The focus of this investigation was ea22, a relatively large gene in the exo-xis region that is common to both λ and Φ24B, a phage that we have used in this and prior studies as a representative of the Stx phages. From developmental assays of ea22 mutant phages, we have observed that unlike other exo-xis genes such as orf60a, orf63, and orf61, expression of ea22 favors the lysogenic over the lytic pathway and acts early in development. As ea22 from both λ and Φ24B is considered to resist lytic development, Stx phages may possess additional genes that work in conjunction with ea22 to produce stronger effect than what we observed for λ bacteriophage. While two-hybrid studies of phage–host  and phage–phage  proteins failed to identify any interactions involving Ea22, they could have been impeded by low expression levels of one partner, weak interactions, the possibility that Ea22 acts in a multiprotein complex, or the possibility that Ea22 is a nucleic acid binding protein.
We suppose, ea22 may act as key regulator of phage lysis vs. lysogeny decision in response to changing ratio of phage particles to bacterial cells. Although the presence of ea22 favored lysogeny in most cases, the only deviation was observed in Stx phage at the lowest applied m.o.i. Based on it, we speculate that there is a putative additional regulation at m.o.i value = 1 that is considered as low and favorable for the lytic development of phages. It appears that additional factor(s) may interact with ea22 under such conditions, abolish its action and even deepen the lytic response. Importantly, this additional regulation seems to be used only by selected phages. Taken together, ea22 serves a role in decision to choose lytic or lysogenic pathway with the observed effects on phage development and host survival being greater for Stx phage.
Sequence analysis of ea22 from λ and Stx phages offer some insight into the functional differences between λ and Stx phages. Among the Ea22 proteins presented, the carboxy terminus of the sequence (starting at 102 amino acid in λEa22) is dissimilar. However, all proteins terminate with a common K/RGE motif. We speculate that Stx− and Stx+ phages have adopted a repertoire of carboxy-terminal domains that may all act upon the same host process but not necessarily the same host proteins. In addition to this, we observed an occurrence of a α-helical coiled-coil region in the N-terminal part of all the analyzed Ea22 protein sequences. Interestingly, a large number of the identified phage integrases (including λ Int) have extended coiled-coil domains which on the one hand, facilitate association and stabilization of the initial complex of recombination, and on the other hand, prevent prophage excision in the absence of the main excision protein .
The sequence divergence also extends to the expression patterns of ea22 in λ and Stx phages during infection of E. coli host. With reference to λ phage, the level of mRNA for ea22 is significantly lower when compared with the number of transcripts for orf60a, orf63, and orf61, whereas in Φ24B and other Stx phages, we observe the opposite relationship. In fact, the level of ea22 gene expression in Stx phages (especially in P32, P27, and P22) was significantly higher than the expression of other tested genes (even the early genes like, N and cro). Surprisingly, in opposite to ea22, the other genes (both early and late) were expressed at almost equal level. As we know from previous report, Stx phage genes present different levels of expression which increase during time-course infection experiment . Due to this, we suspect that the efficient expression of early genes, e.g., N and cro have probably not started yet in the tested phages P32, P27, and P22. Importantly, at the same time, the level of expression of ea22 was high, suggesting that despite belonging to the pL operon, the ea22 gene might be expressed earlier and regulated independently of the studied phage genes. Above that, the expression of two other phage genes ea8.5 and int have been analyzed by us previously in the frame of similar but not exactly the same infection experiment . The obtained results indicate that the ea8.5 presents a level of expression similar to that observed for ea22 leading us to speculate that ea22 has a similar mechanism of regulation. On the other hand, expression of int was decreased compared to most of the analyzed genes from pL operon . This was possible due to the occurrence of double control of the int expression during phage infection. As shown previously, product of the int gene is responsible for an integrative recombination of phage and bacterial DNA during lysogenic pathway and works in conjunction with Xis to achieve excision of prophage DNA after induction . The int gene is located near the ea22 gene and is transcribed from both its own promoter, pI, and the leftward major pL promoter . The pI promoter is positively regulated by cII protein which operates at the early stage of lysogenic infection. Shortly after making the decision to transition into a lytic cycle, when pI is inactive either due to the absence of cII , or by other exo-xis region proteins including Ea8.5 , int expression may still occur via pL . Looking for convergence in the regulation of the int and ea22 genes expression, we came across the data from a comprehensive ribosomal profiling study of phage genes during early lytic infection. These data allowed us to identify int as a gene with a similar to ea22 profile and level of expression in the first twenty minutes of temperature-dependent prophage induction —Fig. 1. In addition, upstream of the λ and Φ24Bea22 genes, there was a predicted but never investigated promoter named p1 . Interestingly, the p1 promoter, like the well-established pI promoter, also contains binding sites for host RNA polymerase sigma factor 70 (RpoD17) and two host arginine-sensitive regulators ArgR and ArgR2, as found by BPROM software . Undoubtedly, at this stage of knowledge, we cannot exclude that similar to that observed in int, the double control of ea22 expression also exists. However, further research are needed to confirm the activity of p1 promoter and to explain the mechanism of this regulation.
The last aspect of this discussion focused on the physiological significance of ea22 expression. We showed that in contrast to other exo-xis genes (orf60a, orf63 and orf61), expression of ea22 favors the lysogenic over the lytic pathway. Wild-type λ and Φ24B phages revealed higher efficiency of lysogenization of bacterial cells and lower efficiency of progeny phage production during the lytic cycle, when compared with Δea22 mutants. Interestingly, differences between wild-type and mutant phages were more pronounced in Φ24B than in λ. In the context of ea22 gene expression, its disparate function relative to other exo-xis region genes, and some similarities in its mechanism of regulation to phage integrase, there is a possibility that Ea22 navigates the lysogenic-lytic decision by working in concert with the phage integrase and is dependent on the physiological state of the bacterial cell. It is worth noting that the function of ea22 that we have proposed is different from earlier assumptions. In one of previous reports, authors concluded that Ea22 from λ is similar to Ehly 2 protein that is associated with an enterohemolysin 2 activity and encoded by phage C3208 in E. coli O26:H11 [40, 41]. This finding was based on the sequence similarity that was estimated at level of 35% . According to the work of Rost and collaborators, such a level of identity is considered as result of twilight zone that is difficult to interpret . In addition, the location of the ea22 gene in the early region of the phage genome does not indicate on its enterohemolysin activity. Importantly, there were no other premises that indicated on similarity of Ea22 to Ehly2.