Dial "V" for Murder

by Christoph
 

If you read this blog frequently and, perhaps, because we "emphasize the unusual and the un­ex­pec­ted phenomena", you may feel a little, Um.., bored to see yet another post on horizontal gene transfer (HGT). But wait, we've actually touched this topic in fewer than 30 of our 1,200+ posts (to be precise: 1,247 as of December 31th, 2017 ). That's less than the estimated average number of genes acquired via HGT over time by a small bacterial genome of, say, 1,200 genes! So, I should not worry too much about redundancy when talking about it again (at length, a fair warning! ). We previously discussed HGT by conjugation as it happens, and by phage transduction. Now I will dis­cuss HGT by transformation, that is, by DNA uptake from the environment. This has been studied in great detail in several systems, and I could have stopped dialing at "B" for Bacillus subtilis, at "H" for Haemophilus influenzae, at "N" for Neisseria gonorrhoeae, or at "S" for Streptococcus pneu­mo­ni­ae (think of Griffith's 'transforming principle' from 1928 ). I skipped dialing "M" for Alfred Hitch­cock, and ultimately chose "V" for Vibrio cholerae. Be prepared for a crime movie par excellence, one that outperforms Hitchcock for thrill and suspense.
 

Keep in mind that the Ten Commandments don't apply for bacteria, and any mention of murder, killers, victims, weaponry, etcetera is, therefore, mere anthropomorphizing. That said, jump for­ward to 'Take #3: Street Action!' if you're keen to witness the actual murder scene. Otherwise, con­tinue reading about natural competence and transformation in 'Take #1: In the Basement', and about Type VI Secretion Systems (T6SS) in 'Take #2: Inside the Armory'. Watch the screenplay un­fold as the plot thickens...
 

Take #1: In the Basement

Figure 1. V. cholerae ComEA binds to DNA in vi­vo. Com­EA-mCherry aggrega­tion and foci for­ma­­tion after the ad­dition of ex­ternal DNA. Com­pe­­tence-induced cells without (no DNA) or with ex­ternal DNA (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, genomic DNA of various length) were imaged in the red (mCherry; lower row) or the phase contrast channel (phase; upper row) to visualize ComEA-mCherry localization. Ex­ter­nal DNA did not lead to foci forma­tion of peri­plas­mic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). Scale bars: 2 µm. Modified from Source

As simple as it sounds, 'DNA uptake' is mechanistically as complex as conjugation or phage transduction. In both Gram-positive and Gram-negative bacteria, 'competence' for DNA uptake requires the coordinated action of rough­ly two dozen genes (including some with regulatory func­tions ) organized within several operons. The picture is si­milar in Vibrio – 'similar' refers to a great deal of protein homology among the numerous components in various species, despite vastly different regulatory circuits – for which the present model for competence involves 19+ genes, and, as core components: 1. a type IV pilus (Tfp) (not to be confused with T4SS as Elio had pointed out here ) that does not attach to a surface for twitching mo­tility but may rather act like a plug for the outer-mem­bra­ne pore, PilQ; 2. a periplasmic protein, ComEA, that binds nonspecifically to double-stranded DNA; and 3. the inner membrane-bound ComEC complex (see here for a sche­ma­tic diagram (Source )). According to this model, 'DNA uptake' starts with retraction of the Tfp pilus and pulling of the DNA through the outer membrane pore, PilQ, by a supposed 'Brownian ratchet' mechanism (despite Richard Feynman having debunk­ed the possibility of a 'Brownian ratchet' ). Upon entry into the periplasm, the incoming DNA is tight­ly bound and compacted by ComEA (so tightly that ComEA overexpressed in the cytoplasm of  E. coli kills the cell by sequestering the nucleoid ), and then shuttled to the ComEC inner membrane-pore complex. As in Bacillus subtilis, the incoming DNA enters the cytoplasm of V. cholerae probab­ly single-stranded with the 3'-end head-on, but the responsible periplasmic (membrane bound? ) exonuclease is still elusive. Upon entry into the cytoplasm, the incoming DNA is bound by single-strand binding protein (Ssb), by DprA (like Ssb an ssDNA-protecting protein ), and by RecA, thus priming it for recombination. While that's Vibrio cholerae transformation in a nutshell, two missing details are still needed for the developing 'screenplay' of the crime story.
 

Figure 2.  Fluorescence microscopy of chitin bead-co­lo­nizing cells of V. cholerae engineered to carry trans­lational fusions for the competen­ce protein ComEA-mCherry (red) and the T6SS sheath pro­tein VipA-sfGFP (green), both encod­ed at their native chromosomal locus. Merged signals from the two fluorescence chan­nels (GFP+mCherry). Three assembled and/or con­trac­ted T6SS (VipA‑sfGFP sheath) structures are indicated by white arrows. Scale bars: 2 µm. Source

For one, synthesis of the transformation apparatus is en­ergetically costly. It would make sense, therefore, to trig­ger its synthesis only under conditions where exogenous DNA was potentially available, for example, in biofilms. In their natural aqueous habitats, the vibrios preferentially colonize the carapaces (=exoskeletons ) of crustaceans. When they sense chitin, the major 'sugar-y' macromole­cu­lar components of the crustaceans' shells which they can degrade to earn their living, they attach to it and make a biofilm (see here: vibrios on chitin particle (Source )). As was found early on, competence follows. The integration of signals from chitin sensing, nutrient limitation (catabo­li­te repression ), and cell density sensing in/for biofilm for­mation (quorum sensing ) triggers the expression of TfoX, a key regulator of competence expression. Despite this complexity, TfoX alone can elicit full competence in high cell density when expressed from an inducible promoter. This is fortunate for the development of this crime story among vibrios because it simplifies their mis-en-scène ex­perimentally.
 

For the second key detail, in competent Vibrio cholerae  cells, ComEA translocates to the periplasm. Luckily, so does a functional ComEA‑mCherry fusion which can be readily visualized as seen in Fi­gures 1 + 2 (slightly brighter fluorescence in the cell periphery is normal when looking at a 2D pic­ture of a rod-shaped 3D object ). Upon adding purified DNA to such cells, the 'periplasmic staining' of the cells by ComEA‑mCherry coalesces into one or a few bright spots that indicate tight binding to and compacting of the incoming DNA (Figure 1). Borgeaud et al. assume that these ComEA ag­gregates represent the 'pawl' of the 'Brownian ratchet' mentioned above. Anyways, ComEA aggre­gation upon binding to DNA is of prime importance for the developing crime story as this 'behavior' provides clear visual  evidence for ongoing DNA uptake.
 

Take #2: Inside the Armory

Figure 3. Overview of the T6SS.  A Electron cryo-to­mo­graph of V. cholerae cells producing T6SS. The arrow points the long cytoplasmic tu­bular structure cor­res­ponding to the T6SS sheath. A magnification of the upper part of the tomo­graph is shown in panel B, em­pha­siz­ing the pre­sence of distinct complexes (green, tail sheath; purple, baseplate; yellow, putative mem­brane com­plex). Pa­n­­els A and B: Source. C Schematic re­presentation of the T6SS based on the elec­tron cryo-tomographs and on gene­tic, bioche­mical, microscopy and struc­tural data (IM, inner membrane; PG, pep­ti­doglycan layer; OM, outer mem­brane). Scale bar is 50 nm. Source

When you peek into the armory of Proteobacteria and Bacteroidetes (=not all Gram-negatives, and also not all species of the Proteos ) you will immediately spot their masterpiece, the conspicuous Type VI Secretion System (Figure 3). As Zoued et al. characterize it: "The T6SS is constituted of an envelope-spanning complex anchoring a cytoplasmic tubular edifice. This tubular structure is evolutionarily, functionally and structurally related to the tail of contractile phages. It is composed of an inner tube tipped by a spike complex, and engulfed within a sheath-like structure. This structure assembles onto a platform called "baseplate" that is connected to the membrane sub-complex." We have mentioned the T6SS earlier in STC here and here, and in both posts focused on its use as a weapon. Zoued et al. continue:"The T6SS functions as a nano‑crossbow: upon contraction of the sheath, the inner tube is propelled towards the target cell, allowing effector delivery." They should have said "propelled into the target cell", because this is what actually happens. T6SS is not a long-range weapon like a "crossbow" : it works only at short distances, when cells sit side-by-side. And in contrast to the tails of phage particles, the "inner tube" does not deliver DNA through it but, like a poison arrow, through "effector" proteins attached to its "spike", the arrow­head. Yet Zoued et al. point out rightly by using a more neutral term that "effector delivery" is not necessarily bad news for the recipient. Although it mostly is, since most known effectors are nuc­leic-acid-degrading, cell wall-degrading, or membrane-degrading enzymes. Think of T6SS as a sort of forced, near-field communication that the recipient cell cannot decline accepting and that can serve various purposes including invasion or defense of a community, biofilm remodeling, killing of phage-infected bacteria, and killing of non-cooperators (the scuffle between Pseudomonas and Vibrio was featured by Spencer Scott & John De Friel here in STC ). However, a T6SS 'notification' can turn into a mere 'signaling' if the recipient cell has cognate immunity factors at its disposal. Note that the "effector" cholera toxin (CT) encoded by the CTXφ prophage of many V. cholerae strains is not delivered to human intestinal cells via T6SS – that's a completely different crime story.
 

The T6SS "nano-crossbow" of Vibrio cholerae  is a multi-protein complex of >20 components that includes not only the mechanical parts (sheath, inner tube, membrane anchors ), but also the "ef­fector" proteins and the respective (auto)immunity factors needed for insurance against harming oneself or one's siblings. The T6SS genes are organized in three clusters, two on chromosome II and one on the main chromosome I. Recurring to the argument made for the competence machi­nery (see 'Take #1: In the Basement' ): synthesis of the T6SS apparatus is energetically costly, and it would make sense to trigger its synthesis only under conditions of high cell density. And indeed, T6SS expression is co-regulated by quorum sensing.  Borgeaud et al. identified, in addition, TfoX as the key regulator, exactly the same factor that triggers competence expression (see their diagram here ). Thus, expression of T6SS and competence are tightly coupled, and the idea that T6SS leads to killing of neighboring cells followed by horizontal gene transfer is, well, straightforward. But how to demonstrate T6SS expression visually ? Basler et al. had found earlier in their study of the T6SS sheath of Vibrio cholerae  that one of its two components, VipA, can be coupled to GFP with­out grossly affecting its properties (actually they used a particularly well 'foldable' version of GFP called 'superfolder GFP' (sfGFP )). This enabled them to visualize sheath formation (=assembly ), contraction, and re-assembly after 'firing' in live cells (see their stunning movie here ). To test their hypothesis, Borgeaud et al. made a V. cholerae  O1 El Tor A1552 strain derivative with the genoty­pe TntfoX vipA-sfgfp comEA-mcherry. Upon induction, this 'predator' strain expresses T6SS and competence, and the cells become detectable in the microscope by green fluorescence (VipA sheaths ) and red fluorescence (periplasmic ComEA ) (Figure 2). To complete the casting for the 'murder scene', they chose as 'prey' V. cholerae strain Sa5Y ΔvipA gfp, which is T6SS-deficient, lacks immunity to the toxins of A1552, and expresses GFP that is then distributed throughout its cyto­plasm.
 

Take #3: Street Action!

I invite you now to the shooting location where filming of the key murder scene is about to begin, and will continue for the next two hours. Camera is in position; the actors have rehearsed their script, have been made up professionally, and are eager to act. They have already taken their po­sitions, gathered in close contact to each other: one 'blue' Vibrio  predator and three 'grey' Vibrio  prey cells (t=0; shown schematically in Fig. 4, bottom row ). Take your seat and keep an eye on the action in Figure 4. Also keep in mind that the players will grow (=elongate ) and divide over the next 120 min unless/until they're killed. Recognize the killer dressed for attack by the faint green GFP fluorescence of one of its T6SS-sheath building blocks, VipA‑sfGFP, and prepared for DNA up­take by the red fluorescence of ComEA-mCherry distributed in its periplasm (compare with Figure 2 ). The three presumptive victims express GFP in their cytoplasm and are thus recognizable by their cell-wide bright green fluorescence.
 

Figure 4.  Induced T6SS leads to the killing of neigh­boring cells followed by horizontal gene transfer. This figure shows a time-lapse micro­scopy image series of a T6SS-dependent attack of nonimmune prey cells (prey, bottom row) fol­lowed by their rounding and lysis and the upta­ke of their DNA by a neighbor­ing T6SS-compe­tent predatory cells (pre­dator; bottom row). A vipA-sfgfp and comEA-mcherry Vibrio cho­lerae strain (preda­tor) was grown to high cell density under T6SS-inducing conditions, mixed with the T6SS-negative and gfp-labeled V. cholerae strain Sa5Y (ΔvipA gfp) (prey), and spotted onto agaro­se pads for ima­ging. White arrows and arrow­heads denote prey cells before and after attack (seen as cell rounding), respectively, and black arrows indicate cell debris after lysis as visuali­zed in the phase contrast images (phase; top row). Accumu­lations of the VipA-sfGFP protein and as­sembled + con­tracted T6SS sheath struc­tures are visible in the green channel (GFP; se­cond row). DNA released by the prey lysis is ta­ken up by the competent preda­tor, as indicated by the gray arrowheads highlighting the re­dis­tri­bu­tion of periplasmic ComEA-mCherry (mCherry; second last row). Scale bar: 2 µm indicated in the upper left ima­ge here and in the original picture, and valid for all ima­ges). Modified from Source

The action starts immediately, at t=0 min, when the killer attacks victim #1 (white ar­rows ). At t=30 min, victim #1 has already been stabbed and lysed (black arrow ). Its GFP fluorescence dilutes into the surround­ing medium and thus becomes invisible. Victim #2 is about to be stabbed (white ar­row ) and by t=60 min (white triangle ) has blown-up to a bleb (=protoplast formation due to peptidoglycan disintegration, prece­ding lysis ). The killer is now busy stabbing victim #3 as seen by bright green streaks and dots (assembled and contracted T6SS sheaths ), while also taking up the former victim's DNA: the red fluorescence coalesces into dots (gray arrows ). Although by t=90 and t=120 min there are no more potential victims within reach, the raging killer keeps wielding its T6SS crossbow while simultane­ously taking up DNA from the deceased. Ac­tually, the killer has already doubled once during the filming, and is about to divide again shortly after t=120 min. From the ci­ne­matographic point of view: no stunts or spe­cial effects required here to make the mur­der scene scarier! Note that although victim #3 got stabbed before t=90 min and has promptly blebbed, it has astonishingly re­mained in this stage for >30 min. Although the screenplay does not demand the imme­diate death of victim #3 during the take, we know that the cell is doomed (one could ea­sily speculate that the burst of victim #3 is delayed due to complete coverage of the 'zombie' protoplast by all the DNA goo of victims #1 and #2 ).
 

Fun aside, the imaginary 'murder scene' I guided you through was just an example of the efforts Borgeaud et al. made to show the T6SS-killing of neighboring cells followed by HGT. Now that you're well versed in the details, you could peek at their 'crowd scene', from which the 'murder scene' is a cutout (the hi-res version was kindly supplied by Melanie Blokesch ). The observed kil­ling of neighbor cells and the taking-up their released DNA are, taken alone, not conclusive proof of horizontal gene transfer (HGT). One must also demonstrate integration of transferred genes into the killer's chromosome(s). RecA‑dependent homologous recombination would be the most efficient route for such integration, and Seitz and Blokesch had shown in an earlier study that in­deed RecA-GFP accumulates (=forms foci ) close to the cytoplasmic site where single-stranded DNA slips from the periplasm into the cytoplasm. To prove HGT, one needs to also demonstrate the recombination of some of this incoming DNA into one of the two V. cholerae  chromosomes and its subsequent expression. To this end, the authors co-cultured a T6SS‑proficent V. cholerae  O1 El Tor A1552 (Rif^R) 'predator' strain with a T6SS‑deficient and non‑immune V. cholerae  Sa5Y 'prey' strain tagged with a lacZ::aph (Kan^R) marker on chromosome II under T6SS- and competen­ce-inducing conditions on LB-plates (=keeping the cells in intimate contact ). After colony forma­tion, they selected transformants on LB‑plates containing kanamycin and rifampicin (note that selection for antibiotic resistance was not applied during the HGT-promoting step, a subtle but important detail reminiscent of the classical selection procedure for transconjugants in Hfr-cross­es ). Double-resistant transformants (Rif^R+Kan^R) were then checked by PCR for the presence of the aph  gene (Kan^R), and for the presence of two V. cholerae  O1 El Tor-specific markers – not present in Sa5Y – on chromosome I and chromosome II, respectively. All transformants turned out to be V. cholerae  O1 El Tor A1552 (Rif^R) having acquired the aph  gene (Kan^R) via homologous recombina­tion from the prey strain (Figure 5). Conclusion: although the authors did not catch the 'predator' after recombination in flagrante delicto on camera, this transformation experiment constitutes a legally incontestable confession. They can thus rightfully claim to have shown HGT of a chromoso­mal antibiotic resistance marker.
 

Take #4: A Side Story (Dial "A" for Murder)

Figure 5. Double-resistant colonies are trans­formants of the predator strain. PCR ampli­fi­ca­tion products con­firming the transformation of the predator strain (lower two images) by the prey-derived aph gene (kanamycin resistance cassette; upper image). The schemes next to each image depict the priming sites of the oli­go­nuc­leotides. Strains used for the colony PCR were the predator (lane 1; WT, A1552-TntfoX Rif^R), the prey (lane 2; Sa5Y ΔvipA Kan^R), and randomly chosen trans­formants (lanes 3 to 8). L, DNA ladder. chrI and chrII abbreviates V. cholerae chromosome I and chromo­so­me II, respectively. Source

T6SS‑mediated neighbor killing and gene acquisition by HGT is by no means confined to the vibrios. Cooper et al. demonstrated in a very recent study that T6SS-proficient and transformation-competent Acinetobacter baylyi  kill co-cultured E. coli  cells (view one of their videos here; scroll to the end of this post for the legend ) and acquire their antibiotic resistance genes. HGT occurred at high frequency within a few hours under the chosen experi­mental conditions (in microfluidic devices or on plate spots ) and newly resistant A. baylyi  cells rapidly displa­ced antibiotic-sensitive kin under selective growth condi­tions. These findings may help to solve the riddle of how so many clinical isolates of the human pathogen Acine­to­bacter baumannii  accumulate multiple antibiotic resist­ance genes from other human pathogens so rapidly (cau­tionary note: A. baylyi is a soil bacterium and the regu­la­tion of competence and T6SS expression might differ con­si­de­rab­ly from that of the human pathogen A. baumann­ii). Likewise, the studies performed over several years by the lab of Melanie Blokesch at the EPFL in Lausanne, Switzerland, do not aim merely for a reductionist ex­pla­na­tion of HGT via transformation in Vibrio cholerae  (see 'Take #3: Street Action!' ), but also at using this knowledge to better understand the mechanisms that drive the repeated re-emergence of this human pathogen from the pool of its extant (sub)spe­cies in their normal marine habitats (think of the recent cholera epidemic after the 2010 earth­quake in Haiti with up to now >700,000 infections and ~9,000 deaths; but In this case, the V. cho­le­rae strain causing the epidemic was apparently imported from Asia by UN peacekeeping forces). That's for the factual background of my fictive 'murder story'.
 

Take #5: in the Postproduction Studio

It's a long-standing notion (hotly debated, of course, because it's science! ) that 'naturally compe­tent' bacteria may take up chunks of DNA to supplement their food stocks, or more precisely, their precious phosphate and nucleoside pools. This might well hold true for environmental DNA, which is often rather degraded (think of the DNAses that are present in any bacteria-laden environment, that is, everywhere ), but less so for DNA 'freshly' released from lysing cells in close proximity to competent ones. In this case, ingested DNA, especially when coming from close relatives, might instead be most useful for genome repair processes and gene acquisition via HGT by a competent bacterium, thus increasing its fitness. Maybe the above mentioned vibrios found a clever way to 'have their cake and eat it, too' ? Any incoming DNA could easily first serve for recombina­tion – re­pair or gene acquisition – and then the 'loose ends' of double-crossovers, along with non‑recom­bined pieces, used to replenish their phosphate and nucleoside pools. Just a thought. Yet it re­mains a fact that the uptake of exogenous DNA is a viable path for HGT that can be visually de­mon­­strated, just like conjugation or transduction by phage. And that was the point of this post, a hap­py ending  for 'Dial "V" for murder'.
 

References

Basler M, Pilhofer M, Henderson GP, Jensen GJ, Mekalanos JJ. 2012. Type VI secretion requires a dy­namic contractile phage tail-like structure. Nature, 483  (7388), 182 – 186. PMID 22367545 (Open Access PDF here)

Borgeaud S, Metzger LC, Scrignari T, Blokesch M. 2015. The type VI secretion system of Vibrio cho­lerae fosters horizontal gene transfer. Science, 347  (6217), 63 – 67. PMID 25554784 (Open Access PDF via free registration)

Cooper RM, Tsimring L, Hasty J. 2017. Inter-species population dynamics enhance microbial ho­ri­zontal gene transfer and spread of antibiotic resistance. Elife, 6, e25950. PMID 29091031 (Open Access PDF here)

Lo Scrudato M, Blokesch M. 2012. The regulatory network of natural competence and trans­for­ma­tion of Vibrio cholerae. PLoS Genet, 8  (6), e1002778. PMID 22737089 (OpenAccess PDF here)

Seitz P, Blokesch M. 2014. DNA transport across the outer and inner membranes of naturally trans­formable Vibrio cholerae is spatially but not temporally coupled. Mbio, 5  (4), e01409-14. PMID 25139903 (Open Access PDF here)

Seitz P, Pezeshgi Modarres H, Borgeaud S, Bulushev RD, Steinbock LJ, Radenovic A, Dal Peraro M, Blokesch M. 2014. ComEA is essential for the transfer of external DNA into the periplasm in na­tu­rally transformable Vibrio cholerae cells. PLoS Genet, 10  (1), e1004066. PMID 24391524 (Open Ac­cess PDF here)
 

 

Legend to linked video: representative micro-colonies to highlight the enrichment of E. coli  cell lysis at the boundaries of micro-colonies, where the cells are in contact with Acinetobacter  cells. E. coli  are shown in green (GFP) and Acinetobacter  in red (mCherry); in some cases the E. coli  micro-colony is eliminated (as in this video ), and in others it is heavily reduced but survives. Left panels: difference images were calculated for the green (E. coli ) channel by subtracting the subsequent time point from the current one. This shows where the GFP signal dramatically decreases, indicating cell lysis between the two time points. These regions of puta­tive cell lysis are shown in yellow. Right panels: the same movies, but without the yellow difference signal and including the transmitted light channel, for comparison. After the mCherry signal from Acinetobacter  fades (due to prolonged laser exposition ), the location of Acinetobacter  surrounding the E. coli  can be seen in the transmitted light channel. Note the timer in the upper left corner.