The Cloning of Bacteriophage T4 Proteins Responsible for Shutoff of Bacterial Synthesis

Many of the nearly 150 otherwise-uncharacterized T4 ORFs seem, by a variety of criteria, to be involved in the transition from host to phage metabolism. They are located downstream of very strong immediate-early promoters and are expressed in high levels shortly after infection. Most kill the host bacteria when one tries to clone them by standard techniques, indicating that each alone is sufficient to block a key host enzyme. Many of them lie in regions which are deletable from the T4 genome under general lab conditions, yet are found in all of the diverse T-even phage analyzed to date, indicating their importance.

We were interested to characterize some of these very early host-lethal genes from two regions of the T4 genome--nrdC-tk and tRNA-e--identifying the specific host functions and pathways affected and the mechanisms of that inhibition. We will discuss the cloning of ORFs e.6, nrdC.4, and tk.-6 under tight control in newly-available pET-16b and pAII-17 vectors designed for lethal gene expression, which use a combination of the lac repressor and operator and terminators before the cloning site to prevent leaky transcription during cell growth. For the extremely lethal Alc protein, the expression of target proteins from this vector was induced by providing the T7 RNA polymerase gene by superinfecting with lambda CE6. Strains of E. coli carrying the polymerase are generally too leaky, even in the presence of pLysS, carrying the T7 lysozyme to inhibit the polymerase.

We are purifying the Alc protein and e.6 protein, the latter through taking advantage of incorporating histidine tails, both using various special features of a powerful BioCad purification system. We will include some aspects of discussion to characterize the targets and mode of action of selected host-lethal T4 proteins and thus develop important new reagants for characterizing key bacterial pathways that are essential to host survival. The results may also suggest interesting new possibilities for antibiotic development.

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