Self-organization of bacterial populations

Fluorescent bacteria in glass microchannels

In many environments, hundreds, sometimes thousands of different microbial species coexist as mixtures of cells of various sizes and shapes. For example, each human being teems with their very own and unique microbial mixture (trillions of cells), as the human microbiome project unveiled a few years ago. Microbial diversity in soil is equally – possibly even more – astounding, with a gram of rich soil capable to host thousands to millions of distinct bacterial and archaeal species, as well as hundreds of fungal and protistan species. A recent study in the magazine Nature highlights the results of the earth microbiome project, which aims at revealing the extent of  microbial diversity on our planet.


Yet, despite this wealth of diversity, microbial communities are not simply soups of species – there is order hiding behind this curtain of complexity. Actually, the more we look, the more we find patterns of microbial organization in the natural world. Some patterns are obvious and have been known for a long time, such as the distinct layers of microorganisms visible with the naked eye in sections of microbial mats. The vast majority of patterns, however, reveal themselves only at the scale of individual microbes, that is, at the microscopic scale. See, for example, the beautiful arrangements revealed in  the lab of Jessica Mark Welch at the University of Chicago. 

Microbial populations are thus often spatially organized at small scale, and in a very defined and refined way. But how does it work? Obviously, microbes don't organize following some kind of blueprint that is imposed on the community!... But what then? Part of the answer seems to reside in so-called self-organization processes. With such processes, patterns emerge from the individual behavior of cells that can only sense the conditions in their local environment and react accordingly. This in appearance simple process at the individual level can lead to seemingly complex patterns of organization at larger scale. Think of bird murmuration, or of how ants can form bridges with their own bodies! I also discussed spatial patterns of bacterial organization triggered by metabolic cooperation in a previous post.

In a recent paper whose lead author is my colleague Benedict Borer, we examined some of the basic processes that can lead to bacterial spatial self-organization. (In that study we were specifically interested in pore networks that mimic the spatial structure of soil aggregates, but the processes that matter here are valid in other kinds of environments as well.) The idea is as follows. In a given habitat, bacterial populations with distinct metabolic capabilities and food preferences would spontaneously arrange in space in order to optimize their use of the available resources. We thought there must be two necessary conditions for that. First, that the bacterial cells have some level of motility (which could be flagellar motility, such as swimming, or simply movement provide by growth and cell division). Second, that gradients of carbon and nutrients are present in the habitat (i.e. there must be some spatial heterogeneity for the microbes to respond to).  


We chose to work with two bacterial species that differ on their ability to respire: one is a strict aerobe (Pseudomonas putida), which only respires using oxygen as final electron acceptor, while the other (Pseudomonas veronii) is a facultative anaerobe that can respire with oxygen but also with nitrate as final electron acceptor. Both species are motile thanks to flagella that they can use to swim within liquid films. To facilitate observation, the two species were each tagged with a distinct fluorescent protein (green or red). The two bacterial populations were inoculated into micrometric pore networks with various structures,  and which allowed us to create gradients of oxygen (coming from the periphery of the network) and of carbon source (citrate, coming from the center of the network). Although initially well-mixed in the center of the network, after a week of incubation the two bacterial species segregated in the network to form two distinct and coexisting populations (see figure below). One grew preferentially where the oxygen was more abundant (the obligate aerobe), while the other could occupy the anoxic niche at the center of the network, which also contained more carbon.
 

The two bacterial populations, initially well-mixed, grow and segregate in the pore network as function of their respiration metabolism. From Borer et al., 2018

Interestingly, this spatial organization did not occur when carbon source and oxygen were provided together (i.e., there was no counter-gradients of oxygen and carbon). Another intriguing result was that the coexistence of the two species, as seen in the figure, could not been achieved in well-mixed liquid environments (vials or flasks), because one of the species would always win over and dominate the community (see below). This illustrates how the habitat spatial structure can help limit competition and maintain species coexistence.

Experimental and modeling results show competitive exclusion in homogeneous cultures (a) and coexistence in structured pore networks (b). The percentage corresponds to the connectivity in the network (100% is highest connectivity).

Admittedly, our system is artificial and our community only composed of two species... Yet,  the basic mechanisms that we observed most probably also apply to more complex environments and could participate to the final structure of the community. Bacteria have a remarkable faculty of optimizing their distribution and activity in complex habitats, which we could demonstrate with our simple mathematical and experimental models.
 

Cooperation shapes the spatial patterns of bacterial organization

Cooperative bacterial strains colonizing a surface

Bacteria colonize surfaces in all environments. That could be the surfaces of soil aggregates, of rocks in a stream bed, of plant leaves, of animal skin, or that could be the surface of your showerhead... On such surfaces microbes establish complex communities ('biofilms') that can contain many different interacting species. These various species are usually not randomly distributed in the biofilm, but rather organized depending on their environmental preferences (for example some like well-aerated areas, others not so much...) and on the type of interactions that they have with each other. This can result in complex patterns of organization that manifest at the microscopic scale and up to the millimeter scale. Such patterns are not trivial, as they can sustain microbial activity and functions that would not be possible in a well-mixed environment, which has importance for biotechnology applications as well.

In a new study published this month, we examined the role of cooperation in shaping spatial patterns of bacterial organization on wet surfaces. The paper is available online and is entitled 'Cooperation in carbon source degradation shapes spatial self-organization of microbial consortia on hydrated surfaces'. Our idea was that a feeding dependency between two partners would directly control their distribution in space, hence imposing a specific pattern. We used a simple model system made of two bacterial strains that could grow using the chemical compound toluene (a hydrocarbon), but only when they were working together as a 'team' (a bacterial consortium in the jargon).

Symplasmata: a curious case of multicellularity in bacteria

Cells in a symplasmatum and surrounded by a capsule,
seen with transmission electron microscopy.

'Curiouser and curiouser’, famously said Lewis Carrol’s Alice, as she was experiencing some very peculiar events in Wonderland. I have sometimes felt like Alice when I was studying the curious behavior of the bacterium Pantoea agglomerans [1], during my time in the Lab Leveau at UC Davis.

At first sight, Pantoea agglomerans looks quite ordinary. It grows as rods a few micrometers long, it can swim with flagella and it feeds on all sorts of sugars. It belongs to the family Enterobacteriaceae, and thus it is a distant cousin of E. coli. You can find P. agglomerans in all sorts of environments, but it is particularly good at colonizing the surface of plants, and in certain cases it competes with pathogens and thus keeps its plant host healthy (that is, it can serve as a biocontrol agent). Because it is a very good leaf colonizer, we have used it in many studies of bacterial life in the ‘phyllosphere’ (the aerial surfaces of plants), such as the one described in this previous post. 

Now here’s what special, and actually seemingly unique, about Pantoea bacteria. Under certain conditions, instead of dividing and spreading as individual cells, the bacteria stay close together and form an aggregate containing up to hundreds of tightly packed cells. Aggregation is not uncommon in bacteria but, in the case of Pantoea, cells are constrained by a fibrillar layer, and surrounded by a thick capsule made of polysaccharides, which indicates some level of cooperation and resources sharing (see image on top of the post). The resulting sausage-shaped structures are called symplasmata [2]. Interestingly, the species name 'agglomerans' (forming into a ball), which was coined by the great Dutch microbiologist and botanist Martinus Beijerinck in a paper dating from 1888, probably refers to the species' ability to form symplasmata [3]. Although symplasmata have been known for a very long time, their importance and function in the environment is still a mystery. We have observed symplasmata on bean leaf surfaces, and others have described them attached to the roots of rice plants (Achouak et al., 1994). What is their ecological role? Does it benefit the plant as well? We do not know yet. 

Modelling the bacterial colonization of leaves

Photo courtesy of Jan Tech

Our world is a quite green world: a sea of trees, bushes, grasses, or, if you happen to live in the Midwest, corn fields… What is less obvious, though, is the fact that this green vastness harbors a huge community of microbes. Yeasts and filamentous fungi are often found on plant surfaces, but the most numerous inhabitants are first and foremost bacteria. Indeed, a centimeter square of leaf can contain as many as 10 millions of them! No worries, most of them are harmless to us or their plant hosts. On the contrary, many are required to maintain a healthy plant environment, by stimulating plant growth or by preventing the plant colonization by pathogens (they compete for the same space and the same resources). 

Because plants are so vital to us (think food, raw materials, landscapes, etc.), there is a real interest in understanding what the microbial contribution to the plant ecosystem is. One lingering question, for instance, is how bacteria colonize the surface of leaves (what we call the phyllosphere). What we do know is that bacteria on leaf surfaces appear as clusters of cells, rather than an even layer of bacteria covering the surface; the mechanisms that lead to this colonization pattern, however, is not well understood. I have already written about this question in a previous post that dealt with the use of bacterial bioreporters. Another way to explore these mechanisms of cluster formation is computer-based modelling, which enables us to test different scenarios and compare it with what has been observed on real plants. 

Indole teaches persistence to bacteria

Indole molecule (C8H7N). Source: wikimedia commons

When a bacterial infection is treated with antibiotics, bacteria that are in a so-called dormant, inactive state may escape death – this because antibiotics only kill growing bacteria. It becomes a serious problem when these sleeping beauties start to grow again, in particular when they do so after the period of antibiotic treatment has ended… Thus, an infection that was apparently cured could be followed by a secondary infection days or weeks later. This problematic phenomenon is called bacterial persistence, and it should not be confused with bacterial resistance, in which growing bacteria are immune to one or several antibiotics.

Now what about indole? (The molecule displayed on top of this post.) Actually indole is present in very common and important biomolecules, such as the amino acid tryptophan, the animal hormone serotonin and the plant growth hormone auxin. We have known for more than a century that E. coli produces indole in stationary phase (Lee, 2010), and it does so thanks to an enzyme called tryptophanase, which cleaves tryptophane into indole, pyruvate and ammonia. 

But E. coli is not the only bacterium capable of that: more than 85 species (both Gram-negative and Gram-positive) can synthesize indole (Lee, 2010).  For a long time the biological functions of indole were overlooked, but now we know that indole can act as an extracellular signal and can for instance increase antibiotic resistance and control biofilm formation in E. coli.

Science publishes two reports that contradict the 'arsenic life' story

This is a short follow-up post about the bacteria from Mono lake (California) that allegedly incorporate arsenic instead of phosphorus in their DNA. 

The journal Science just published two papers on the topic. The first report is from Rosie Redfield's lab in Canada, the second from Julia Vorholt's lab in Switzerland. Together, they claim that the arsenic bacteria in fact must have some phosphate to grow, and that arsenic is not incorporated into DNA—thus refuting the main conclusions of the original arsenic bacteria paper.

I only read the abstracts so far, but at first sight it seems to bury the arsenic story for good... It's not that often that we see researchers repeating someone's experiment to challenge it, so Redfield and Vorholt should be praised for the effort. 

The invention of multicellularity

Multicellular yeast cluster. Photo courtesy of William Ratcliff.

In January, William Ratcliff and his colleagues from the University of Minnesota caused quite a stir with their study on the experimental evolution of multicellularity in yeast, published in PNAS. Many media covered the story, including the New York Times, Wired and Scientific American. Briefly, what they did was using artificial selection on unicellular yeast (the baker's yeast Saccharomyces cerevisiae) in order to create an obligate multicellular organism after many generations of selection. What is impressive is that it worked pretty well in all their different test cultures!

Multicellularity was invented several times during the history of life (Rokas, 2008), but since it happened a long time ago it is difficult to reconstruct the exact sequence of events. Experimentation on today's unicellular organisms, however, allows researchers to test mechanisms (and associated mutations) that could lead to a multicellular lifestyle. Of course, this cannot decide for good how the phenomenon occurred many millions years ago – which is not at all the author's claim – but this can prove that such mechanisms can occur, given that an appropriate selection pressure is present.

Reporter bacteria to monitor arsenic concentration in groundwater

Vials containing bioreporter bacteria. Photo courtesy of UFZ.

It is not widely known, but analytical measurements with bacterial bioreporters can 
compete with commercially available detection kits, inasmuch as the bioreporters' efficiency (sensitivity, detection limit, etc.) is often comparable - if not better - than the chemical-based systems. So why are bioreporters not more widespread? Well, the leap from the laboratory to the field is a difficult one, which demands a tight collaboration between fundamental research and engineering. In addition, economical and policy challenges need to be overcome. Today, most developed bioreporters have never left the lab.

But this may change, and a recent study by Konrad Siegfried et al. published in Environmental Science & Technology is a remarkable demonstration of the usefulness of reporter bacteria in the field. [Among the authors of this paper are three scientists with whom I had the privilege to work: Antonis Chatzinotas and Hauke Harms from the UFZ in Leipzig and Jan Roelof van der Meer from the University of Lausanne (HH directed my master thesis and JRvdM directed my PhD thesis).]

In this study, the authors used a strain of E. coli that produces bioluminescence when it is exposed to arsenite and arsenate (oxidized forms of arsenic). Bacteria detect the poison thanks to a specific activator protein which binds arsenic and triggers the expression of specific genes. The result is the production of a set of proteins, including the bacterial luciferase, which makes the bacteria glow. 

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology

Green and red fluorescent bacteria on the surface of a leaf

Our new publication is out there as an early view in Environmental Microbiology, and I shamelessly take this opportunity to write about it here!

Here's the story. The plant foliage is colonized by a crowd of microbes (with bacteria on the front line—up to 10⁸ bacteria per gram of leaf has been reported!). Some of them can be pathogenic, hence a threat, but most of them are harmless or even favorable inhabitants of the plant ecosystem.

Bacteria form large clusters (aggregates) of cells on the surface of leaves, but the mechanism of formation of such structures is not very well understood. What we want to know is how these bacteria grow and colonize this specific environment at the microscale, that is, at their own scale.

First, bacteria have to land on the leaf. Wind, rain, insects can all contribute to bring microbial visitors onto the leaf surface (what we call the phyllosphere), usually few cells at a time. Once on the leaf, these immigrants will grow at the expense of the plant sugars available on the leaf and, if the conditions are favorable, rapidly multiply to form clusters of up to thousands of cells. Do these clusters result from the random aggregation of cells or do they result from the reproduction of a single bacterium? The two mechanisms—that we call aggregative and replicative—are fundamentally different but not necessarily mutually exclusive. 

Thanks to fluorescence microscopy it is possible to visualize glowing bacteria on the surface of a leaf. One difficulty, however, is that we cannot follow the same microarea of leaf overtime… We are limited to a snapshot view, and so it's impossible to decide whether a given cluster was formed through aggregative or replicative behavior. For this reason we developed techniques that would enable us to deduce a posteriori what was the mechanism of formation. 

What news from the ‘arsenic life’ front?

Mono Lake, CA, photo by NASA

This story has already become sort of a case study. In December 2010, NASA held a press conference about its astrobiology research program and the discovery of a bacterium from Mono Lake (CA) allegedly capable of substituting phosphorus by arsenic in its DNA. The research was published in ScienceExpress and attracted a lot of attention from the media - mostly as a consequence of the ‘hype’ factor brought by NASA, which went as far as saying that “The definition of life has just expanded”. All life on Earth shares the same DNA, whose backbone is made of sugar and phosphate. If any organism could replace phosphate by arsenate, that indeed would be very surprising and exciting.