Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications.
M Paula Goetting-MineskyJordan KimDuane T WhiteMichael A L HayashiAlexander H RickardJ Christopher FennoPublished in: Letters in applied microbiology (2024)
Oral Veillonella species are among the early colonizers of the human oral cavity. We constructed a small, single-selectable-marker shuttle plasmid, examined its ability to be transformed into diverse oral Veillonella strains, and assessed its potential use for expressing a gene encoding an oxygen-independent fluorescent protein, thus generating a fluorescent Veillonella parvula strain. Because tetracycline resistance is common in Veillonella, we replaced genes encoding ampicillin- and tetracycline-resistance in a previously described shuttle plasmid (pBSJL2) with a chloramphenicol acetyltransferase gene. The resulting plasmid pCF1135 was successfully introduced into four strains representing V. parvula and V. atypica by either natural transformation or electroporation. We then modified this plasmid to express a gene encoding an oxygen-independent fluorescent protein in V. parvula SKV38. The resulting strain yielded a fluorescence signal intensity ∼16 times higher than the wild type in microplate-based fluorimetry experiments. While fluorescence microscopy demonstrated that planktonic cells, colonies, and biofilms of fluorescent V. parvula could also be imaged, photobleaching was a significant issue. In conclusion, we anticipate this genetic system and information provided here will facilitate expanded studies of oral Veillonella species' properties and behavior.
Keyphrases
- escherichia coli
- genome wide
- single molecule
- quantum dots
- living cells
- crispr cas
- copy number
- genome wide identification
- label free
- wild type
- energy transfer
- induced apoptosis
- endothelial cells
- high resolution
- dna methylation
- healthcare
- gene expression
- protein protein
- transcription factor
- genome wide analysis
- optical coherence tomography
- oxidative stress
- binding protein
- cell cycle arrest
- cell death
- risk assessment
- fluorescent probe
- high throughput
- social media
- climate change
- endoplasmic reticulum stress
- mass spectrometry
- genetic diversity
- pluripotent stem cells