laboratory that they provide in the wild and that are non-essential mutant] and the closeness to each other of the two genes for in the laboratory. metal insertion [D(181,412–189,531) mutant] are explained by Indeed, the data in Figure 2 and Table 2 show that other co-selection for common function. In the case of the metal bacteriophages have DNA translocation operons. The DNA insertion function of the D(181,412–189,531) mutant, we do integration/recombination function of the translocation operon- not have data that further constrain the function, non-essential proximal genes of lysogenic homologs (Fig. 2, rightmost genes in in the laboratory. However, we propose that, in a not-known the bottom three rows) further supports a linking of the roles way, metals are less available for protein insertion in the of translocation and recombination for these bacteriophages. environment than they are in the laboratory. Parenthetically, In the case of the lytic bacteriophage, 0305w8-36, a separate, both deletions include an orf that encodes an AAA ATPase neighboring gene for recombination is not present for the DNA (orfs 201 and 66), for reasons, if any, that are unknown. translocation operon. Recombination may not require a separate However, the island-forming agglomeration of apparently gene in the case of some long-genome lytic bacteriophages, as unrelated non-essential genes, as observed here, is in need of found for the well-studied, smaller lytic bacteriophages, T3 and additional explanation. One hypothesis is that gene insertions via T7.43 To test the possibility of biofilm formation-associated horizontal transfer are most readily made in non-essential, multi- translocation/recombination linking for 0305w8-36, we need to gene islands. By this hypothesis, the multi-gene islands are sites determine the functions of the various 0305w8-36 genes in a at which the probability of lethal damage is minimized during laboratory biofilm-forming system. initial gene insertion and also during subsequent selection for Unusual aspects of the propagation of bacteriophage 0305w8- optimization of the insertion. The edges of our deletions could 36. In the absence of a mitigating factor, the implied biofilm- have been generated either by an accident of the deletion process ©A associated possib2lemit0igating host/bacteriophage 1factor 2isa co-existence quLoruma-sensinng,indfectione-islsands, is problematic, given or by the onBcenuclieateod,will presence of genes sbe at the seclf-proipageating deletion edge ninthe that are csense essential ethat . that 0305w8-36 is both genotypically and phenotypically lytic.8 with the current host. The above hypothesis predicts that the inhibiting event triggered by increase in the concentration of genes introduced by horizontal transfer will be preferentially bacteriophage particles. The simplest possibility is that, as the introduced in a previously initiated island of non-essential genes. bacteriophage concentration increases, the tail-mediated dimer- This hypothesis is independent of the position within the genome ization observed here increasingly inhibits infection by obscuring of the various deletable segments. of infection would also D(1) porovide timnefor osome tbacteridalcellsisgenes tconrstituite bathemue,vatriatieons o.nwhich will be found tail tip components that adsorb to the host cell. This inhibition Thus, we conjecture that islands of unrelated, non-essential to evolve resistance to bacteriophage infection and (2) explain among most, if not all, long-genome bacteriophages. The presence why bacteriophage 0305w8-36 does not propagate sufficiently of a DNA translocation operon in bacteriophage genomes is in liquid culture to cause visible lysis, even though 0305w8-36 already a theme, as established here. is a lytic bacteriophage (Introduction). Dimerization is possibly a stage in the more extensive aggregation4 that subsequently occurs Materials and Methods for the wild type bacteriophage. The agarose-instigated increase in the level of 0305w8-36 host Bacteriophage propagation and purification. Bacteriophage cell clearing (Introduction) is additional evidence of environ- 0305w8-36 was preparatively propagated in a 0.1% agarose mental signaling of bacteriophage lytic activity, possibly via raising overlay, as previously done,7,9 by use of stationary phase plating of the critical concentration for tail tip-tail tip dimerization. A bacteria, grown with aeration in liquid culture. The medium was bacteriophage adaptation to hydrated polymer makes sense in that 10 g Bacto tryptone, 5 g KCl in 1000 ml water supplemented environmental, hydrated polymer is likely to emerge whenever post-autoclaving with 0.002 M autoclaved CaCl2. The agarose rain converts a polymer-rich, natural environment (a cattle pen in overlay was then removed from Petri plates (plate lysate). the case of 0305w8-36) from a dry, hot (summer soil temperature Bacteriophage particles were extracted by shaking and, then, the usually reaching 49–62°C in the case of 0305w8-36) condition to preparation was clarified by relatively low-speed centrifugation. a wet condition in which both bacteriophages and hosts can Bacteriophages were concentrated by higher-speed centrifugation. propagate. Thus, hydrated polymer can, in theory, be a signal to a The procedures of reference 7 were used. Subsequently, con- bacteriophage to become more aggressive in propagating. An centrated, resuspended bacteriophage particles were purified either objective for the future is further testing of the effects of various by ultracentrifugation in a cesium chloride step gradient8 or by polymers on bacteriophage propagation, with potential applica- revised procedure developed here to avoid the tail tip-initiated tail tion to managing environmental bacteria. The bacteriophage as sheath contraction and loss of infectivity observed after purifica- biofilm component hypothesis discussed above can be subjected tion by centrifugation in a cesium chloride density gradient.8 to further testing either by further developing a bacteriophage/ The revised procedure of purification was the following. A host-based laboratory biofilm-forming system or by characterizing sucrose density gradient was pre-poured in a centrifuge tube for additional environmental biofilms by electron microscopy of thin the Beckman SW41 rotor. The gradient had the following sections, as done in reference 13. composition: (1) 1.0 ml of 65% sucrose at the bottom, (2) 9.3 ml Islands of non-essential genes. The closeness to each other of of a 10–35% linear sucrose gradient above the bottom layer of the various genes for DNA translocation [D(118,397–124,982) 65% sucrose. The buffer for the sucrose solutions was 0.01 M 32 Bacteriophage Volume 2 Issue 1