Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator).
The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL1) ORF8 (bacL2), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL1, bacL2, bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL1 and -L2 and bacA mutants. bacL1 and -L2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL1-encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL1-encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.
Tomita
Haruyoshi
H
Department of Bacteriology and Bacterial Infection Control, Gunma University Graduate School of Medicine, Showa-machi 3-39-22, Maebashi, Gunma 371-8511, Japan.
Kamei
Elizabeth
E
Ike
Yasuyoshi
Y
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000378:metabolism*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics* / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001430:Bacteriocins; D004251:DNA Transposable Elements; D009088:Mucoproteins; D010675:Pheromones; C010393:lysin, gastropoda; D004262:DNA Restriction Enzymes