Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.

<h4>Background</h4>In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and...

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Autores principales:	Marie Dozot, Sandrine Poncet, Cécile Nicolas, Richard Copin, Houda Bouraoui, Alain Mazé, Josef Deutscher, Xavier De Bolle, Jean-Jacques Letesson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6bc0d01cef304af3ac9018d6e5ac8036
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spelling	oai:doaj.org-article:6bc0d01cef304af3ac9018d6e5ac80362021-11-18T06:35:17ZFunctional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.1932-620310.1371/journal.pone.0012679https://doaj.org/article/6bc0d01cef304af3ac9018d6e5ac80362010-09-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20844759/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions.<h4>Methodology/principal findings</h4>In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase.<h4>Conclusions/significance</h4>The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.Marie DozotSandrine PoncetCécile NicolasRichard CopinHouda BouraouiAlain MazéJosef DeutscherXavier De BolleJean-Jacques LetessonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 9 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marie Dozot Sandrine Poncet Cécile Nicolas Richard Copin Houda Bouraoui Alain Mazé Josef Deutscher Xavier De Bolle Jean-Jacques Letesson Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
description	<h4>Background</h4>In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions.<h4>Methodology/principal findings</h4>In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase.<h4>Conclusions/significance</h4>The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism.
format	article
author	Marie Dozot Sandrine Poncet Cécile Nicolas Richard Copin Houda Bouraoui Alain Mazé Josef Deutscher Xavier De Bolle Jean-Jacques Letesson
author_facet	Marie Dozot Sandrine Poncet Cécile Nicolas Richard Copin Houda Bouraoui Alain Mazé Josef Deutscher Xavier De Bolle Jean-Jacques Letesson
author_sort	Marie Dozot
title	Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
title_short	Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
title_full	Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
title_fullStr	Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
title_full_unstemmed	Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
title_sort	functional characterization of the incomplete phosphotransferase system (pts) of the intracellular pathogen brucella melitensis.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/6bc0d01cef304af3ac9018d6e5ac8036
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Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.

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