Molecular properties of the two-component cell lysis system encoded by prophage φgaY of Lactobacillus gasseri JCM 1131 T: cloning, sequencing, and expression in Escherichia coli

Abstract

Shotgun cloning of the Lactobacillus gasseri JCM 1131 T 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 φadh, 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 (φgaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY–LysgaY), closely resembling that of Lj928 as well as Lj965.