent disassembly of the T6SS sheath structures shown in (B). The line for generating the kymogram was drawn along the long axis of the highlighted structure.

(B) Three examples of time-lapse imaging of T6SS assembly, contraction, and subsequent disassembly by ClpV. The first frame on the left shows a merge of phase contrast, GFP (in green), and mCherry (in magenta) channels. The frames in the upper rows show fluorescence in the GFP channel (sheath), and the bottom rows show fluorescence in the mCherry channel (ClpV). The arrows indicate the sites where new T6SS sheath structures are forming. The scale bars represent 1 μm.

(A) Large field of view of the parental A. baylyi ADP1 vipA-sfGFP clpV-mCherry2. The images show: the merge of phase contrast, GFP (in green), and mCherry (in magenta) channels on the left, the GFP channel in the middle, and the mCherry channel on the right.

Discussion

R cassette ( Weber et al., 2016 Weber B.S.

Hennon S.W.

Wright M.S.

Scott N.E.

de Berardinis V.

Foster L.J.

Ayala J.A.

Adams M.D.

Feldman M.F. Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis. We show that a markerless in-frame deletion of ACIAD2693 only has a partial effect on the T6SS function, and in many assays, the deletion strain displayed a phenotype similar to that of the wild-type A. baylyi ( Figures 2 A–2C; Movie S1 ). A likely explanation for the discrepancy with the previous results is a potential polar effect on the downstream vipA (tssB) gene resulting from generating insertion mutants using a Tdk-Kancassette ().

Weber et al., 2016 Weber B.S.

Hennon S.W.

Wright M.S.

Scott N.E.

de Berardinis V.

Foster L.J.

Ayala J.A.

Adams M.D.

Feldman M.F. Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis. Weber et al., 2016 Weber B.S.

Hennon S.W.

Wright M.S.

Scott N.E.

de Berardinis V.

Foster L.J.

Ayala J.A.

Adams M.D.

Feldman M.F. Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis. Santin and Cascales, 2017 Santin Y.G.

Cascales E. Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system. ACIAD2685 and TagX were proposed to be essential for T6SS-mediated Hcp secretion (). Interestingly, we show that the strains lacking ACIAD2685 or TagX occasionally assemble sheath structures that display dynamics similar to that of the parental strain ( Figure 2 A; Movie S1 ). Importantly, the prey cell lysis assay shows that those assemblies are functional, which suggests that ACIAD2685 and TagX influence the frequency of T6SS sheath assembly rather than the function of the individual T6SS structures ( Figure 2 B). This is consistent with the fact that TagX is an L,D-endopeptidase cleaving the peptide crosslinks of the peptidoglycan, which was proposed to form holes in the peptidoglycan to allow assembly of the T6SS (). Similarly, the lytic transglycosylase MltE was recently shown to be recruited by the TssM of the Sci-1 in E. coli EAEC 17-2 to fulfil the same purpose (). Therefore, the low number of T6SS assemblies detected in the ΔtagX strain may be due to the formation of holes in the peptidoglycan during its remodeling or aging. Although the phenotype of the ΔACIAD2685 strain is similar to that of the ΔtagX strain, the lack of conserved domains prevents predicting its function.