To Stay or To Go: A Dilemma

By: Thao Nguyen

“Should I stay or should I go now? If I go there will be trouble. And if I stay it will be double” – The Clash

“Should I stay or should I go?”, we’ve all had that one question that makes us feel like pulling our hair out in the midst of frustration; be it leaving our family, our loved ones, our career, etc. It might even be a life-changing decision that determines your survival if you’re a bacterium. Motility in rhizobia is thought to be critical for the establishment of symbiosis with host plants, but also is the formation of biofilms. This post’s main subject will be the Sinorhizobium meliloti’s mechanism that drives biofilm formation and its importance in the symbiotic relationship with the host plant, but the link between motility and biofilm formation of this bacterium will also be called into discussion.

Symbiosis between the S. meliloti and Medicago sativa

S. meliloti is a soil bacterium that lives on the roots of many plants, one of which is Medicago sativa (alfalfa). Each can grow separately from each other, but they choose to form a symbiotic relationship with each other. Why, you might ask. It turns out that S. meliloti is capabel of fixing nitrogen in the atmosphere into a form that the plants can use for growth. Upon its entrance to the root tissues of the plants via infection threads, the bacterium forms nodules on the alfalfa roots, in which it can begin fixing nitrogen for the plant to produce essential proteins (Fig. 1). In return, the plants provide the bacteria with an ample carbon and energy source.

Figure 1. Wild-type Sinorhizobium meliloti nodules on its host Medicago sativa (alfalfa). Source

In order to establish the symbiotic relationships with legumes, S. meliloti produces two types of rhizobial exopolysaccharides, succinoglycan and EPS II. Both are secreted into two major fractions relecting different degrees of subunit polymerization: high molecular weight (HMW), and low molecular weight (LMW). LMW succinoglycan is essential for nodule-invasion, and also is LMW EPS II in succinoglycan-deficient strains. The production of both succinoglycan and EPS II is activated by the ExpR/Sin quorum-sensing system.

Biofilm formation by S. meliloti

In addition to facilitating nodule invasion, exopolysaccharides and other surface components (pili, lipopolysaccharides) may also play important roles in bacterial biofilm formation. A biofilm is a group of bacterial cells attaching to each other on the surface, all embedded within a self-produced matrix of extracellular polymeric substance (slime). Exopolysaccharides enclosing the bacterial biofilms are thought to have many benefits, such as protecting the bacteria against environmental stress, determining host specificity, participating in the early steps of plant infection, such as adhesion of bacteria to the roots, inducing infection thread formation, modulating the plant defense responses, and acting as a plant developmental signal molecule.

As mentioned above, the S. meliloti ExpR/Sin quorum-sensing system is responsible for the production of exopolysaccharides essential for nodule invasion. Rinaudi and Gonzalez (2009) have also investigated the importance of this system in biofilm formation by using two methods: a microtiter plate assay to quantify biofilm formation and a glass chamber assay to study the different stages of biofilm structure by confocal laser scanning microscopy (CLSM). They also used fluorescence microscopy to evaluate biofilm formation directly on alfalfa roots.

Figure 2. Quantification of biofilm formation (bars) and bacterial growth (squares) of S. meliloti. Source

Wild type S. meliloti strains with an intact ExpR/Sin system are capable of producing both succinoglycan and EPS II. The expR mutant, however, does not produce EPS II due to a insertion mutation in expR, a gene responsible for encoding ExpR regulator of EPS II synthesis. By using microtiter plate assay, they showed that the wild-type strain produced larger mass of biofilm than the expR mutant, but not the expR (pSWExpR) strain, in which the pSWExpR plasmid was transferred to the expR mutant to restore the expR gene function. This suggests that the ExpR quorum-sensing regulator plays a role in biofilm formation (Fig. 2). Similarly, the sinI strain with a defect in the gene producing signaling molecules involved in quorum-sensing also showed a reduction in biofilm formation (Fig. 2). This indicates that an intact quorum-sensing system is required for maximal biofilm formation.

Figure 4. Images of S. meliloti strains on the root hairs
and main root of alfalfa at 2 dpi. Panel A (wild-type) and
B (exoY) show confluent biofilms that covered the entire
surface of the root, including the root hairs, where the
initial symbiotic interaction takes place. Source

Succinoglycan does not play a major role in this process, as the exoY mutant which inhibits succinoglycan synthesis showed no reduction in biofilm formation (Fig. 2). However, the expA mutant whose EPS II production was blocked showed a decrease in biofilm development. The exoY expA strain which eliminates succinoglycan production in the expA strain showed no further decrease in biofilm formation (Fig. 2). Thus, EPS II (but not succinoglycan) is crucial in S. meliloti for the formation of biofilms. More specifically, the LMW fraction of EPS II is required for biofilm formation by S. meliloti, as the mutant that still producing EPS II but lacking the LMW fraction showed a significant decrease in biofilm formation compared to that of the wild-type.

Using the same microtiter plate assay technique, they also showed that extracellular complementation of quorum sensing is sufficient to restore biofilm formations to wild-type level. The same result was achieved for extracellular complementation of EPS II production. EPS II-producing strains were also shown to develop more structured and highly organized biofilms, and colonize root surfaces more efficiently (Fig. 4). All in all, in order to develop an efficient symbiosis with alfalfa, S. meliloti must have either an intact quorum-sensing system that is capable of producing LMW EPS II, or extracellular complementation of quorum sensing or EPS II production.

So stay or go?

The role of biofilms in a successful symbiosis is still under question, but the fact that biofilms protect bacteria from stress and host responses and to meditate attachment could impact the overall fitness of rhizobia in the soil and in the rhizosphere microenvironments and therefore contribute to the symbiotic process. Now that we know only the LMW fraction of EPS II is required for biofilm formation in S. meliloti, another question regarding the importance of the HMW fraction of EPS II for the bacterium might arise.  Another study has shown that the HMW EPS II (but not the LMW fraction) facilitates the initial stages of swarming in S. meliloti. This supports the hypothesis that bacteria have to select between motility, such as swarming, and biofilm formation at certain stages. But how exactly do bacteria know when to switch from sessile to motile lifestyle and vice versa? Identification of crucial environmental and intracellular signals and the regulatory mechanisms that lead to sessile or motile bacterial behavior are still under research. It seems like there are still many things we have to learn from bacteria regarding this “to stay or to go” dilemma. 

References

Rinaudi L V. and Gonzalez J. E. 2009. The Low-Molecular-Weight Fraction of Exopolysaccharide II from Sinorhizobium meliloti is a crucial determinant of Biofilm Formation. Journal of Bacteriology 191: 7216-7224