John J. Dennehy, Stephen T. Abedon, Paul E. Turner. HOST DENSITY IMPACTS RELATIVE FITNESS OF BACTERIOPHAGE Φ6 GENOTYPES IN STRUCTURED HABITATS. Evolution (OnlineEarly Articles). doi:10.1111/j.1558-5646.2007.00205.x
Citation classic? Ah...not quite. Just kidding. No, this is just my latest published paper, now appearing online early in Evolution. The work stems from a side project I conducted while I was a postdoc in Paul Turner's laboratory at Yale. What originally began as a simple experiment in the spring of 2005 to determine whether the density of hosts in a habitat affected parasite competition quickly snowballed as additional experiments were conducted, data analyses and reanalyses performed, revisions made and resubmitted (3x! The first submission was on 1/31/06), authors added (the estimable Stephen Abedon) and much sweat, angst and time sacrificed. This paper is the fruit of the most difficult effort I've undertaken in my fledgling career. (However, as Homer Simpson might say, "The most difficult effort of your career... SO FAR!").
Here we competed two Phi6 strains over a range of host densities in two separate habitats: in liquid culture and on agar plates. In liquid culture, the results were not surprising. The more fit phage out-competed the less fit phage over all host densities. This result was expected because, in a well-mixed liquid culture, the phages are not spatially limited in their access to hosts, and their net reproduction should be a product of the number of hosts and the reproduction per host. Since the more fit phage produced more babies per host (in the paper, greater burst size), its advantage over the less fit phage should be consistent over all host densities, and the total number of babies produced by both strains should increase with increasing host density (See Fig. 5 below. Note: the slopes are not significantly different despite appearing to converge.). By contrast, in agar, phage dispersal is limited by its ability to diffuse through the viscous agar. This causes a shift from direct competition to indirect competition between the phage strains. That is, in the liquid culture, phage compete globally for hosts, whereas in the agar culture, competition is limited locally. Here the results were surprising; the relative fitness of the less productive phage strain increased with increasing host density (up to a point where it leveled off). (See Fig. 4 below with fitness comparisons to unstructured habitat).
This result caused us considerable consternation and its cause is still under debate. Since what is actually occurring in a growing plaque (i.e., the region on a lawn of bacteria where hosts are infected and lysed) is somewhat of a black box, we can only speculate as to why we observe increasing relative fitness with increasing host density for the less productive strain. One possible explanation, as described in our paper, is:
...bacteria may differ physiologically over space depending on their initial densities. This phenomenon may be attributed to the fact that bacteria form microcolonies on a lawn, and microcolony size depends on initial density (Kaplan et al. 1981). Lower initial inocula lead to larger microcolony sizes. This outcome makes intuitive sense if we assume that microcolonies are spheres that are packed within a constant volume (the top-agar layer). Each microcolony is initiated by a single bacterial cell seeded in the top agar. Thus, if fewer bacteria are seeded, then microcolonies must grow to a larger size in order to attain the same cumulative volume.
Large microcolonies contain relatively fewer outer-surface bacteria with access to oxygen and nutrients, and with relatively unobstructed diffusion of wastes. For these reasons, large microcolonies may contain lower numbers of bacteria that are competent for phage infection. Thus, the final 20% of infections at low initial bacteria densities likely result in reduced burst sizes per cell (due to the larger microcolony size) and, therefore, less particles per plaque. This effect could be substantial with a Pseudomonas host given that it is an obligate aerobe, and that bacteria in the center of Pseudomonas microcolony may be particularly physiologically inappropriate for phage infection. To summarize, greater input of bacteria into a habitat may lead to smaller microcolonies that contain greater numbers of bacteria competent for phage infection, and this may lead to better phage growth.
Why does the less fit strain display even greater increase in phage density as plaques form under greater bacterial densities? We speculate that the presumptive poorer host physiology with larger microcolony size has a greater impact on Φ6M relative to wild type Φ6. Alternatively, Φ6M may be less able to efficiently penetrate into larger microcolonies, resulting in fractionally fewer bacteria infected within the confines of the plaque, rather than fewer phage produced per bacterium infected.
Whatever the cause of the changes in relative fitness, one thing is clear: the strength of selection against the less productive phage strain was reduced at greater host densities. Consequently these genotypes may gain time to adapt to the habitat conditions, and may eventually out-compete the more productive phage strain in evolutionary fitness. This result is relevant in a practical sense with regard to phage therapy, and is consistent with the general finding that biofilms are more resistant to phage or antimicrobial attachment than are planktonic bacterial populations. It may also be relevant to the emergence of infectious diseases where habitat structure and increased host densities permit the persistence of less fit genotypes.
Photo credit: Dennis Bamford. Phi6 adsorbing to Pseudomonas phaseolicola pili.