Perspectives and Directions

In recent years remarkable strides have been made in our understanding of the molecular basis of phage lysis and its regulation. The progress has raised many new questions. That there are at least two general strategies for lysis is clear. All complex phages seem to use holin-endolysin lysis, whereas two of the prototype small ssDNA and ssRNA phages encode proteins which act as inhibitors of cell wall synthesis. The diversity of holins has always been a stunning, and somewhat daunting feature of lysis, suggesting that there may be several fundamentally different lysis mechanisms. Nevertheless, the basic features of all these systems are still comfortingly common, especially the ability to be triggered by energy poisons. It will be useful to assess whether class II and class III holins share the ''kill without warning'' property of the S holin, as they should if our understanding of what drives holin evolution is correct. In addition, although the discovery of the secretory endolysins means that the muralytic enzymes can no longer be regarded as ''dumb'' reporter functions for the activity of holins, it has perhaps further brought into focus how important the timing function is for the lytic event that terminates all dsDNA phage infections. Holins apparently have evolved, perhaps independently and at multiple times, to provide a temporal schedule for lysis and to allow the optimization of that schedule. Competition experiments to test the fitness of various holin genes and series of timingmutant alleles of a particular holin should be instructive. In terms of the mechanism of holin function, it is clear that the next step must include cytological localization of the holins to assess whether patches or rafts are formed in the membrane during the vegetative cycle. In vitro experiments in which holin function is reconstituted in artificial lipid vesicles should help clarify the roles of concentration, oligomerization, and membrane energization in the holeformation process, as well as providing structural insight into the nature of the lesions. These same systems can also be exploited for investigating the mechanisms by which antiholins block holin function. The diversity of topologies available to the known antiholins suggest that a wealth of specific interactions underlie the regulatory properties of these molecules, interactions which should be fertile areas for genetic analysis.

With regard to the single-gene systems, it will be interesting to see whether MS2 L is also an amurin, or if a third general strategy is available, perhaps a ''magic button'' that allows the induction of so-called autolysis without disturbance of murein synthesis. It should be noted that a wide variety of ssRNA phages have been isolated against a number of different R factors (23). It is a reasonable expectation that some of these ssRNA phages may have evolved amurins against targets other than MurA. Recently, MH2K, a lytic Microvirus of Bdellovibrio, was isolated and sequenced. MH2K lacks a scaffolding protein gene equivalent to D (25) and, consequently, lacks the E lysis cistron embedded in D in fX174 and its coliphage relatives. Instead, the candidate lysis genes are short open reading frames embedded in other essential MH2K genes. It will be interesting to test whether this independently evolving lysis gene also targets MraY. Interestingly, a Microvirus sequence for the wall-less intracellular bacterium Chlamydia has no obvious reading frame available for a lysis gene (103). It is unknown how a phage can cause lysis of a host cell that grows, without a cell wall, in the iso-osmotic environment of a mammalian cell cytoplasm.

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