pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

Abstract

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5=-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola. Treponema denticola is an obligatory anaerobic and highly motile bacterium that is associated with human periodontitis (for a review, see references 1-3), which is a chronic inflammatory disease that damages the supporting connective tissues around the teeth and ultimately leads to tooth loss. The use of targeted mutagenesis followed by phenotypic characterizations in vitro and in vivo is a routine method for identifying bacterial virulence factors and elucidating their roles in bacterial pathogenicity. Due to its fastidious growth requirements, only a few virulence factors have been characterized in T. denticola (for a review, see references 4 and 5). During the last decade, although tremendous efforts have been devoted to the identification of genetic tools, only a few have been developed for the genetic manipulation of T. denticola (6-10). Even now, genetic manipulation (e.g., gene deletion) of T. denticola is still inefficient and cumbersome (7,11). In addition, all of the current genetic tools for T. denticola are built upon positive selection by inserting an antibiotic resistance marker into a targeted gene on the chromosome (6,8,9,12). The drawback of this method is that the insertion of an antibiotic resistance cassette often impairs downstream gene expression, particularly for a gene within a large polycistronic gene cluster. To precisely interpret the function of a targeted gene, the cognate mutant has to be genetically complemented by reintroducing the targeted gene either back into the chromosome or to the cells through a shuttle vector. However, T. denticola is not amenable to the introduction of exogenous genetic el...

Atypical chemoreceptor arrays accommodate high membrane curvature

“…The primers used here are listed in Table S3 . To delete TDE1492 , the plasmid of TDE1492::ermB was transformed into Td wild-type competent cells via heat shock for 1 min at 50 °C 34 . Erythromycin-resistance colonies that appeared on the plates were screened by PCR for the presence of ermB and absence of TDE1492 (781 – 1308 nt) gene.…”

Atypical chemoreceptor arrays accommodate high membrane curvature

“…The primers used here are listed in Table S3. To delete TDE1492, the plasmid of TDE1492::ermB was transformed into Td wild-type competent cells via heat shock, as previously described 4 . Erythromycin-resistance colonies that appeared on the plates were screened by PCR for the presence of ermB and absence of TDE1492 (781-1,308 nt) gene.…”

Transcriptional and functional characterizations of multiple flagellin genes in spirochetes

“…The resultant gels were subjected to Coomassie blue staining or immunoblotting analyses. The antibodies against T. pallidum FlaBs and T. denticola FlaA and DnaK are described in our previous publications (Kurniyati et al, 2017; Kurniyati & Li, 2016). The stoichiometry of each flagellar filament protein was analyzed using Image Lab software from Bio‐Rad (Bio‐Rad).…”