Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e.
The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide. These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli. Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.
Kakikawa
Makiko
M
Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan. kakikawa@eng.toyama-u.ac.jp
Yokoi
Ken-ji
KJ
Kimoto
Hisashi
H
Nakano
Masataka
M
Kawasaki
Ken-Ichi
K
Taketo
Akira
A
Kodaira
Ken-Ichi
K
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015870:Gene Expression; D007763:Lac Operon / Q000235:genetics; D007778:Lactobacillus / Q000821:virology*; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017384:Sequence Deletion; D019810:Sodium Azide / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C117327:Lys protein, Oenococcus oeni temperate bacteriophage fOg44; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D014764:Viral Proteins; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins