| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Tail-lysozyme, gp5^<**>, is a 42 kD structural protein of the baseplate which has a lysozyme activity and is located under the baseplate. Upon adsorption of the phage to Escherichia coli, it penetrates into the outer membrane together with the tail tube and digs a whole on the peptidoglycan layr so that the tail tube can reach the inner membrane. We have previously isolated the tail-lysozyme and shown that the tail-lysozyme has the same substrate specificity as that of T4 lysozyme, gp e, namely N-acetyl muramidase (1). The expected molecular weight from the nucleotide sequence was 62 kD and in fact it is expressed as a 62 kD protein from the cloned gene 5, but the lysozyme activity is absent in the precurser protein (2). In the present study, we have determined the N-and C-terminal sequence of the mature protein. The N-terminal sequence coincided with that expected from the nucleotide sequence and the the C-terminus was Val390. A region with high homology (44% identical) to gp e, is located towards the C-terminus of the mature gp 5. N-terminal region is likely to assume a domain which constitutes the integral part of the baseplate. From the result, it was concluded that the 20 kD fragment is cleaved off from the precurser protein to become mature 42 kD tail-lysozyme.
On the other hand, we have mapped the mutation sites of 12 mutants which have a mutation in gene 5. These include five ts (temperature sensitive), two hs (heat sensitive) and five amber mutants. All the mutation sites were mapped upstream of Val390. Among the ts mutants, 5ts1 (3) is a bypass e mutant wich does not require gp e to lyse from within. This was Gly322Asp mapped in the lysozyme domain. Another ts mutant, 5DH6318, is a bypass 63 mutant which does not require gp63 for efficient attachment of the tail fibers. This was Ala65Thr mapped in the "structural" domain. Including these mutations, other mutations will be discussed in respect to the structure-function relation of the tail-lysozyme.
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