Warfare, competition, survival of the fittest. We hear these terms often, but usually in relation to humans. However, competition exists for all living organisms, even bacteria. Let’s see how the plant pathogen, Agrobacterium tumefaciens, and one of the most well-known bacteria, Escherichia coli, engage in a primal war for survival.
But first some background. A. tumefaciens is a Gram-negative, rod-shaped soil bacterium. It is the causative agent of crown gall disease in plants. The bacteria’s parasitization of plant tissue and its use for genetically engineering plants are well studied, but less is known of the harm it can levy onto other bacteria in the environment. E. coli is also a Gram-negative, rod-shaped bacteria usually found in the lower intestine of warm-blooded organisms, but can also be found in soil. While it is mostly harmless, some strains are notorious for causing severe stomach pain, diarrhea and vomiting.
But how do these two bacterial species engage in a battle with each other? A recent study by Yu et al. sheds some light on this. Many Gram-negative bacteria make use of a powerful antibacterial weapon known as the type VI secretion system (T6SS). T6SS delivers effectors to target cells and the surrounding environment. Effectors are proteins secreted by pathogenic bacteria, usually for the purpose of invading host cells. A. tumefaciens employs T6SS to engage in interbacterial competition. Through a combination of multiple effectors, A. tumefaciens tries to gain a competitive advantage. Responsiveness to changing environmental cues, and attacking cells in the vicinity is what allows A. tumefaciens to survive.
Three effectors from the T6SS system help A. tumefaciens in the quest for survival: Tde1, Tde2 and Tae. Tde1 and Tde2 are nucleases, and key players in competition between bacteria when environmental conditions are not ideal. They trigger cell elongation and DNA degradation in the target cell. Tde effectors specifically display a dominant killing effect when the environment has low carbon sources. On the other hand, Tae is a highly conserved peptidoglycan amidase effector that cleaves the peptidoglycan (PG) bonds of a target’s cell wall. It leads to cell enlargement but not DNA damage. This combination of Tde1, Tde2 and Tae improves A. tumefaciens chances of survival in an ever-changing environment.
To examine this microbial warfare between A. tumefaciens and E. coli more closely, the researchers dove deep into the plan of attack by A. tumefaciens. It was found that E. coli’s T6SS-dependent susceptibility can be increased through two major conditions: carbon starvation and peptidoglycan disturbance. To set the stage, researchers looked to a previous study that has shown that antimicrobial activities of A. tumefaciens can only be observed in planta but not on the in vitro agar plates. This suggested that the T6SS activity of A. tumefaciens is dependent on certain environmental signals. After a series of experiments, the Murashige and Skoog agar medium, a plant culture medium that does not contain any carbon source, was found to be the environment that most optimized the T6SS activity for A. tumefaciens. It has been concluded that the lack of carbon in the agar mimics the acidic environment that is common in the apoplast of plants and is a more competitive environment for all the microbes. Therefore, under these carbon-poor conditions, A. tumenfaciens is more likely to induce the killing actions of T6SS to gain a competitive advantage.
Additionally, the researchers also discovered that when E. coli (and potentially other recipient microbes) are induced to overexpress certain genes that control cell wall synthesis, they are more susceptible to the killing actions of T6SS. To confirm this observation, researchers experimented with E. coli cultures with disturbances to the cell wall through cleavage of PG bonds. This alteration to cell wall structure makes them vulnerable to the outside environment. Thus, E. coli became more susceptible to Tae effectors and displayed phenotypical cell elongation as a result.
Recall that A. tumefaciens can secrete different effectors to maintain its competitiveness within a mixed bacterial population. Now let us take a look at the opposing side of this bacterial warfare. How do the recipient cells react to the effectors? Lucky for us, we can actually observe the morphological change on recipient cells with the help of a microscope!
The researchers from this study conducted an experiment that featured three effectors secreted by A. tumefaciens, Tde1, Tde2, and Tae. Each A. tumefaciens strain had the choice of containing functional Tde1, Tde2, Tae, non-functional Tde1 (Tde1M) or non-functional Tde1 (TaeMX). Each strain contained a mix-and-match of these different effectors. These different flavors of A. tumefaciens were subsequently co-inoculated with recipient E. coli cells at a 9:1 ratio. After growing on 523 agar plates for 3 hours at 28 ℃, the recipient cells were observed with fluorescence microscopy. Since the recipient E. coli cells can express green fluorescent proteins, we can see the shape and size of these recipient cells easily. If we look at group A in Figure 3, E. coli cells that have not received any effectors retained their normal short rod shape. When E. coli cells received all three functional effectors (see group b), the cells became long and filamentous. However, when the E. coli cell received functional Tde1, Tde2 and non-functional Tae (see group c), the cells were still long and filamentous. By comparing group b and c, it appears that while Tde1 and Tde2 effectors can significantly increase E. coli cell size, the Tae effector can only result in a slight increase in cell size. This was further supported by the fact that the E. coli cell increased in size ever so slightly when it received functional Tae and Tde1M (see group d).
Figure 3. The effect on the morphology of E. coli cells after being incubated with different effectors secreted by the A. tumefaciens C58 strain. Tde1, Tde2, and Tae are functional effectors, where Tde1M is a non-functional Tde1 effector, and TaeMX is a non-functional Tae effector. E. coli recipient cells are marked by green fluorescent proteins. Image Source.
Figure 2. Crown gall on an oak tree caused by A. tumefaciens. Source.
Three effectors from the T6SS system help A. tumefaciens in the quest for survival: Tde1, Tde2 and Tae. Tde1 and Tde2 are nucleases, and key players in competition between bacteria when environmental conditions are not ideal. They trigger cell elongation and DNA degradation in the target cell. Tde effectors specifically display a dominant killing effect when the environment has low carbon sources. On the other hand, Tae is a highly conserved peptidoglycan amidase effector that cleaves the peptidoglycan (PG) bonds of a target’s cell wall. It leads to cell enlargement but not DNA damage. This combination of Tde1, Tde2 and Tae improves A. tumefaciens chances of survival in an ever-changing environment.
To examine this microbial warfare between A. tumefaciens and E. coli more closely, the researchers dove deep into the plan of attack by A. tumefaciens. It was found that E. coli’s T6SS-dependent susceptibility can be increased through two major conditions: carbon starvation and peptidoglycan disturbance. To set the stage, researchers looked to a previous study that has shown that antimicrobial activities of A. tumefaciens can only be observed in planta but not on the in vitro agar plates. This suggested that the T6SS activity of A. tumefaciens is dependent on certain environmental signals. After a series of experiments, the Murashige and Skoog agar medium, a plant culture medium that does not contain any carbon source, was found to be the environment that most optimized the T6SS activity for A. tumefaciens. It has been concluded that the lack of carbon in the agar mimics the acidic environment that is common in the apoplast of plants and is a more competitive environment for all the microbes. Therefore, under these carbon-poor conditions, A. tumenfaciens is more likely to induce the killing actions of T6SS to gain a competitive advantage.
Additionally, the researchers also discovered that when E. coli (and potentially other recipient microbes) are induced to overexpress certain genes that control cell wall synthesis, they are more susceptible to the killing actions of T6SS. To confirm this observation, researchers experimented with E. coli cultures with disturbances to the cell wall through cleavage of PG bonds. This alteration to cell wall structure makes them vulnerable to the outside environment. Thus, E. coli became more susceptible to Tae effectors and displayed phenotypical cell elongation as a result.
Recall that A. tumefaciens can secrete different effectors to maintain its competitiveness within a mixed bacterial population. Now let us take a look at the opposing side of this bacterial warfare. How do the recipient cells react to the effectors? Lucky for us, we can actually observe the morphological change on recipient cells with the help of a microscope!
The researchers from this study conducted an experiment that featured three effectors secreted by A. tumefaciens, Tde1, Tde2, and Tae. Each A. tumefaciens strain had the choice of containing functional Tde1, Tde2, Tae, non-functional Tde1 (Tde1M) or non-functional Tde1 (TaeMX). Each strain contained a mix-and-match of these different effectors. These different flavors of A. tumefaciens were subsequently co-inoculated with recipient E. coli cells at a 9:1 ratio. After growing on 523 agar plates for 3 hours at 28 ℃, the recipient cells were observed with fluorescence microscopy. Since the recipient E. coli cells can express green fluorescent proteins, we can see the shape and size of these recipient cells easily. If we look at group A in Figure 3, E. coli cells that have not received any effectors retained their normal short rod shape. When E. coli cells received all three functional effectors (see group b), the cells became long and filamentous. However, when the E. coli cell received functional Tde1, Tde2 and non-functional Tae (see group c), the cells were still long and filamentous. By comparing group b and c, it appears that while Tde1 and Tde2 effectors can significantly increase E. coli cell size, the Tae effector can only result in a slight increase in cell size. This was further supported by the fact that the E. coli cell increased in size ever so slightly when it received functional Tae and Tde1M (see group d).
So the Tae effector can result in increased E. coli cell size. But how effective is it? To answer this question, the researchers took the E. coli cells from the co-inoculation experiment described above, normalized each sample to the same optical density such that each sample contained the same amount of cells, added in supplements to kill A. tumefaciens, and recovered the E. coli cells in Lysogeny Broth (LB) growth medium, a nutrient-rich medium that is beneficial for bacterial growth. E. coli cells receiving functional Tae effectors (∆2tdei*) took more time to recover to the same optic density than Wild-Type (WT) E. coli cells receiving no effectors (∆3Tls*). This is exciting because this piece of data shows that the Tae effector can repress E. coli recipient cell growth to give A. tumefaciens the upper hand when competing in the pool of bacteria for space and resources.
E. coli recipient cells were further recovered in optimized Agrobacterium kill-triggering medium supplemented with glucose (AKG medium). The AKG medium is optimized to induce a basal level of killing activity of A. tumefaciens, which in result inhibits the growth of E. coli. Interestingly, there was no change in E. coli cell density before and after 16 hours of recovery in AKG medium. However, E. coli cell density increased when it was recovered in LB medium under the same conditions. This result suggests that Tae is only effective in suppressing the growth of E. coli cells when it is in a growing state.
Before this study, it was thought that only the Tde effectors played a role in killing recipient cells. A better understanding of Tae’s role emerged when researchers showed that T6SS in A. tumefaciens have an enhanced ability to kill target cells in conditions of carbon starvation or target cell peptidoglycan disturbance. Tae, not Tde, is key in maintaining competitiveness when a target cell is growing. This is because Tae provides A. tumefaciens with a growth advantage depending on the growth status of target cells in a mixed population. However, effectors like Tae with less observable traits in laboratory conditions, may be understudied and overlooked, despite their importance in maintaining competitiveness in a mixed bacterial environment.
Future studies can focus on how a bacterial species like A. tumefaciens maintains an advantage over fast growing opponents like E. coli when nutrients are available. It would also be interesting to see how other bacterial species use conserved effectors from the T6SS system to defeat competing species when nutrients are abundant. Moreover, as Tde is produced under nutrient-poor conditions and Tae is produced under nutrient-rich conditions, how A. tumefaciens regulates their production and secretion is another area worth exploring. Is the T6SS system also able to suppress and kill species other than E. coli? Furthermore, what happens in an environment with multiple bacteria possessing a T6SS system? Who wins? Such studies can improve our understanding of T6SS in bacteria and contribute to the field of microbial ecology. The major impacts like improvement to crop health in agriculture, microbe usage in environmental restoration and measuring the effects of climate change are all possible. What this research can achieve is the basic scientific understanding of T6SS mechanisms that can eventually be translated into the applications mentioned above.But for now A. tumefaciens is the winner of “The Hunger Games” against E. coli, and it is the lethal combination of Tde and Tae that equips it to win.
About the authors:
Maha Mapara ‘21 is a Biology and Statistics major from Karachi, Pakistan. She is looking forward to starting grad school in the fall. She has been active in SGA and Residential Life during her time at Mount Holyoke.
About the authors:
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