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https://www.quantamagazine.org/the-beautiful-intelligence-of-bacteria-and-other-microbes-20171113/ November 13, 2017

Seeing the Beautiful Intelligence of MicrobesBacterial biofilms and slime molds are more than crude patches of goo. Detailed time-lapsemicroscopy reveals how they sense and explore their surroundings, communicate with theirneighbors and adaptively reshape themselves.

By John Rennie and Lucy Reading-Ikkanda

All images by Scott Chimileski and Roberto Kolter (except where indicated)

The slime mold Physarum polycephalum forms a network of cytoplasmic veins as it spreads across a surface.

Intelligence is not a quality to attribute lightly to microbes. There is no reason to think that bacteria,slime molds and similar single-cell forms of life have awareness, understanding or other capacitiesimplicit in real intellect. But particularly when these cells commune in great numbers, their startlingcollective talents for solving problems and controlling their environment emerge. Those behaviorsmay be genetically encoded into these cells by billions of years of evolution, but in that sense thecells are not so different from robots programmed to respond in sophisticated ways to theirenvironment. If we can speak of artificial intelligence for the latter, perhaps it’s not too outrageousto refer to the underappreciated cellular intelligence of the former.

Under the microscope, the incredible exercise of the cells’ collective intelligence reveals itself withspectacular beauty. Since 1983, Roberto Kolter, a professor of microbiology and immunobiology at

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Harvard Medical School and co-director of the Microbial Sciences Initiative, has led a laboratorythat has studied these phenomena. In more recent years, it has also developed techniques forvisualizing them. In the photographic essay book Life at the Edge of Sight: A PhotographicExploration of the Microbial World (Harvard University Press), released in September, Kolter andhis co-author, Scott Chimileski, a research fellow and imaging specialist in his lab, offer anappreciation of microorganisms that is both scientific and artistic, and that gives a glimpse of thecellular wonders that are literally underfoot. Imagery from the lab is also on display in the exhibitionWorld in a Drop at the Harvard Museum of Natural History. That display will close in early Januarybut will be followed by a broader exhibition, Microbial Life, scheduled to open in February.

High magnification of the slime mold Physarum polycephalum shows the cytoplasm pumping furiously through itshuge single cell. This cytoplasmic streaming allows the slime mold to push forward toward nutrients and potentiallycarpet a surface.

The slime mold Physarum polycephalum sometimes barely qualifies as a microorganism at all: Whenit oozes across the leaf litter of a forest floor during the active, amoeboid stage of its life cycle, it canlook like a puddle of yellowish goo between an inch and a meter across. Yet despite its size,Physarum is a huge single cell, with tens of thousands of nuclei floating in an uninterrupted mass ofcytoplasm. In this form, Physarum is a superbly efficient hunter. When sensors on its cell membranedetect good sources of nutrients, contractile networks of proteins (closely related to the ones foundin human muscle) start pumping streams of cytoplasm in that direction, advancing the slime moldtoward what it needs.

But Physarum is not just reflexively surging toward food. As it moves in one direction, signalstransmitted throughout the cell discourage it from pushing counterproductively along less promising

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routes. Moreover, slime molds have evolved a system for essentially mapping their terrain andmemorizing where not to go: As they move, they leave a translucent chemical trail behind that tellsthem which areas are not worth revisiting.

After Physarum explores an area and finds it lacking in nutrients, it leaves behind a chemical trail as a kind ofexternalized memory that tells the slime mold not to go back there.

When bacteria were first observed through a microscope, suspended in liquid on slides, in theirsimplicity they seemed like the archetypes of primitive, solitary cells. The truth, however, is that inthe wild, most bacteria are highly gregarious. Some bacteria do swim through their environment aslonely individuals but most bacterial cells — and most species of bacteria — prefer to live in compactsocieties called biofilms anchored to surfaces. (The individual swimmers often represent offshoots ofbiofilms, seeking to colonize new locations.)

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Roberto Kolter and Steve Minsky (Bacillus)

In a high-magnification scanning electron micrograph of a Pseudomonas aeruginosa biofilm (left), the individualrod-shaped bacteria are interlinked by hairlike structures called pili. Bacillus bacteria secrete an extracellularmatrix that encases the cells and helps them form a more structured community (right).

Moreover, biofilms are not just dense accumulations of bacterial cells. They have elaboratefunctional structures, inside and out, that serve the cells’ collective destiny, as can be seen in theimages below of Pseudomonas aeruginosa. The biofilm is stained with Congo red dye, which bondsto the extracellular matrix proteins that the bacteria secrete as a scaffolding for their community.The deeply wrinkled surface of the biofilm maximizes the area through which the bacteria canabsorb oxygen; it also probably helps them collect nutrients and release waste products efficiently.

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As this Pseudomonas biofilm expands, it develops a more complex internal structure. Bacteria in different parts ofits mass may also develop more specialized functions.

Within the biofilm, the bacteria divide the labor of maintaining the colony and differentiate intoforms specialized for their function. In this biofilm of the common soil bacterium Bacillus subtilis, forexample, some cells secrete extracellular matrix and anchor in place, while some stay motile; cells atthe edges of the biofilm may divide for growth, while others in the middle release spores forsurviving tough conditions and colonizing new locations.

The wrinkled structure of this Bacillus subtilis biofilm helps to ensure that all the bacteria in it have access to

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oxygen (left). A digital scanned model of the biofilm helps illustrate how the bacterial community can vary itsstructure in three dimensions (right).

One might wonder why natural selection would have favored this collective behavior instead of morerampant individualism among the cells. Part of the answer might be what evolutionary theorists callinclusive fitness: In so far as the bacteria within a biofilm are related, individual sacrifices are offsetby the increases in fitness to each cell’s millions of cousins. But it may also be that every role withinthe biofilm has its advantages: Cells at the edge are most exposed to dangers and must reproducefuriously to expand the biofilm, but they also have access to the most nutrients and oxygen. Cells onthe inside depend on others for their vital rations but they may survive longer.

The surfaces that biofilms grow across are not always solid. These B. subtilis are forming a pellicle— a kind of floating biofilm at the interface between water and air. The genetic pathways involved informing a pellicle are essentially the same as those used in growing across stones, though they mayrespond to the changes in their habitat by altering the precise mix of proteins in the extracellularmatrix as needed.

Bacteria can grow across nonsolid surfaces, too, as this B. subtilis culture shows by forming a pellicle, or floatingbiofilm, across the air-liquid interface in a beaker.

Expansive growth is not the only way in which microbial communities can move. Below, B. subtilis isengaging in a behavior called dendritic swarming, in which cells rapidly push outward in branchingcolumns that can efficiently pave a surface. Biofilms swarm when they detect that they are inenvironments rich in nutrients: Swarming helps a biofilm exploit this valuable territory before anycompeting communities can.

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At least two important changes in the differentiation of the cells in a biofilm take place to enableswarming. First, motile cells on the periphery of the film develop extra flagellae, which enables themto swim more energetically. Second, some edge cells also begin to secrete surfactant, a slipperymaterial that helps the motile cells slide more rapidly over the surface.

When biofilms grow in flat laboratory dishes, the dendritic columns of swarming biofilms remainneatly distinct: They extend and coil in and around one another but they do not cross. That seems tobe in part because the surfactant piles up around the biofilm branches as a barrier. Similarly, somebacteria can swarm in more terraced structures under laboratory conditions. What the implicationsof that option are for bacteria in nature is still a mystery.

These bacteria are engaging in the behavior called dendritic swarming, which allows a microbial community toexpand rapidly into desirable, resource-rich environments.

Another type of behavior demonstrated by biofilms growing under laboratory conditions is spiralmigration, demonstrated in the time-lapse video below of Bacillus mycoides. These bacterial cellsgrow in long chains or filaments that curl either clockwise or counterclockwise. The specificadvantages of this spiraling movement are still under investigation, according to Chimileski, but theymust be considerable because B. mycoides excels at taking over available environments. “Bacillusmycoides is one of the easiest bacterial species to cultivate from the soil,” he explained. Whenscientists isolate microbes from soil and grow them on agar dishes, particularly at roomtemperature, “the mycoides will often spread across the entire plate and overtake all of the otherorganisms. For this reason, it is considered if anything a kind of ‘nuisance species’ for manymicrobiologists.”

What’s curious is that the direction of the spiraling migration — clockwise or counterclockwise —seems to be a hereditary trait: Different strains of bacteria, even within the same species, spiral indifferent directions. It is yet another example of how bacteria, obeying instructions in theirindividual DNA, can manifest problem-solving behaviors that are surprisingly complex and adaptiveat the collective level of biofilms.

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These geometric and presumably functional patterns that biofilms produce in culture areintriguingly beautiful. Yet Chimileski notes that there is much left to discover when it comes totranslating behaviors seen in the lab to natural microbial communities.

Chimileski points out that “most natural biofilms are multi-species ecosystems and cells insidenatural biofilms usually grow more slowly.” He continued, “I like to think of the way we growbacteria in a petri plate, where a single species is by itself and has everything it needs to grow atoptimal temperatures, as ‘turning up the volume’ on the biology of the organism.” Under laboratoryconditions, researchers can study which genes are involved in complex multicellular behaviors andthey can measure the benefits to the fitness of the bacterial species. But in natural environments,biofilms don’t usually get to form exactly the same patterns as in the lab because of limited nutrientsor competition with other species. “So the same biology might be occurring on a particle of soil inyour backyard at smaller size scales and over longer time periods,” he said, even if it is less easy tovisualize.

Spiral migration is a behavior favored by the highly successful soil bacterium Bacillus mycoides. Communities ofthese cells expand by forming long filaments of cells that coil either clockwise or counterclockwise — an orientationthat is strain-specific and genetically determined.

Biofilm behaviors testify to the capacity and openness of bacterial to form collectives — but thatopenness has limits, as shown in this culture with several cohabiting biofilms. Here, adjacentbiofilms that consist of the same bacteria or closely related strains comfortably merge. But the

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adjacent biofilms made up of more divergent bacteria keep themselves distinct and may even try toeliminate or control each other.

Biofilms are so intolerant of other strains and species because they invest considerably in theproduction of surfactant, extracellular matrix and other molecules that bacteriologists classify aspublic goods — ones that the bacteria secrete for other members of their community. The bacteriaguard these jealously because unrelated freeloading cells could benefit strongly by using them first.

Biofilms rebuff such freeloaders in different ways. For example, the B. subtilis colonies in this imageadopt a strategy of “kin discrimination,” in which they secrete antibiotic compounds that are toxic toother species but not to their own. Proteus mirabilis bacteria defend their interests in a differentway based on “self-recognition”: The P. mirabilis biofilms examine encroaching cells, stab any from adifferent species with a spearlike structure and inject them with poisons that will kill almost all butclosely related species.

Several different strains of B. subtilis grow side by side in this dish. Because the biofilms discriminate againstdissimilar strains of bacteria, they may merge compatibly with close relatives but form boundaries against others.

The colors appearing in the biofilm culture of Streptomyces coelicolor in the video below reflectnatural pigments that the bacteria produce. The value of the pigments for the biofilms is not entirelyclear, but it is probably not tied to their color. Rather, these pigment molecules are often bioactive invarious ways. “The blue pigment seen in this video is actinorhodin, which is technically anantibiotic,” Chimileski said, but added that the term is misleading in this context. “Killing or growthinhibition usually occurs only at very high concentrations relative to what is out in nature.” For thatreason, he said, there is “an emerging view that killing is probably not the ecological function of

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many or most antibiotics. Rather, these bioactive molecules act as signals or developmental cues” toother cells.

That view is echoed in a note from Gleb Pishchany, another research fellow in Kolter’s laboratorywho studies how diverse types of bacteria cohabit. “An intriguing possibility is that in naturalecosystems, Streptomyces use pigments and other bioactive molecules” at “lower concentrations assignals that are exchanged among multispecies microbial communities,” he wrote. The pigmentsmay help cohabiting assortments of bacteria rein in one another’s less neighborly instincts, andthereby maintain a more cooperative and fruitful communal existence.

In this powdery colony of Streptomyces coelicolor, the pigmentation comes from actinorhodin, a molecule withantibacterial effects. Biofilms may use bioactive pigments as signals for controlling the behaviors of othermicroorganisms in their shared environment.

These striking photographs of microbe communities were captured by DSLR cameras. Chimileskicollects his still images with macro lenses while working at the bench, while the videos are made inan incubator dedicated to time-lapse microscopy. He sets the camera to snap a picture every 10minutes, although he increases the frequency to every minute or two for behaviors happening morequickly, such as the movements of slime molds. As a result, the movements of the microbes in thesevideos are typically accelerated between 5,000 and 50,000 times their actual speeds.Chimileski does not use false color to beautify the images: Aside from using dyes to stain theextracellular matrix in some cultures, he shows the natural coloration of the microorganisms.

Chimileski typically grows bacterial colonies at 30°C, a temperature at which he can collect imagesof slower growing species for several weeks. Although the heat and humidity suited to biofilm

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growth are less than ideal for cameras, he said the equipment is rated for more extreme conditions.The few cameras that have malfunctioned did so for a mechanical reason: The number of shots thathe needs to document microbial behaviors is so large that the shutters on the cameras eventuallybreak down after hundreds of thousands of clicks.

This article was reprinted on Wired.com.