Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product

Abstract

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gpl8) that is encoded by gene 18, gpl8 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N-and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined. * Corresponding author. "Achromobacter lyticus" M467-1, was purchased from Wako Junyaku Co. Polynucleotide kinase and all of the restriction enzymes used were purchased from Takara Shuzo Co. Methanesulfonic acid (4 M) was purchased from Pierce Chemical Co., and all other reagents were analytical grade, from either Nakarai Chemicals or Wako Junyaku Co. Nutrient broth, tryptone, yeast extract, Bacto-Agar, and Casamino Acids were purchased from Difco Laboratories. Medium and buffer. M9A medium was used to grow E. coli. It contained, per liter of water, 6 g of Na2HPO4, 3 g of KH2PO4, 0.2 g of MgSO4. 7H20, 0.5 g of NaCl, 1 g of NH4C1, 4 g of glucose, and 10 g of Casamino Acids. Phages were stored in B+ buffer, which contained, per liter of water, 7 g of Na2HPO4, 4 g of NaCl, and 3 g of KH2PO4, supplemented with 1 mM MgSO4 for use. Phage, plasmids, and bacterial strains. T4D.23amH11 is from our collection. T4D.18am mutants (Fig. 1) were kindly supplied by W. B. Wood and J. King. E. coli BE was used as a nonpermissive host for amber mutant phages, and E. coli CR63 was the permissive host. E. coli JM103 was used to propagate M13 phages and to prepare replicative-form DNA of M13 phage. Three E. coli BE strains, which carry plasmids p662, p655, and p664, respectively, were kind gifts from Tom Mattson. Each of the three plasmids contains an EcoRI fragment which forms approximately one-third of gene 18 (Fig. 1). Marker rescue test. Marker rescue of T4D.18am mutants by plasmids was performed as described by Mattson et al. (21). The results are also shown in Fig. 1. DNA preparations for sequencing. DNA preparations for the Maxam-Gilbert method have been described by Christensen and Young (8). DNA was 5' end labeled with polynucleotide kinase, and the strands were then separated by polyacrylamide gel electrophoresis, or alternatively, the double-stranded DNA was cut with a restriction enzyme and the desired fragment was purified by polyacrylamide gel electrophoresis. DNA sequencing procedures. The Maxam-Gilbert method was done essentially as described by Maxam and Gilbert (22). The dideoxy method was carried out as described by Messing (23). Preparation of gpl8. Gpl8 was prepared as described ...

Structural Studies of the Phage G Tail Demonstrate an Atypical Tail Contraction

“…In phi812, the cryo-EM density in the domain IV region has been described to contain 2 major helices [11]. The region has not been described from the T4 studies because of the limited structural information [8,31,32], but we have also observed these two helices in our phage G gp178 cryo-EM density (Figures 6 and 7).…”

“…It is also the least re portion of the sheath protein density (Figure 7A). The density for domain II is not a as it is described for T4 [33,31], which could be due to phage G's sheath protein, (579 amino acids), being smaller than T4′s gp18 (659 amino acids) [31]. In phage G At the domain level, phage G's sheath protein appears to be similar to the sheath protein of T4, gp18 [6], as labeled in Figure 7B.…”

Structure of the bacteriophage T4 baseplate as determined by chemical cross-linking

“…A complex between gpl8 and gpl9 was also seen, but considering the extensive overlap between the core and sheath, we expected to see a much higher yield of complexes involving these two proteins. It seems unlikely that the lack of cross-linking would be due to inaccessibility to the center of the tail, given the apparently open structure of the sheath as revealed by optical diffraction (14,31,44), or to the lack of reactive groups, since both proteins contain numerous lysine residues (2,3). This may indicate that once the sheath is fully polymerized, there is only limited interaction with the core in readiness for the opposing movements of the sheath and care during contraction.…”

Morphological Changes in Escherichia coli Phages Treated with Ethanol.

“…Bar,100nm. hydrophobic balance of the amino acids of phage head proteins in ethanol sensitive-phages indicates hydrophilicity whereas that in ethanol-tolerant phages indicates hydrophobicity (Yamashita et al, 2000) . Arisaka et al (1988) reported the amino acid composition of the tail sheath protein of the T-4 phage. We calculated the hydrophilic-hydrophobic balance of the tail sheath protein of the T-4 phage of Arisaka by our method.…”

“…It was reported that the contracted tail sheath could be prepared by heat treatment at 55°C for 5 min (Yamamoto and Uchida, 1973) . It was reported that the tail sheath protein is a GTP-binding protein (Serysheva, et al, 1984) , nucleoside triphosphate and Ca binding protein (Wahl and Kozloff, 1962) and that it is a region homologous to the corresponding region in Ca2+binding proteins, indicating that Ca2+ might be important for stabilizing the native structure of the sheath protein (Arisaka et al, 1988) . From these data, it is suggested that ethanol at 30% (v/v) acts on Ca2+ and GTP or ATP of the sheath proteins, resulting in the shrinkage of such proteins.…”