Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.
Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.
Yokoi
Ken-ji
KJ
Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan.
Shinohara
Masayuki
M
Kawahigashi
Nobutaka
N
Nakagawa
Kazuaki
K
Kawasaki
Ken-Ichi
K
Nakamura
Shogo
S
Taketo
Akira
A
Kodaira
Ken-Ichi
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D001435:Bacteriophages / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D008242:Lysogeny / Q000235:genetics*; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D019936:Probiotics; D039002:Prophages / Q000502:physiology*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009238:N-Acetylmuramoyl-L-alanine Amidase