Enhanced transformation efficiency in Treponema denticola enabled by SyngenicDNA-based plasmids lacking restriction-modification target motifs.
Treponema
Treponema denticola
transformation
Journal
Molecular oral microbiology
ISSN: 2041-1014
Titre abrégé: Mol Oral Microbiol
Pays: Denmark
ID NLM: 101524770
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
revised:
25
09
2023
received:
09
08
2023
accepted:
08
10
2023
medline:
30
11
2023
pubmed:
26
10
2023
entrez:
26
10
2023
Statut:
ppublish
Résumé
Oral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated that Treponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oral Treponema have been detected, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus, T. denticola is a key model organism for studying spirochete metabolic processes, interactions with other microbes, and host cell and tissue responses relevant to oral diseases, as well as venereal and nonvenereal treponematoses whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using an Escherichia coli-T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction-modification (R-M) systems and designing a high-efficiency R-M-silent "SyngenicDNA" shuttle plasmid resistant to all T. denticola ATCC 35405 R-M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R-M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R-M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.
Substances chimiques
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Pagination
455-470Subventions
Organisme : NIDCR NIH HHS
ID : R01 DE025225
Pays : United States
Organisme : NIDCR NIH HHS
ID : R01 DE027850
Pays : United States
Informations de copyright
© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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