A tightly controlled gene induction system that contributes to the study of lethal gene function in Streptococcus mutans

Abstract

Effective control of gene expression is crucial for understanding gene function in both eukaryotic and prokaryotic cells. While several inducible gene expression systems have been reported in Streptococcus mutans, a conditional pathogen that causes dental caries, the significant non-inducible basal expression in these systems seriously limits their utility, especially when studying lethal gene functions and molecular mechanisms. We introduce a tightly controlled xylose-inducible gene expression system, TC-Xyl, for Streptococcus mutans. Western blot results and fluorescence microscopy analysis indicate that TC-Xyl exhibits an extremely low non-inducible basal expression level and a sufficiently high expression level post-induction. Further, by constructing a mutation in which the only source FtsZ is under the control of TC-Xyl, we preliminarily explored the function of the ftsz gene. We found that FtsZ depletion is lethal to Streptococcus mutans, resulting in abnormal round cell shape and mini cell formation, suggesting FtsZ's role in maintaining cell shape stability.

Keywords: FtsZ; Gene induction system; Streptococcus mutans; lethal gene function; xylose-induction.

Figure 1.

Schematic and sequence of the TC-Xyl. (a) Illustration of the genetic organization of induction elements in the TC-Xyl and Xyl-S1, the schematic diagram of Xyl-S1 was adapted from ref 10. (b) The sequence of the repetitive sequence of xylAo, the xylAo are marked in red box, promoter elements are shown in blue. (c) Displays the schema for TC-Xyl construction. Xyl-S1 and TC-Xyl are marked by green and blue dot line box, respectively. (d) The plasmid map of pYL01. The proportions in the schematic do not represent the actual proportions.

Figure 2.

TC-Xyl shows tightly controlled gene-inducible expression. (a) the changes in relative mRNA expression of EGFP and mNeongreen in UA159 wild-type strains were investigated via real-time PCR. F-E: FtsZ_EGFP; F-m: FtsZ_mNeongreen. WT: wild type. ‘***’ p value < 0.001; ‘**’ p value < 0.01. ‘ns’ no significance. The p value was determined via student’s t-test. (b) the protein expression levels of exogenous FtsZ (exo FtsZ) under the control of TC-Xyl, Xyl-S1, and Xyl-S2 in UA159 wild-type strains were examined by Western blot. Endogenous FtsZ (endo FtsZ) was used as reference. For group of Xyl-S1, the whole cell proteins were set as reference and detected by anti-sm. (c) the expression of FtsZ_mNeongreen under the control of TC-Xyl in the presence of different concentration xylose were analyzed by Western blot. (d) the expression of FtsZ_mNeongreen in WT was analyzed by the fluorescent microscope. The cell morphology pictures were collected via bright field illumination. mNeongreen was excited by a 488 nm laser. Scale bar: 2 μm. (e) the signal to background ratios before and after induction were analyzed. The mean with SD were shown.

Figure 3.

Growth phenotypes of FtsZ depletion in S. mutans. (a) Growth of different dilutions of strain YL001 and YL002 on BHI plate with or without xylose. (b) The cell morphology change after FtsZ depletion were analyzed by live cell microscope. Red dot line box marked the enlarged area. Scale bar: 2 μm.