Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages

Abstract

The major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoeleetric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4.70 to 5.92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6.1 to 6.3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M r and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeriaphages were compared. The base composition of listeriaphage DNAs was between 37 % and 39 % G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.

Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh

“…A511 DNA was extracted and purified by described methods (31,38). Enzymatic digestions with various endonucleases (BspHI, HindIII, XbaI, MunI, SpeI, NdeI, EcoRV, SnaBI, Psp1406I, and Bsp1407I) were carried out according to the suppliers' recommendations (United States Biochemical, Stratagene, and New England Biolabs).…”

“…Phage structural proteins were separated by horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes as outlined elsewhere (24,31). Coomassie blue-stained protein bands (Cps and Tsh) were excised from the membranes and used for determination of the N-terminal amino acids with an ABI 477A protein sequencer.…”

“…Upon recognition of its cell surface receptor (42), the sheath is contracted to twothirds of its original length and the linear double-stranded DNA molecule of approximately 116 kb (31) is injected into the host cell. After a latent period of 55 to 60 min, the multiplication cycle is terminated by cell lysis through the ply gene product, a Listeria cell wall-specific N-acetylmuramoyl-L-alanine amidase (35).…”

“…Listeria bacteriophage A511 is the only known virulent phage for this genus and is unrelated to all other Listeria phages (31,43). A511 has an extremely broad host range, being capable of lysing approximately 95% of all L. monocytogenes strains of serovars 1/2 and 4b (30,33).…”

Bacteriophage Morphological Characterization by Using Transmission Electron Microscopy

“…Early papers on bacteriophages are dated around the 20's. At the beginning, phages were employed as diagnostic tools in bacteria [3,[7][8][9][10][11][12][13][14][15]. Lately they started to be used for prophylaxis and therapy both in animals and humans in Eastern Countries.…”