Fruiting bodies also form during predation (see ). This is enigmatic since fruiting body formation has long been known to be inhibited by growth on nutrient rich media and induced when exposed to low or no nutrients (Dworkin, 1962
). The ability to construct large multicellular structures from populations of essentially independent individuals, has been a major focus of study on the M. xanthus
model system (Shimkets, 1999
). Fruiting bodies are aggregates that typically consist of ~106
cells, and in most species of Myxobacteria mature fruiting bodies contain cells that have differentiated into metabolically dormant spores (Lee et al., 2005
). In some species, spore-filled fruiting bodies are embedded within the biofilm matrix, in other species the fruiting bodies protrude up from the surface in a complex morphology consisting of stalks and appendages (Shimkets, 1999
). But a common trend is the separation of cell type that is demarked by the boundaries of the fruiting body structure. Cells within the fruiting body differentiate into spores, while cells outside of the fruiting body remain in the vegetative state.
Multicellular development during predation in M. xanthus
In the laboratory, fruiting body formation can be rapidly induced through plating cells with a combination of high cell density and low nutrient availability. This shift in nutrient conditions results in typically randomly distributed fruiting aggregates where cells rapidly differentiate into spores. During predation, however, fruiting bodies form in predictable patterns around the edges of a prey colony, such that fruiting bodies can be observed at the boundary between predatory rippling populations and non-rippling populations (see ) (Berleman and Kirby, 2007
). Non-random fruiting body formation was also observed during mono-culture analysis of rippling behavior (Welch and Kaiser, 2001
). In this case, fruiting bodies were also observed to form in a circle surrounding the rippling region. If rippling behavior is a result of predatory feeding on macromolecules from prey or lysed M. xanthus
sister cells, then detection of a sudden loss in macromolecule availability could stimulate a change in behavior that results in fruiting body aggregation. In support of this, fruiting bodies were induced by a step-down in prey availability, but not by a corresponding step-up (Berleman and Kirby, 2007
). This pattern was observed across a wide range of prey cell densities and basal nutrient levels, indicating that the decision to aggregate into fruiting bodies during predation results from relative changes in nutrient availability rather than a single, absolute starvation threshold. Additionally, relA
mutants, while unable to aggregate into fruiting bodies in mono-culture conditions (Cho and Zusman, 1999
; Harris et al., 1998
; Singer and Kaiser, 1995
), were shown to form fruiting bodies when co-incubated with prey. relA
code for proteins essential for producing intracellular ppGpp and extracellular A-signal, respectively. These signals are the earliest known required steps of fruiting body formation and sporulation in mono-culture conditions. Although fruiting body structures were formed, neither the relA
strain showed any significant differentiation into spores. Thus, during predation the multicellular fruiting structure can be stimulated by changes in extracellular nutrient availability, but the final conversion to spores requires sensation of an absolute starvation threshold and production of the appropriate cellular signals. Interestingly, while starvation has often been thought of as inducing a program of fruiting body formation and subsequent sporulation, it may be that in natural settings, induction of fruiting aggregates occurs in response to a relative decrease in prey and/or nutrient availability, followed later by an absolute starvation threshold that induces sporulation of cells within the aggregate. This could explain why relA
mutants remain competent for fruiting body formation in the presence of prey, yet are unable to sporulate.
The identification of rippling as a form of multicellular development utilized during predation leads to some exciting possibilities. There are several hypotheses that are worth considering. The first model to consider is that the rippling pattern observed provides no significant group benefit, that it only arises through the repetitive behavioral pattern elicited as each individual moves tactically in response to a similar prey stimulus (see “Autonomous Behavior Model” ). Synchronization of cells could result from a combination of factors including proximity to prey for nutrients, access to oxygen for respiration, and contact with the agar surface for movement. Integration of all these factors could lead to the emergence of a multicellular pattern, without providing any additional benefit to the group. Another model to consider is that the synchronized multicellular movement of M. xanthus cells may result in a physical disruption of prey biofilms that could not be accomplished by uncoordinated individuals (see “Grinder Model” ). If true, then non-rippling mutants should be inefficient predators, particularly in situations where prey are embedded in a sturdy biofilm.
A third model to consider is that M. xanthus cells utilize rippling to regulate cell density and cellular differentiation (See “Population Control Model”, ). It is important to consider that direct contact with prey macromolecules may be required in order to utilize this nutrient source. In other words, there might not be a substantial pool of diffusible nutrients as proposed by the original wolfpack model, but rather a limited number of nutrient-rich prey macromolecule access sites. If contact with prey is essential for nutrient access, then the rippling pattern may allow for a greater number of direct contacts with prey as a wave creates a greater surface area than a flat plane. As prey are consumed and the number of prey contact sites diminishes, individual cells may have to range farther for sufficient prey cell access. Although this change in range may be on the order of a few microns of extra movement between reversals, this can be observed at the population level as the distance between rippling waves of M. xanthus cells increases over time during predation. This change in ripple spacing effectively decreases the local cell density of M. xanthus and forces the excess M. xanthus cells out and away from the remaining available prey. Cells that are forced out of macromolecule-rich areas aggregate into fruiting bodies, where prolonged nutrient depletion will result in sporulation. Thus, the population is segregated into rippling cells that maximize growth, and aggregating cells that maximize survival by differentiating to spores, rather than a single population that promotes growth unchecked until nutrient exhaustion.
As with any social process, it is possible that M. xanthus
predation is susceptible to the presence of cheater sub-populations that reap nutrients without a corresponding cooperative contribution to the group (Velicer, 2009
). This could occur, for instance, in mutants deficient in exo-enzyme production, that expend less energy but benefit from the exo-enzymes of neighboring cells. It will be interesting to see if the complex behavior and development of M. xanthus
cells during predation provides a mechanism for insulating the population from cheater phenotypes. Further experiments will be required to distinguish between group and individualistic tendencies in predatory M. xanthus