in all experiments , we exposed bacterial strains to the phage t4rii , which targets lipopolysaccharides embedded in the bacterial outer membrane .
as our altruistic suicidal strain , we used e. coli carrying the prophage  ( henceforth e. coli  ) , which encodes the rex abortive infection system that is triggered by several phages including t4rii . as the phage - sensitive strain , we used e. coli carrying the abi - free control prophage hk97 ( henceforth e. coli hk97 ) .
finally , we used an e. coli hk97 strain that has evolved resistance against t4rii ( henceforth e. coli t4 ) . while we compared the performance of phage - sensitive e. coli hk97 vs. suicidal e. coli  in our previous study
, we here compare the competitive abilities of phage - sensitive e. coli hk97 vs. phage - resistant e. coli t4 , and relate it to our previous findings .
we first examined the performance of e. coli hk97 and e. coli t4 in an unstructured environment .
we did this by growing the two strains together in shaken liquid medium at various frequencies ( e. coli t4 varied from 0.1% to 99% ) in the presence of t4rii ( moi = 0.001 ) and followed population growth and the change in relative strain frequency over time .
we found that t4rii eradicated monocultures of e. coli hk97 , whereas e. coli t4 could thrive even when initially rare ( fig .  1 )
moreover , e. coli t4 significantly outcompeted e. coli hk97 under all conditions ( fig .  2 , one - sample t - tests for fitness > 0 : t5 > 6.9 , p < 0.001 ) .
these results demonstrate that individual - based resistance mechanisms can easily spread in a completely unstructured environment , a finding that strongly contrasts with the suicidal host - defense strategy , which either could not prevent population collapse ( with e. coli  frequencies  90% ) or did not result in fitness benefits ( fig .  2 ) .
this comparison reveals that individual - based resistance is the only way of how bacteria can defy bacteriophages in unstructured environments .
population growth of mixed cultures containing e. coli t4 ( resistant against the obligately lytic phage t4rii ) and e. coli hk97 ( t4rii sensitive ) in shaken liquid medium .
while phage t4rii eradicated monocultures of e. coli hk97 , cultures containing e. coli t4 could always grow even when e. coli t4 was initially rare ( percentage of e. coli t4 is indicated on the right ) .
figure  2 . comparing the performance of suicidal e. coli  and resistant e. coli t4 in competition against e. coli hk97 in the presence of phage t4rii in an unstructured environment ( shaken liquid medium ) . while e. coli t4 significantly outcompeted e. coli hk97 under all conditions ( malthusian fitness > 0 , p < 0.001 ) , e. coli  either experienced no relative fitness benefit or the entire culture went extinct ( these latter results were initially published in refardt et al.,10.1098/rspb.2012.3035 ) .
these findings demonstrate that individual - based resistance is the only way of how bacteria can defy bacteriophages in unstructured environments .
we found a different pattern when competitions were performed in a structured environment ( fig . 
when we added 1.5% agar to the medium to limit bacterial dispersal , 
 e. coli hk97 was significantly outcompeted by both suicidal e. coli  ( one - sample t - test for fitness > 0 : t29 = 11.17 , p < 0.0001 ) and e. coli t4 ( t11 = 9.52 , p < 0.0001 ) .
this comparison shows that both altruistic host - defense and individual - based resistance mechanisms are efficient means to defy bacteriophages in a structured environment .
however , e. coli t4 seemed to be slightly but significantly more efficient than e. coli  ( fig . 
3 ; two sample t - test : t40 = 4.20 , p = 0.0001 ) .
a plausible explanation is that suicide always takes its toll , especially at high moi , which reduces the efficiency of suicidal host defense compared with an individual - based resistance mechanism .
additionally , we also found that the relative fitness of e. coli t4 was greatly reduced in the structured compared with the unstructured environment , an observation that is compatible with the view that medium viscosity also reduces phage dispersal , such that some fraction of e. coli hk97 colonies remain unharmed .
figure  3 . comparing the performance of suicidal e. coli  and resistant e. coli t4 in competition against e. coli hk97 in the presence of phage t4rii in a structured environment ( 1.5% agar plate ) .
both strains significantly outcompeted e. coli hk97 , indicating that suicidal host defense as well as individual - based resistance were efficient in withstanding phages .
a direct comparison between the two strategies reveals that individual - based resistance is slightly but significantly better under these conditions . but note that although the experiments were performed following the exact same protocol , they were performed at different dates , which might explain some of the differences . to be able to put our findings in an ecological context
, we need to become clear about the level of structuring that occurs in natural bacterial populations .
while an unifying answer is not possible owing to the fact that bacteria populate an enormous range of different habitats , it seems justified to conclude that some degree of population structure always exists .
consequently , we can deduce that altruistic host defense likely represents an efficient strategy to withstand bacteriophages in natural habitats .
while our comparison revealed that both host - defense strategies can be efficient in laboratory settings , it remains unclear , which of the two strategies is more prevalent in nature .
individual - based resistance might be more common because it is easier to evolve , since a single point mutation can lead to resistance .
in contrast , suicidal defense systems are often based on complex machineries that eventually have first evolved for other purposes , and have only later been coopted into suicidal systems .
although more complex , we propose that suicidal host - defense systems might be cheaper to maintain in the long run because they are often decoupled from other functions , and might therefore entail little pleiotropic cost .
individual - based resistance , on the other hand , is typically achieved by altering a molecule on the bacterial cell surface that is recognized by a phage and to which the phage adsorbs .
such an adaptation may impair the original function of the molecule and consequently reduce bacterial fitness ( as observed in t4 phages ) , even more so if further modifications are necessary to resist different phages , or to respond to phage counter - resistance .
these potential cost differences might also affect the co - evolutionary dynamics between phages and bacteria .
moreover , it would be interesting to see whether both resistance mechanisms can occur in the same individual , or whether they are mutually exclusive , with one resistance mechanisms eventually replacing the other one over evolutionary time scales . while speculative at this stage , these considerations will hopefully stimulate future work . finally , another unresolved issue about abi systems is that they often involve the action of prophages .
a plausible explanation for this is that prophages already possess mechanisms that can kill bacteria during induction ( i.e. , when a prophage enters the lytic phase ) , which can eventually be coopted into suicide machineries .
more sinister is the possibility that bacteria are not always free to choose their doom .
for instance , is has been observed that competition between two prophages in the same bacterial cell leads some prophages to induce premature cell death , possibly to prevent the other prophage from replicating .
to obtain e. coli t4 , we diluted overnight cultures of e. coli hk97 100-fold , and incubated 100 l together with 10 pfu of phage t4rii overnight in a plate reader .
cultures whose growth pattern indicated the evolution of resistance were selected and serially passaged ( three times ) in 3 ml medium . before every passage , we diluted cultures 1,000-fold and added 10 pfu of phage t4 .
this was done to select for full resistance , and to reduce possible resistance costs through compensatory mutations .
we then streaked out cultures , and picked a single colony to establish a stock culture , stored at 80c .
we verified resistance by cross - streaking against lysates of both phages t4 and t4rii .
we chose this procedure because we were interested in obtaining a strain that exhibits a different defense mechanism against phage t4rii , but otherwise grows comparably to e. coli  and e. coli hk97 ( which do not differ in their growth rate in the absence of phages ) . indeed , when grown in mixed cultures , we found that neither e. coli t4 nor e. coli hk97 experienced a selective advantage in the absence of phages ( t4 = 0.48 , p = 0.66 , difference in malthusian fitness = 0.02 ) .
the downside of this procedure is , however , that we can not infer the initial cost of phage resistance .
we performed all experiments following the same protocol as described previously . in short , experiments shown in figures 1 and 2 were performed in 96-well microtiter plates in 100 l of medium .
initial concentration of both competing strains together was 5  10 cfu / ml , initial concentration of phage t4rii was 10 pfu / ml .
experiments for figure  3 were performed in 24-well plates with wells filled with 1.5 ml lb 1.5% agar each .
between 10 and 10 cfu of e. coli  and 10 cfu of e. coli t4 were added together with 10 pfu of phage t4rii . to be able to distinguish bacterial strains in mixed competition
, we marked all strains with plasmids pgfp and pdsred carrying genes for fluorescent proteins under an inducible promoter as described previously .
competition assays in unstructured and structured environments followed protocols previously described , except that experiments in structured environments were performed on agar instead of agarose .
 adaptations by hosts in response to parasitism are generally believed to reduce the susceptibility of the adapted individual . 
 however , recent work on escherichia coli showed that bacteria can fight deadly phage attacks by committing altruistic suicide upon infection , in order to prevent parasite transmission to nearby relatives . here , we compare the efficiency of suicidal host defense with individual - based resistance . 
 we show that in unstructured environments suicidal host defense is futile since suicide can not preferentially protect relatives , whereas individual - based resistance is highly efficient in defying phages . in contrast 
 , we found that in structured environments suicidal host defense and individual - based resistance were both efficient in withstanding phages , with the latter type performing slightly better . 
 we propose that the putative lower efficiency of suicidal host defense might be compensated by the fact that suicidal systems usually do not bear pleiotropic costs of resistance , as it is usually the case for individual - based resistance mechanisms . 
Results and Discussion
Methods