LES phages exhibit different immunity profiles Each phage conferred inhibition of superinfection by the same phage, although the Mu-like phage, LESφ4 was observed to infect LESφ4 lysogens at a very low frequency. This may represent the development of rare mutations that affect immunity functions. There are several examples of such mutations in phage Mu [31]. Repressor/operator coevolution has been suggested to be the driving force for the evolution of superinfection immunity groups of lambdoid phages [32]. The same may hold true for Mu-like phages. For example, mutation of the operator region has been shown to affect binding of the repressor

in Mu vir mutants [33]. Sequential infection of PAO1 with different

LES phages revealed an interesting superinfection hierarchy. LESφ3 selleck chemical lysogens remained susceptible to LESφ2 and LESφ4; and LESφ4 lysogens were susceptible to LESφ2 and LESφ3. However, LESφ2 prevented infection by LESφ3 and greatly reduced susceptibility to LESφ4. Such uni-directional infection exclusion has been reported between other phages, and is commonly associated with super-infection exclusion genes such as the lambda rex genes [34] Proteasome inhibitors in cancer therapy and sieA, sieB and a1 in the Salmonella phage, P22 [35–38]. It is likely that LESφ3 and LESφ4 prophages would have been acquired before LESφ2, because the infection hierarchy suggests that prior acquisition of LESφ2 would have prevented subsequent LESφ3 and LESφ4 infection. LES prophages in PAO1 undergo spontaneous activation to the lytic cycle at a far higher rate than in LESB58 High

levels of spontaneous induction were observed in PLPLs, suggesting that lysogeny is relatively unstable in the PAOl genetic background. We show that phage production remained high between PLPLs containing one, two or three LES prophages, suggesting that polylysogens were no more or less stable than any single lysogens. Southern analysis confirmed that LESφ2 and LESφ3 integrated into the same position in PLPLs as they did in LESB58. Therefore, the instability of PLPLs was not not due to prophage integration into unstable sites. LESφ4 integrated in several alternative sites in PLPLs. The sequence of this phage shares a high level of genome synteny and homology with the transposable Mu-like phage D3112 [16], whose random integration has been demonstrated to create mutations within the host chromosome. LESφ4 may play a similar role in LES genome evolution. The LES phages exhibit a narrow host-range Our investigation of the LES phage host range revealed narrow, overlapping host specificity. No association between bacterial clone-type and phage susceptibility was observed, although testing more strains may have identified a pattern. Despite the high proportion of resistant clinical isolates, our data show that LES phages are capable of infecting some P. aeruginosa strains isolated from keratitis patients and non-LES infected CF patients.