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Insight into the Lytic Functions of the Lactococcal Prophage TP712
The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832245/ https://www.ncbi.nlm.nih.gov/pubmed/31546996 http://dx.doi.org/10.3390/v11100881 |
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author | Escobedo, Susana Campelo, Ana Belén Wegmann, Udo García, Pilar Rodríguez, Ana Martínez, Beatriz |
author_facet | Escobedo, Susana Campelo, Ana Belén Wegmann, Udo García, Pilar Rodríguez, Ana Martínez, Beatriz |
author_sort | Escobedo, Susana |
collection | PubMed |
description | The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria. |
format | Online Article Text |
id | pubmed-6832245 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-68322452019-11-21 Insight into the Lytic Functions of the Lactococcal Prophage TP712 Escobedo, Susana Campelo, Ana Belén Wegmann, Udo García, Pilar Rodríguez, Ana Martínez, Beatriz Viruses Article The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria. MDPI 2019-09-20 /pmc/articles/PMC6832245/ /pubmed/31546996 http://dx.doi.org/10.3390/v11100881 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Escobedo, Susana Campelo, Ana Belén Wegmann, Udo García, Pilar Rodríguez, Ana Martínez, Beatriz Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title | Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title_full | Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title_fullStr | Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title_full_unstemmed | Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title_short | Insight into the Lytic Functions of the Lactococcal Prophage TP712 |
title_sort | insight into the lytic functions of the lactococcal prophage tp712 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832245/ https://www.ncbi.nlm.nih.gov/pubmed/31546996 http://dx.doi.org/10.3390/v11100881 |
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