Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications

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-in...

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Bibliographic Details
Published in:	Letters in applied microbiology Vol. 77; no. 8
Main Authors:	Goetting-Minesky, M Paula, Kim, Jordan, White, Duane T, Hayashi, Michael, Rickard, Alexander H, Fenno, J Christopher
Format:	Journal Article
Language:	English
Published:	England 05-08-2024
Subjects:	Biofilms - growth & development Electroporation Fluorescence Genetic Vectors Humans Luminescent Proteins - genetics Microscopy, Fluorescence Mouth - microbiology Plasmids - genetics Tetracycline Resistance - genetics Veillonella - genetics bacterial biofilms transformation anaerobes plasmid biology molecular genetic
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1472-765X 1472-765X
DOI:	10.1093/lambio/ovae069