Table 1.
Bacterial strains, plasmids and primers used in this study.
Fig 1.
Growth study under different pHs.
S. mutans wild-type (UA159, solid squares), PBP1a-deficient mutant (JB467, open circles), and the complement strain (JB467C, solid circles) were grown in BHI adjusted to pH 7.2 (A), pH 6.2 (B), and pH 8.5 (C) in Bioscreen C with sterile mineral oil overlay, and the culture optical densities were monitored every 30 minutes at 600nm. The results suggest that PBP1a-deficeincy affects growth rate and cell yield in optical density at 600 nm, especially in BHI adjusted to higher pH. The data presented here are representatives of three independent experiments.
Fig 2.
Results of pH drop experiments showed that relative to S. mutans UA159 (triangles), the PBP1a-deficient mutant, JB467 (squares), had a slower pH drop and a higher resting pH after 20 minutes (#, P<0.01). Complementation with wild-type pbp1a in JB467C (open cycles) was able to restore the phenotype to the wild-type, UA159.
Fig 3.
Acid (A) and hydrogen peroxide (B) killing assays.
S. mutans wild-type (UA159, squares), PBP1a mutant (JB467, circles), and the complement strain (JB467C, triangles) were grown in BHI until mid-exponential phase (OD600nm≅0.3), washed and then subjected to acid and hydrogen peroxide killing assays by incubating the cells in buffer of pH 2.8 and buffer containing 0.2% (w/v) hydrogen peroxide, respectively, for periods of time as indicated. Results showed that PBP1a deficiency in JB467 weakens the ability of the deficient mutant to tolerate low pH (A) and hydrogen peroxide (B), when compared to the wild-type. Data presented here are representatives of three independent experiments. Significant difference is indicated by * and # at P<0.001 and P<0.01, respectively.
Fig 4.
Growth study in the presence of paraquat.
S. mutans wild-type (UA159, squares), PBP1a mutant (JB467, open circles), and the complement strain (JB467C, triangles) in the presence of increasing amounts of paraquat, and growth was continuously monitored using Bioscreen C with sterile mineral oil overlay. The data presented here are representative of three independent experiments, showing the increased susceptibility of the PBP1a-deficient strain, JB467, to 0.1 mM (A) and 5 mM (B) of paraquat (P<0.001, as indicated by *).
Fig 5.
Overnight cultures of S. mutans wild-type (UA159, diamonds), PBP1a mutant (JB467, open circles), and its complement strain (JB467C, triangles) were washed and resuspended in phosphate buffered saline, pH 7.2 with and without inclusion of Triton X-100 (TX, 0.2%), and the optical density of these cells was then monitored using Bioscreen C. Relative to wild-type UA159, PBP1a mutant JB467 displayed a significantly quicker and more severe reduction of the optical density over the time of incubation (indicated by a asterisk, P<0.01). The graph shows results with the presence of Triton X-100 and is representative of more than three independent experiments.
Fig 6.
S. mutans wild-type (UA159), PBP1a-deficient mutant (JB467), and the complement strain (JB467C) were grown in 96-well plates or HA discs in biofilm media with glucose (G), sucrose (S), and glucose plus sucrose (GS). Biofilms in 96-well plates were quantified by spectrophotometry following crystal violet staining (A) and 24 hour biofilms on HA discs were analyzed using FE-SEM (B). The results demonstrate that PBP1a-deficiency significantly decreased biofilm formation by the deficient mutant (* indicates differences at P<0.001 when compared to the wild-type, UA159 under the same conditions). Unlike the robust, evenly distributed biofilms of the wild-type, UA159, biofilms of PBP1a mutant, JB467 were sparse and featured with elongated giant cells. In addition, the biofilms of the PBP1a mutant also possessed significantly bigger and seemingly more eDNA nanofibers (indicated by arrows). FE-SEM micrographs were taken at 20k× magnification.
Fig 7.
S. mutans strains wild-type (UA159), PBP1a-deficient mutant (JB467), and the complement strain (JB467C) were grown in BHI, pH 7.4 to mid-exponential phase (OD600nm≈0.3). Panel A shows differences in cell morphology with JB467 displaying elongated cells, defects in cell separation and cells with multiple septa. Images were taken at 20,000x (20k) and 50,000x (50k). Panel B highlights JB467 with a thinner layer of peptidoglycan (indicated by arrows), when compared to the wild-type and the complement strain.