Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans.
Reporter genes have become standard genetic tools used to evaluate either the transcriptional or the translational activity associated with genes of interest, whose products cannot be easily assayed. The lacZ gene from Escherichia coli has been used very effectively to quantify such regulated activities in many different organisms. This report describes the construction of a pair of plasmids that may be used for either transcriptional or translational lacZ gene fusions in Streptococcus mutans. The translational E. coli beta-galactosidase gene (lacZ) fusion plasmid, pALH109, as well as the transcriptional lacZ gene fusion plasmid, pALH122, have been used successfully in S. mutans to measure the activity of various PTS genes. Both plasmids employ fusions with the E. coli lacZ gene that can be easily quantified using standard O-nitrophenyl-beta-D-galactopyranoside (ONPG) based enzyme assays or the more sensitive fluorometric assays using 4-methyl-umbelliferyl beta-D-galactopyranoside (MUG) as the enzyme substrate. Currently, there has been only one other report of the use of lacZ as a gene reporter in S. mutans. The plasmids described in this paper will provide new tools and techniques for the analysis of S. mutans gene regulation. In addition, we have compiled the complete nucleotide sequences of these gene reporter plasmids.
Honeyman
Allen L
AL
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA. ahoneyma@hsc.usf.edu
Cote
Christopher K
CK
Curtiss
Roy
R
D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D017930:Genes, Reporter / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000138:chemical synthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013295:Streptococcus mutans / Q000737:chemistry / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis
D004269:DNA, Bacterial; D001616:beta-Galactosidase