Deficiency of MecA in Streptococcus mutans Causes Major Defects in Cell Envelope Biogenesis, Cell Division, and Biofilm Formation

Abstract

MecA is an adaptor protein that guides the ClpC/P-mediated proteolysis. A S. mutans MecA-deficient mutant was constructed by double-crossover allelic exchange and analyzed for the effects of such a deficiency on cell biology and biofilm formation. Unlike the wild-type, UA159, the mecA mutant, TW416, formed mucoid and smooth colonies, severely clumped in broth and had a reduced growth rate. Transmission electron microscopy analysis revealed that TW416 grows primarily in chains of giant "swollen" cells with multiple asymmetric septa, unlike the coccoid form of UA159. As compared to UA159, biofilm formation by TW416 was significantly reduced regardless of the carbohydrate sources used for growth (P < 0.001). Western blot analysis of TW416 whole cell lysates showed a reduced expression of the glucosyltransferase GtfC and GtfB, as well as the P1 and WapA adhesins providing an explanation for the defective biofilm formation of TW416. When analyzed by a colorimetric assay, the cell wall phosphate of the mutant murein sacculi was almost 20-fold lower than the parent strain (P < 0.001). Interestingly, however, when analyzed using immunoblotting of the murein sacculi preps with UA159 whole cell antiserum as a probe, TW416 was shown to possess significantly higher signal intensity as compared to the wild-type. There is also evidence that MecA in S. mutans is more than an adaptor protein, although how it modulates the bacterial pathophysiology, including cell envelope biogenesis, cell division, and biofilm formation awaits further investigation.

Keywords: Clp protease complex; MecA; Streptococcus mutans; biofilms; cell envelope biogenesis; stress tolerance response.

FIGURE 1

The schematic diagram of the mecA flanking region (A) and RT-PCR analysis (B). (A) Schematic diagram of the flanking regions of mecA in the genome of S. mutans UA159. Numbers underneath represent the size of the respective genes. Arrows above and underneath indicate the location and orientation of the primers used for reverse transcription (5) and cDNA amplification (F and R). (B) RT-PCR analysis of the mecA/rgpG cluster. cDNA generated from reverse transcription using total RNA extract of S. mutans was PCR amplified using primer set PmecARgpG (lane 1), which shows a single amplicon indicative of co-transcription of mecA and rgpG. Negative control (lane 2) with no reverse transcriptase shows no DNA amplification. M, for molecular marker.

FIGURE 2

Colony morphology of the mecA mutant. S. mutans wild-type (UA159), the mecA mutant (TW416) and its complement strain (TW416C) were grown on BHI agar plates. Relative to the rough, dry colonies of UA159, the colonies of TW416 were round, mucoid, and smooth. Images were taken using Samsung Galaxy Note V.

FIGURE 3

Growth characterization of the mecA mutant. (A) Growth phenotypes of S. mutans wild-type (UA159), the mecA mutant (TW416) and its complement strain (TW416C) when grown in BHI broth overnight. Arrow indicates severe clumping of the mecA mutant at the tip of the tooth pick. (B) S. mutans strains grown in BHI broth, BHI broth with pH adjusted to 6.0 and BHI broth with inclusion of methyl viologen at 12.5 mM. The optical densities of the cultures at 600 nm were recorded continuously using a Bioscreen C. Data presented in panel (B) are representatives of three separate experiments.

FIGURE 4

TEM analysis. S. mutans wildtype (UA159 and A&D), the mecA mutant (TW416 and B&E) and its mecA complement strain (TW416C and C&F) were grown in BHI broth until mid-exponential phase (OD_600nm ≈0.4). (A,B,C) were images taken at magnification of 10,000 × g, and (D,EF) at 25,000×, respectively. Scale bars represent 500 nm. Images in (G) are inserts of blow-up regions of the cell envelope of the different strains with the mutant showing a fuzzy, loose cell envelope (ce) and a thin cytoplasmic membrane (cm), as indicated. The arrows in (E) indicate the fuzzy, loose cell envelope and the asterisks indicating low-density patches of the deficient mutant.

FIGURE 5

Biofilm formation. S. mutans wildtype (UA159), the mecA mutant (TW416) and its complement strain (TW416C) were grown in BM medium with glucose and sucrose (BMGS), glucose (BMG) or sucrose (BMS). Biofilms were grown on polystyrene surface in 96 well plates and analyzed using a spectrophotometer. Results presented here represent mean absorbance at 575 nm (±standard deviation in error bars) from three independent experiments and ^∗ and ^#P < 0.01, P < 0.05, respectively.

FIGURE 6

Confocal microscopic analysis of biofilms. S. mutans wildtype (UA159), the mecA mutant (TW416) and its complement strain (TW416C) were grown in BM medium with glucose and sucrose, glucose or sucrose. Biofilms were grown on HA disks vertically placed in 12 well plates for 24 h, and analyzed using a laser scanning confocal microscope. Panel shows representatives of the compressed confocal images at xy, yz, and xz axis of biofilms of UA159, TW416, and TW416C grown in BM plus glucose and sucrose.

FIGURE 7

SEM analysis of biofilms. S. mutans wildtype (UA159), the mecA mutant (TW416) and its complement strain (TW416C) were grown in BM medium with glucose and sucrose, glucose or sucrose. Biofilms were grown on HA disks vertically placed in 12 well plates for 24 h, and analyzed using a scanning electron microscope (SEM). Panel shows images of UA159 and TW416 biofilms grown in BM plus glucose and sucrose, which were taken at magnification of 5,000 and 10,000 × g as indicated, with arrows indicating extracellular polymeric substances and asterisks indicating broken cells and cell debris, respectively.

FIGURE 8

Acid and hydrogen peroxide challenge assays. S. mutans strains were grown until mid-exponential phase (OD_600nm ≈0.3) and then subjected to an acid or hydrogen peroxide killing. (A) shows survival rate of wildtype (UA159), the mecA mutant (TW416) and its complement strain (TW416C) following incubation in glycin buffer of pH 2.8, while (B) shows survival rate of the strains in the presence of hydrogen peroxide. Data represented here are means (±standard deviation) of at least three independent experiments, with ^∗P < 0.001 as comparing to the wild-type.

FIGURE 9

Western blot and slot blot assays. For Western blot analysis of AtlA and WapA, whole cell lysates (10 μg total protein) were separated using 12% SDS–PAGE (A) Commassie Blue-stained gel, and following blotting onto a PVDF membrane, probed using polyclonal antibodies against autolysin AtlA (B) and WapA (C). For analysis of GtfB, GtfC an SpaP, total proteins (10 μg) were separated using an 8% resolving gel (D) Commassie Blue-stained gel and probed using polyclonal antibodies against GtfB (E) and GtfC (F) and mixture of monoclonal antibodies against SpaP (G). (H) For analysis of cell envelope antigens, preps extracted from murein sacculi were blotted onto a nitrocellulose membrane using a Slot Blot and probed using rgpG/brpA/psr triple mutant adsorbed whole cell antiserum. Lanes 1, 2, and 3 represents cell lysate (A–G) or cell envelope associated antigen extract (H) from the wildtype, TW416, and TW416C, respectively.