You've probably heard of spiders that fly without wings. They climb onto flowers or leaves leaves, stand on their tiptoes, and push gossamer threads of silk from their spinnerets, glands in their abdomen, and take off (Figure 1; see also this video). They do that to reach a nearby twig when weaving their webs or to make long-distance flights, sometimes over hundreds of kilometers. This behavior has been called 'ballooning' or 'kiting,' referring to passive aerial movements. (recent research suggests that also atmospheric electrostatics plays a role.) But have you ever heard of 'ballooning' or 'kiting' bacteria, marine cyanobacteria, to be precise, and underwater? Me neither, that is, until last week. Here goes...
It has long bothered microbiologists that the highly abundant marine picocyanobacteria of the Synechococcus and Prochhlorococcus families are found preferentially at defined depths in the water column. The high-light adapted (HL) Prochlorococcus MED4 ecotype, for example, inhabits the uppermost layer of the photic zone while the low‑light adapted (LL) MIT9313 ecotype is found at depths below 150 m. The picocyanobacteria lack flagella for swimming or gas vacuoles for flotation. How then can the planktonic picocyanobacterial cells in suspension − they rarely encounter any kind of surface in their natural habitat − combat the downward pull of gravity? Aguilo-Ferretjans et al. addressed this issue in a study just published (Open Access.)
The vexing question was not helped much by their finding that a quarter of the picocyanobacteria have in their genomes gene sets for the synthesis of Type IV pili (T4P). These gene sets are similar to those known from freshwater cyanobacteria. Synechocystis and Synechococcus elongatus use pili for transformation and twitching motility. Picocyanobacteria don't surf around on surfaces!
While the similarity to S. elongatus PCC 7942 pili genes helped annotate the pili genes set of picocyanobacteria, what has really helped were the findings, by screening of metatranscriptomics data sets, that the major structural component of pili, the PilA1 protein, is expressed in seawater samples worldwide. Likewise, PilA1 is expressed in relatively easy-to-cultivate Synechococcus sp. WH7803 and readily detected in its exoproteome. And finally, they saw complete pili of WH3801 in the electron microscope (Figure 2, left and middle).
The logical next step was, of course, to "make" a Δpili mutant to find out what function(s) they have. "Making" any kind of mutant is a matter of days, if you work with E. coli, Pseudomonas, Vibrio, or Caulobacter. Not so for the picocyanobacteria. First, they are slow-growers with generation times in the range of 1−2 days under optimized conditions and transformations can take several months. Second, their transformation cannot be done with 'naked' DNA but requires conjugation, which is a tad more tedious and has low yields. Third, as purely planktonic cells the picocyanobacteria fiercely resist colony formation and need a helper bacterium that prevents their self-intoxication by metabolic waste. These helper bacteria must be removed in a counter-selection step (in liquid culture) to finally obtain axenic mutant isolates. But they were able to obtain the mutant. And yes, they didn't see pili in the mutant and found no PilA1 in its exoproteome (Figure 2, right).
Flow cytometry analyses confirmed that wild-type and pili mutant cells have virtually the same size. How then do wild type and Δpili mutant cells differ other than having or not having pili? In static cultures, wild-type cells easily stayed suspended for 3–4 days while Δpili cells rapidly sank to the bottom (Figure 3A). The authors found that this is not a property of cell aggregates, but a property of single cells. They followed the trajectories of hundreds of cells (for details see the methods section of the paper) for 1, 2, or 5 minutes each, with a recording rate of 1 fps. Δpili cells followed a pretty straight downward path, driven by gravity (Figure 3B). Wild-type cells, however, hovered more-or-less horizontally. They defied the pull of gravity and tumbled horizontally due to Brownian motion of the small molecules (H2O!) in the medium. Since pili are not propelling like the 'motorized' flagella, the cells were not actively 'swimming' but rather 'kiting,' thus the title of this post (no 'ballooning' here as thermals weren't involved).
What could cause the cells to make pili in the first place? A valuable hint came from their observation that wild-type cells of Prochlorococcus MIT9313 sank when grown for 2−3 days in nutrient-poor medium but remained suspended when grown in nutrient-enriched medium (Figure 3C). In addition, their PilA1 protein was only detected in the exoproteome of suspended cultures. So, could nutrients trigger the formation of pili, or the extension of already existing ones?
They tested this in a simple yet elegant approach by placing a layer of pre-cultured Synechococcus sp. WH7803 wild-type and Δpili mutant on top of a medium gradient in which the cells would, without shaking, first sink into a nutrient-poor zone due to gravity. Sinking cells would subsequently encounter a nutrient-enriched zone before sinking further into an again nutrient-poor bottom zone. The Δpili mutants sank continuously through the three zones, and reached the bottom after 4 days (Figure 4B). In contrast, wild-type cells sank less rapidly overall and accumulated predominantly in the nutrient-rich zone after 4 days (Figure 4B), even visibly (Figure 4A). An important control by flow cytometry revealed no aggregates but only planktonic cells for both the wild-type and the Δpili mutant. From these results, the authors conclude: "This pilus, widely distributed and expressed amongst dominant marine oligotrophic cyanobacteria, allow these purely planktonic organisms to increase cell drag and, consequently, maintain an optimal position in the water column. This mechanism responds to discrete stimuli e.g. nutrients, which may vary depending on the ecological adaptation and preferred niche of each individual organism." But there's more to it than just flying kites...
Aguilo-Ferretjans et al. thought it likely that, these Synechococcus cell surface appendages would modulate ecological interactions with other organisms, namely bacterivorous protozoa − known in the field as "grazers" − and cyanophages. Both groups are considered key population regulators of the picocyanobacterial populations in aquatic environments − besides death by sinking below the photic zone, of course. Regulators, because it's a long-standing observation that picocyanobacteria double every 1-2 days yet their concentration (cells/ml) is fairly constant over shorter time periods at a given sampling site. "Someone" must be responsible for the disappearance of half of the picocyanos every 2nd day, right? The role of phages as regulators could not be tested because no cyanophage is (yet) known to bind to pili. But what's about the "grazers"?
The authors showed that the pili reduce the susceptibility of wild-type cells to grazing by the ciliate Uronema and the flagellate Cafeteria, likely by interfering with the way these predators access their prey. They found in co-cultures of wild-type cells and Uronema that the bacteria increased in population size roughly 20‑fold before efficient grazing reduced them to concentrations below the inoculum within 12 days (Figure 5). In sharp contrast, the Δpili mutant in co-culture with Uronema declined in population size right from the start and was reduced to concentrations below the inoculum within 6 days. Also in co-cultures, Cafeteria flagellates didn't succeed in hunting down wild-type bacteria for up to 300 h but happily snacked on Δpili mutant cells when these had increased in population size 20‑fold, then rapidly reduced them (Figure 5). Apparently, the pili‑lacking prey had to be present in greater numbers and thus in closer proximity to the Cafeteria predators to whet their appetite. So, despite differences in the 'predation kinetics', the pili protected Synechococcus significantly from grazing by both protozoans tested.
Kiting under water and fending off predators add to the impressive list of functions Type IV pili play in bacteria. Craig et al. summarized in their recent review: "The T4P are unique in that they are dynamic: filaments polymerize and depolymerize, leading to rapid cycles of extension and retraction that generate considerable mechanical force. This feature enables T4P to pull adherent bacteria along mucosal surfaces [twitching motility] into close association with host cells and other bacteria, to exert forces on host cells, to pull bound substrates like DNA [see here in STC, and do not miss Courtney Ellison's movie of Vibrio T4P 'at work'] and bacteriophages into the periplasm and to export exoproteins across the outer membrane. T4P extend and retract at rates of ~1,000 subunits per second, requiring a complex protein machinery that spans both membranes of Gram-negative bacteria [see here for a diagram. Source]. T4P are present on a diverse range of bacteria, including M. xanthus, C. crescentus and T. thermophilus, as well as important human pathogens such as N. gonorrhoeae, P. aeruginosa and V. cholerae." Now we know that pili are also present and functional in the 'classical' way in freshwater cyanobacteria and, with a novel, so far unknown function in a quarter of the marine picocyanobacteria. Of course, and because it's science after all, this leaves open the question of what tricks the other three quarters of picocyanobacteria have in store to stay put in their comfort zone and avoid sinking in the water column below the photic zone...