Bdellovibrio bacteriovorus: King of the (Micro)biome

By: Stella Grill-Dubois '19

When you think of a predator, what comes to mind? A fierce lion, swiftly chasing a zebra across the savanna? A hawk, snatching rodents and snakes from the ground and taking flight again? Or perhaps even a great white shark, swimming proudly at the top of the oceanic food chain?

See the resemblance? (On the left is Bdellovibrio bacteriovorus, and on the right are two lions.) Source and source.

Having sat through dozens of elementary school nature documentaries, we all understand that some animals attack and kill prey to ensure a continuous food supply. What those nature documentaries leave out is the constant warfare being conducted all around us, from the soil to the riverbed, and even inside us. Bacterial predation involves species so small that we don’t even notice them - and yet their methods of attack are incredibly sophisticated.

Bdellovibrio bacteriovorus is a model predator for microbiologists, which means they study it in order to understand other predatory microbes and bacterial predation in general. Interestingly, it has two very distinct life phases, where its physiology and behavior are very different. In its attack phase (AP), B. bacteriovorus uses a high-powered flagellum to swim through water or soil in search of prey. The jury is still out on how it recognizes prey cells, which are other bacteria, much larger than B. bacteriovorus itself. When B. bacteriovorus collides with a prey cell, a pilus, which is an appendage the predator extends, mediates membrane contact between the two cells. After that, proteins within B. bacteriovorus’ cell wall act as enzymes, modifying the prey’s cell wall to create an opening large enough for it to slip inside. You can see how this brutal invasion looks in real life in the image below.

A false color micrograph of B. bacteriovorus (top) attacking a larger soil bacterium (bottom). It is in the process of reshaping the prey’s cell wall so it can gain entry. Source.

Once safely in the periplasm of a prey cell, that is, between its inner and outer cell membrane, B. bacteriovorus enters the intraperiplasmic growth phase (IP) of its life cycle, shown in the bottom part of the below diagram. In this stage, the predator-in-prey combination is referred to as a bdelloplast. The predator stops cellular respiration in the prey and remodels the prey cell, killing it. It uses the macromolecules (carbohydrates, lipids, proteins, and nucleic acids) inside the prey to fuel its growth and eventual division - which takes place right inside the prey cell! Because B. bacteriovorus lacks the metabolic pathways to synthesize and break down many amino acids, it is dependent on external sources for them, which the prey can provide. Finally, having emptied the prey of all useful substance, the prey cell membrane is broken and mature attack-phase B. bacteriovorus cells are released to locate prey cells of their own. (This part is referred to as “lysis” in the below diagram.) 

The life cycle of B. bacteriovorus. Note the obvious differences between the attack phase and the (intraperiplasmic) growth phase. Source.

But how is it that B. bacteriovorus can switch its activities so drastically when it enters a prey cell, transforming from a fearless hunter to a hungry (and carnivorous) caterpillar? That is precisely what Dwidar and his team investigated and published in their 2017 paper, “Attack-Phase Bdellovibrio bacteriovorus Responses to Extracellular Nutrients Are Analogous to Those Seen During Late Intraperiplasmic Growth.” Based on prior research, they understood that B. bacteriovorus secretes proteases during intraperiplasmic growth, and that a host-independent strain (HIB) growing alone in nutrient media does the same. (The HIB strain is a mutant strain that is not reliant on its prey for growth. You can read about its discovery here.) They wanted to learn more about how wild type attack-phase B. bacteriovorus (not the HIB strain) behaves growing alone in nutrient media, and specifically whether it would also secrete proteases in that scenario. Although this laboratory condition may seem irrelevant to the microbe’s activities in nature, this research helps us understand the molecular processes that switch B. bacteriovorus from attack phase to intraperiplasmic growth phase. Essentially, Dwidar’s team simulated certain conditions of the intraperiplasmic growth phase, and observed responses in attack-phase B. bacteriovorus that are similar to typical physiology of the intraperiplasmic growth phase bacteria.

Dwidar’s team hypothesized that either protease secretion would only occur in HIB mutants growing alone or attack phase B. bacteriovorus growing in a nutrient-rich environment would also secrete proteases. In the end, their data showed that the wild type bacteria does secrete proteases in nutrient media. Moreover, they found that B. bacteriovorus also secreted proteases in 1% peptone, a medium with only amino acids and small peptides. This suggests that it is the presence of amino acids that generates protease secretion, rather than the other nutrients in the nutrient media. Given this protease secretion, they surmised that attack-phase B. bacteriovorus cells do not solely synthesize proteins using their amino acid stockpiles, as previous research had suggested. Rather, they have the ability to break down external proteins using proteases and use them to fuel growth.

Building on that result, the researchers analyzed changes in the transcriptome of attack-phase B. bacteriovorus in nutrient media. (The transcriptome tells scientists which genes in the cell are being transcribed at a given point in time.) They noted that transcription of protease genes increased under these conditions to levels similar to intraperiplasmic growth phase levels. Thus, they concluded that the breakdown of proteins inside the prey likely helps provoke a massive transcriptional change, leading to protein synthesis and growth. (These researchers did not actually confirm that a corresponding protein synthesis occurred, which would certainly help to validate their model.) However, although AP B. bacteriovorus elongated, it did not experience population growth under these conditions. This suggests that other signals from the prey are necessary to induce cell division.

Take a look at the results for yourself. Panel A shows B. bacteriovorus protease secretion over time. HEPES is a buffer, not a nutrient medium, so you can see very little change in protease secretion over time when B. bacteriovorus was grown in it. On the other hand, you can see that protease secretion significantly increases when Dwidar’s team grew B. bacteriovorus in 1X NB and 5X NB, which are concentrated nutrient broths. This effect is particularly pronounced in 5X NB. The differential protease activity started to become apparent after six hours in the buffer or nutrient broth, suggesting that if and only if nutrients are present, they trigger biological cascades that, with time, promote protease secretion. 

B. bacteriovorus protease secretion over time. Each color represents the solution in which the microbe was cultured. In a strong nutrient media (marked 5X NB in the legend), B. bacteriovorus secretes high levels of proteases. Source.

Moreover, panel C shows that after 24 hours, protease activity increased not only in nutrient broth, but also in 1% peptone, which consists only of protein building blocks. This suggests that it is the protein component of the nutrient broth that provokes increased protease activity. (This makes sense, since proteases are necessary to break down proteins.) As these are simulated intraperiplasmic conditions, this implies that proteins within the prey cell’s periplasm may be part of what generates the massive regulatory change that switches B. bacteriovorus from the attack phase to the intraperiplasmic growth phase. 

Protease secretion is significantly greater in both nutrient broth and 1% peptone, which contain proteins, than in HEPES buffer. Source.

Excitingly, nutrient broth and 1% peptone stimulate growth in attack-phase B. bacteriovorus, normally reserved for intraperiplasmic growth phase. You can see this visually in the Panel E. On the left are images of B. bacteriovorus grown in HEPES buffer; on the right is a sample grown in concentrated nutrient broth. On average, the bacteria incubated in nutrient broth grew to lengths almost double those of the bacteria incubated in buffer! (You can see the numerical data here.) 

Bdellovibrio becomes longer over time in nutrient broth (at right), but not in HEPES buffer (at left). Source.

Thus, it is clear that something about the nutrient composition inside the prey (likely the presence of proteins, based on the graphs we just discussed) triggers growth. This elongation shows that attack-phase B. bacteriovorus does not only use the nutrients in nutrient broth for protease production, but also uses them to fuel the processes necessary for growth.

But proteins can’t be the end of the story, because of what you can see in panel B. This shows the number of bacteria in the buffer and nutrient broths of different strengths initially and then after 24 hours of growth. Under all conditions, there was virtually no change in number of bacteria over time. 

The number of B. bacteriovorus in the population studied stayed constant over time regardless of whether it grew in HEPES buffer or nutrient broth. Source.

Yet, if you consider the life cycle we discussed earlier in this article, you will recall that B. bacteriovorus uses prey cell components and molecules to fuel growth and division. Thus, although proteins are partially what trigger intraperiplasmic growth, and they are sufficient to fuel that growth, other conditions in the interior of a prey cell are necessary for B. bacteriovorus to divide. Scientists are not yet sure what conditions provoke cell division, but it is an interesting direction for further research.

Dwidar and his team also analyzed what genes were transcribed in attack-phase B. bacteriovorus growing in nutrient media. They wanted to find out how the increase in protease secretion occurred, and what other genetic changes came with it. They found that incubating attack-phase B. bacteriovorus in nutrient media drastically changed which genes were transcribed. In fact, transcription of over 60% of B. bacteriovorus genes increased! Dwidar’s team compared these changes with some of the transcriptome changes other studies have reported in intraperiplasmic growth phase B. bacteriovorus, and results were similar but not identical. For example, in nutrient media attack-phase B. bacteriovorus still transcribes the genes used for chemotaxis, which it uses to track down prey. When B. bacteriovorus is actually inside a prey cell, those genes are turned off, because seeking prey is no longer necessary.

So what does this all mean, and why should you care? Well, aside from learning more about a unique method of growth and development, B. bacteriovorus and other bacterial predators have the potential to be useful to humans. Maybe you’ve heard that some pathogens are developing resistance to antibiotics. Scientists are working to find alternate ways to defeat resistant bacteria. One of them is to release bacterial predators into humans infected by resistant pathogens, so that they can attack and kill those pathogens. It may sound counterintuitive to infect humans with another bacterium in order to cure them, but results from in vitro and animal studies have been promising. (If you’re interested in learning more about this research, check out this article from NPR.) Of course, it gets more complicated than that, but understanding predators such as B. bacteriovorus could help us kill antibiotic-resistant bacteria that cause pneumonia and other diseases. In order to figure out how to make B. bacteriovorus work for us, and to mitigate risks associated with treating humans using bacteria, we first need to know how B. bacteriovorus works. This research is another step toward unmasking the mystery of this tiny, but potent, killer.

3-dimensional rendition of B. bacteriovorus. We have a lot left to learn before we develop effective B. bacteriovorus treatments for human illness! Source.

Sources: 

Chatterjee, A. (2009). Bdellovibrio bacteriovorus: Life cycle and potential as a predatory renaissance. Advanced Biotech 8, 27-29.

Dwidar, M., Im, H., Seo, J.K. and Mitchell, R.J. (2017). Attack-phase Bdellovibrio bacteriovorus responses to extracellular nutrients are analogous to those seen during late intraperiplasmic growth. Microbial Ecology 74: 4, 937-946.

Nuñez, M., Martin, O.M., Duong, L.K., Ly, E. and Spain, E.M. (2003). Investigations into the Life Cycle of the Bacterial Predator Bdellovibrio bacteriovorus 109J at an Interface by Atomic Force Microscopy. Biophysical Journal 84: 5, 3379-3388.


About the Author:


Stella Grill-DuBois ‘19 is a history major and a biology minor. She plans to work in eating disorder treatment after graduation and eventually become an eating disorder dietitian. She hopes to help transform the field of nutrition into one that supports nourishment and a positive relationship with food, rather than restriction, for people in all bodies. Her favorite place on Mount Holyoke’s campus is the path around Upper Lake.