Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Escobedo, Susana, Campelo, Ana Belén, Wegmann, Udo, García, Pilar, Rodríguez, Ana, Martínez, Beatriz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
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
_version_ 1783466126217314304
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
work_keys_str_mv AT escobedosusana insightintothelyticfunctionsofthelactococcalprophagetp712
AT campeloanabelen insightintothelyticfunctionsofthelactococcalprophagetp712
AT wegmannudo insightintothelyticfunctionsofthelactococcalprophagetp712
AT garciapilar insightintothelyticfunctionsofthelactococcalprophagetp712
AT rodriguezana insightintothelyticfunctionsofthelactococcalprophagetp712
AT martinezbeatriz insightintothelyticfunctionsofthelactococcalprophagetp712