mCherry fusions enable the subcellular localization of periplasmic and cytoplasmic proteins in Xanthomonas sp.
Arabinose
/ pharmacology
Bacterial Proteins
/ metabolism
Chromosomes, Bacterial
/ genetics
Cytoplasm
/ metabolism
Genetic Vectors
/ metabolism
Microbial Viability
/ drug effects
Periplasm
/ metabolism
Protein Transport
/ drug effects
Recombinant Fusion Proteins
/ metabolism
Starch
/ metabolism
Subcellular Fractions
/ drug effects
Xanthomonas
/ metabolism
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2020
2020
Historique:
received:
23
03
2020
accepted:
30
06
2020
entrez:
31
7
2020
pubmed:
31
7
2020
medline:
23
9
2020
Statut:
epublish
Résumé
Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.
Identifiants
pubmed: 32730344
doi: 10.1371/journal.pone.0236185
pii: PONE-D-20-08193
pmc: PMC7392301
doi:
Substances chimiques
Bacterial Proteins
0
Recombinant Fusion Proteins
0
Starch
9005-25-8
Arabinose
B40ROO395Z
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0236185Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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