revised comprehensive mutational analysis of sucrose-metabolizing

1

REVISED 1 2 3 4 5

COMPREHENSIVE MUTATIONAL ANALYSIS OF SUCROSE-METABOLIZING

PATHWAYS IN STREPTOCOCCUS MUTANS REVEALS NOVEL ROLES FOR THE

SUCROSE PTS PERMEASE

Lin Zeng and Robert A. Burne*

Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, 6 Florida 32610. 7

Running title: Engineered sucrase-deficient S. mutans

Key words: Sugar:phosphotransferase system, carbon catabolite repression, biofilm,

polysaccharide, dental caries

* Corresponding author

Mailing address: Department of Oral Biology, University of Florida, College of

Dentistry, P.O. Box 100424, Gainesville, FL 32610.

Phone: (352) 273-8850

Fax: (352) 273-8829

E-mail: [email protected].

8

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.02042-12 JB Accepts, published online ahead of print on 7 December 2012

on January 31, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

2

Abstract 9 Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries 10

in humans, and the primary caries pathogen Streptococcus mutans encodes multiple 11 enzymes involved in the metabolism of this disaccharide. Here, we engineered a series 12 of mutants lacking individual or combinations of sucrolytic pathways to understand 13 control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for 14 sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD 15 (glucan exopolysaccharide synthases), ftf (fructan exopolysaccharide synthase) and the 16 scrAB pathway (sucrose-PTS permease, sucrose-6-PO4 hydrolase) constitute the 17 majority of the sucrose-catabolizing activity; however mutations in any one of these 18 genes alone did not affect planktonic growth on sucrose. The multiple sugar metabolism 19 pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking 20 gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic 21 growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on 22 fructooligosaccharides, due in large part to increased expression of 23 the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction 24 system as a model for carbohydrate-dependent gene expression in strains lacking 25 extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with 26 sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose 27 through components of the LevQRST complex, but not for activation by the cognate 28 LevQRST sugars, fructose or mannose. Sucrose-dependent catabolite repression was 29 also evident in strains containing an intact sucrose PTS. Collectively, these results 30 reveal novel regulatory circuitry for control of sucrose catabolism, with a central role for 31 ScrA. 32


.org/D

ownloaded from

http://jb.asm.org/

3

Introduction 33 Sucrose is among the most cariogenic carbohydrates (1, 2) and this disaccharide, 34

composed of β2,1-linked fructose and glucose, influences the development of caries in 35 multiple ways. First, sucrose can serve as a readily metabolizable carbon and energy 36 source for many members of the oral microbiome, and is a particularly effective 37 substrate for generation of organic acids via glycolysis by the abundant oral streptococci 38 and Actinomyces spp. that comprise a large proportion of the oral microbiome. Many 39 oral bacteria possess transport systems for sucrose, but can also hydrolyze sucrose 40 outside the cell. Sucrose is a substrate for a variety of glucosyltransferase enzymes 41 (GTFs) that are secreted mainly by oral streptococci. For example, the GTF enzymes of 42 Streptococcus mutans, the primary etiologic agent of human dental caries, release free 43 fructose and form high molecular mass α1,3- and α1,6-linked homopolymers of glucose, 44 commonly called glucans or mutan, which act as an adhesive scaffolding to promote the 45 formation of oral biofilms, particularly on smooth surfaces of the teeth (3, 4). Many oral 46 streptococci and certain Actinomyces spp. also produce fructosyltransferase (FTF) 47 enzymes that convert sucrose into homopolymers of fructose (fructans) and free glucose 48 (5, 6). Fructan polymers appear to serve mainly as extracellular energy storage 49 compounds that can be hydrolyzed by fructanase enzymes to extend the depth and 50 duration of oral biofilm acidification (7-9). 51

In S. mutans, sucrose can be internalized by the sugar:phosphotransferase system 52 (PTS) via a high-affinity permease, EIIScr, encoded by the scrA gene (10, 11). 53 Intracellular metabolism of sucrose-6-PO4 is then initiated by ScrB, a sucrose-6-PO4 54 hydrolase, to produce glucose-6- PO4 and fructose. A fructokinase (ScrK) encoded 55 immediately downstream of scrA can channel the fructose into the glycolytic pathway 56 (see Figure S1 of supplementary material for diagram) (12, 13). The expression of both 57


.org/D

ownloaded from

http://jb.asm.org/

4

scrA and scrB is under the control of a negative regulator, ScrR, encoded downstream of 58 scrB that binds to the promoter regions of scrA and scrB, and sucrose-grown cells 59 showed the highest expression levels of scrAB (12). Another PTS permease EIITre that 60 appears to be the primary transporter for trehalose has been suggested to internalize 61 sucrose in S. mutans GS-5 (14). Besides the PTS, two binding-protein-dependent 62 carbohydrate transport systems, the multiple sugar metabolism system (Msm) (15, 16) 63 and the maltose/maltodextrin ABC transporter (17) have been implicated in sucrose 64 uptake by S. mutans. The metabolism of sucrose after internalization via ABC 65 transporters likely involves the sucrose phosphorylase GtfA, which converts sucrose into 66 glucose-1-PO4 and fructose (18). 67

Three major GTFs are encoded in S. mutans: GtfB and GtfC produce predominantly 68 α1,3-linked water-insoluble glucans and GtfD produces an α1,6-rich, water-soluble 69 glucan (4). The FTF (6) of S. mutans converts sucrose into a predominantly β2,1-linked 70 inulin-type fructan polymer, although many oral bacteria make a levan-type β2,6-linked 71 fructan. Both types of polymers can be hydrolyzed by S. mutans by a single secreted 72 exo-β-D-fructosidase (FruA)(7, 19, 20). Expression of fruA is governed by an unusual 73 four-component regulatory system that is composed of a conventional histidine kinase 74 (LevS) and response regulator (LevR) and two membrane-associated carbohydrate-75 binding proteins (LevQ and LevT) that work in concert with LevS and LevR (21-24). The 76 same regulatory system also controls expression of the levDEFG operon that encodes 77 the A, B, C and D domains of a fructose/mannose-PTS EII permease (24). 78

Prioritization of carbohydrate utilization in the human oral cavity is a critical function 79 and is achieved in large part through carbon catabolite repression (CCR), the selective 80 expression of genes involved in the uptake and metabolism of preferred carbohydrates 81 coupled with repression of alternative catabolic pathways (25). In most low-G+C Gram-82


.org/D

ownloaded from

http://jb.asm.org/

5

positive bacteria, CCR is primarily effected by catabolite control protein A, CcpA, which 83 regulates gene expression by binding to conserved catabolite response elements (CRE) 84 in the promoter regions of CCR-sensitive genes, although many alternative mechanisms 85 to selectively regulate carbohydrate uptake and catabolic pathways exist. In S. mutans, 86 CcpA does play an important role in global control of gene expression (26), but CcpA-87 independent mechanisms involving the PTS phospho-carrier protein HPr and various EII 88 permeases play dominant roles in CCR; regulating catabolic pathways such as the fruAB 89 operon, the fructose/mannose-PTS encoded by levDEFG (22, 27), and transport and 90 catabolism of cellobiose (28) and lactose (29). There are data to support that sucrose 91 may be a preferred carbohydrate source that can elicit CCR in S. mutans (30-33). For 92 example, a microarray-based transcriptomic analysis (30) indicated that growth in 93 sucrose resulted in two- to three-fold lower expression of the maltose/maltodextrin ABC 94 transporter (SMU.1568-1571) and the msm operon. In contrast, expression of the scrAB 95 cluster was found to be relatively constant in cells growing on various carbohydrates 96 (30). 97

The inherent complexity of multiple overlapping systems for sucrose dissimilation has 98 been the primary impediment to the development of a clear understanding of control of 99 sucrose-metabolism and how the presence of sucrose exerts its effects on gene 100 expression and virulence of S. mutans. In particular, there have not yet been any 101 mutants of S. mutans constructed or identified that completely lack the ability to grow on 102 sucrose. In addition, GTFs, FTF and FruA are capable of releasing free fructose and 103 glucose in the extracellular environment, which can affect the expression of PTS porters 104 and trigger CCR. Further, production of glucan leads to clumping of bacterial cells, which 105 makes it difficult to monitor planktonic growth and to be confident that factors other than 106 the growth carbohydrate, for example cell density or quorum sensing pathways, are 107 inducing the observed changes in gene expression and cell behavior. In this study, we 108


.org/D

ownloaded from

http://jb.asm.org/

6

overcame many of these challenges by engineering a series of mutants deficient in 109 various combinations of sucrose-utilizing enzymes. The results provide some definitive 110 answers for long-standing questions about sucrose metabolism and its role in gene 111 expression, and provide new insights into the ability of S. mutans to regulate gene 112 expression in response to sucrose availability. 113 114

Materials and Methods 115 Bacterial strains and culture conditions. S. mutans strain UA159 and various mutant 116 derivatives of this strain were maintained on brain heart infusion (BHI; Difco 117 Laboratories, Detroit, MI) agar plates and routinely cultured in liquid BHI medium at 37°C 118 in a 5% CO2 - 95% air incubator. Escherichia coli strain DH10B was maintained in Luria-119 Bertani medium at 37°C in air. BHI was supplemented with antibiotics when needed at 120 the following concentrations: kanamycin (Km) 1 mg/mL, erythromycin (Em) 10 µg/mL, 121 spectinomycin (Sp) 1 mg/mL, and tetracycline (Tc) 10 µg/mL. 122

When preparing cells for chloramphenicol acetyltransferase (CAT) assays (34) and 123 RNA extraction, S. mutans strains were first cultured overnight in tryptone-vitamin (TV) 124 base medium (21) supplemented with various carbohydrates, sub-cultured in the same 125 fresh medium by diluting 50 fold and then incubated till the optical density (OD600) 126 reaches ≈ 0.5. Two different types of fructooligosaccharides (FOS) were purchased: an 127 FOS powder (NutraFlora FOS) from The Vitamin Shoppe (North Bergen, NJ) that was 128 enzymatically prepared from sucrose, hence the major component of this FOS was of 129 the GFn-type (35); and an inulin-based FOS (Fn-FOS) from Jarrow Formulas (Los 130 Angeles, CA) that was derived from partial hydrolysis of long-chain inulins. To eliminate 131 sucrose contamination, a 10% (w/v) GFn-FOS solution was treated with 1 mg/mL of 132 invertase (from yeast; Sigma, St. Louis, MO) at 37°C for 1 h, passed through a sterile 133 0.2 µm filter and added to TV base medium at a concentration of 0.5% (w/v). For 134


.org/D

ownloaded from

http://jb.asm.org/

7

monitoring of growth, S. mutans strains were pre-cultured overnight in BHI, sub-cultured 135 in BHI until the OD600 reached 0.4~0.5, and inoculated at a dilution of 1:300 into fresh TV 136 medium containing the desired carbohydrates. The optical density of the cultures was 137 monitored over a period of 24 to 48 h using a Bioscreen C (OyGrowth Curves AB, 138 Helsinki, Finland) with individual wells covered with sterile mineral oil and maintained at 139 37°C. 140

To explore effects of carbohydrates on catabolite repression and gene expression in 141 strains carrying reporter gene fusions, cells were cultivated overnight in a 5% CO2 142 aerobic atmosphere at 37°C with TV-base medium supplemented with 0.5% of 143 galactose. Subsequently, the cultures were diluted 1:12 into fresh TV-galactose medium 144 and incubated for 2 to 2.5 h to allow the cultures to reach an OD600 of 0.2. Various 145 concentrations of sucrose, fructose, mannose or glucose were then added to the culture 146 medium and cells were incubated for an additional 3.5 h before harvesting and 147 immediately freezing at -80°C. 148 149 DNA manipulation and mutant strain construction. Standard techniques were 150 applied for preparation and manipulation of DNA (36). All DNA restriction and modifying 151 enzymes were purchased from New England Biolabs (Beverly, MA) and used as 152 recommended by the supplier. Oligonucleotides for PCR amplifications were custom-153 synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). 154

Multiple mutagenesis steps were carried out to construct various recombinant S. 155 mutans strains, each achieved by transformation using competent cells prepared by 156 growth in the presence of 10% horse serum and 100 nM competence-stimulating 157 peptide (CSP) in BHI medium (37). First, an allelic-exchange mutagenesis was 158 performed in the wild-type strain UA159, using ligation products of DNA fragments 159 containing the flanking sequences of gtfBC and a Tc-resistant marker inserted in 160


.org/D

ownloaded from

http://jb.asm.org/

8

between. Briefly, DNA oligonucleotide primers (for upper flanking fragment: gtfBC-1, 5’- 161 GCT AAA GTT GGA GTT TGT AAT CTC C -3’ and gtfBC-2, 5’- GAC GAT ACC AAC 162 TTT CGG CTG TCA A -3’; for lower fragment: gtfBC-3, 5’- GGG ACT CCT GTT GCA 163 GGA AGT CA -3’ and gtfBC-4, 5’- CTT AGA TGT CCA TCT GCA TCT CTG ATA -3’) 164 were used to PCR-amplify two DNA fragments. After digestion of the upper and lower 165 fragments with BclI and BamHI, respectively, the DNA fragments were purified and put 166 into a ligation mixture together with a Tc cassette released from plasmid pLN2 (9) with 167 BamHI digestion. After transformation, Tc-resistant colonies were confirmed by PCR 168 coupled with DNA sequencing to rule out the introduction of any unintended mutations 169 into the regions flanking gtfBC. Similar deletions of the gtfA (em), scrA (em, km or sp), 170 scrB (km), treB (sp), fruA (km) and fruB (sp) were constructed. All three markers, em, km 171 and sp have been used commonly in our genetic study of S. mutans and none have 172 displayed any apparent polar effects (23). On the other hand, a polar km marker (Ωkm) 173 was also used in certain cases when specified. 174

In addition, a PCR-based site-directed mutagenesis technique was applied to UA159 175 to convert the Met9 codon (ATG) of the gtfD coding sequence into a stop codon (TAG). 176 As detailed elsewhere (23), a recombinant PCR reaction was conducted to produce an 177 approximately 2-kbp DNA fragment containing a single gtfDM9stop mutation located 178 near the middle of the fragment. This DNA product (mutator DNA) was used to transform 179 UA159 along with a suicide plasmid (pLacG-em) that inactivates the lacG gene encoding 180 a phospho-β-galactosidase that is required for growth on lactose (29) via a single cross-181 over recombination. After screening the Em-resistant transformants for gtfDM9stop 182 mutations using MAMA-PCR (mismatch amplification mutation analysis) and an allele-183 specific primer (gtfDM9stop-3’MAMA, 5’- ACC CAG TGC TTT TTA ACC TTG TAG AT -184 3’) (38), positive clones were further confirmed by DNA sequencing. 185


.org/D

ownloaded from

http://jb.asm.org/

9

A confirmed clone of gtfDM9stop/lacG:em mutant was then subjected to a second 186 round of site-directed mutagenesis, this time targeting the Met9 codon of the ftf 187 sequence. Similarly, a homologous mutator DNA fragment was created by recombinant 188 PCR to include a ftfM9stop mutation (to TAG); another PCR product was generated 189 using the chromosomal DNA of the gtfBC:Tc mutant (see above) as template to include 190 the gtfBC:Tc marker. After co-transformation of gtfDM9stop/lacG:em with the mutator 191 DNA and gtfBC:Tc marker, Tc-resistant colonies were then screened by MAMA-PCR to 192 identify ftfM9stop mutants. After confirmation by sequencing, 193 gtfBC/gtfDM9stop/ftfM9stop/ lacG:em mutants were patched onto TV-lactose agar plates 194 to select for Em-sensitive revertants that had lost the suicide plasmid pLacG-em, 195 resulting in strain MMZ950 (gtfBC/gtfDM9stop/ftfM9stop). Similarly constructed was a 196 fruAM12stop point mutation (in strains MMZ952 and MMZ966) that had its Met12 codon 197 replaced with a stop codon (TAG). Also a gtfDM9stop strain (MMZ948) was created by 198 curing pLacG-em from gtfDM9stop/lacG:em. All antibiotic replacement and point 199 mutations maintained in various mutant strains in this report have been confirmed by 200 PCR and sequencing. 201 202 RNA preparation and quantitative RT-PCR. Total RNA was extracted from 203 exponentially-growing S. mutans cells using an RNeasy minikit (Qiagen, Germantown, 204 MD), according to procedures detailed elsewhere (39). For preparation of first-strand 205 cDNA, random hexamers were used along with 1 µg total RNA in each 10-µL reverse 206 transcription reaction, following instructions from the supplier (Invitrogen by Life 207 Technologies, Carlsbad, CA). The levels of various mRNA transcripts were quantified by 208 Real-Time PCR using specific primers: for levD, 5’- GGA AGC CCT TTG ACA ACA GC -209 3’ (forward) and 5’- CTG CCA TTG GTA AGT TCA TCC C -3’ (reverse); for gtfD, 5’- 210 CAC AGG CAA AAG CTG AAT TAA CA -3’ (forward) and 5’- GAA TGG CCG CTA AGT 211


.org/D

ownloaded from

http://jb.asm.org/

10

CAA CAG -3’ (reverse); for ftf, 5’- AAA TAT GAA GGC GGC TAC AAC G-3’ (forward) 212 and 5’- CTT CAC CAG TCT TAG CAT CCT GAA -3’ (reverse); and for 16S rRNA as a 213 control, 5’- CAC ACC GCC CGT CAC ACC -3’ (forward) and 5’- CAG CCG CAC CTT 214 CCG ATA CG -3’ (reverse). 215 216 Biochemical assays. When preparing cells for PTS assays, bacterial strains were 217 cultured overnight in BHI medium, diluted 1:50 into fresh BHI and grown to mid-218 exponential phase (OD600 ≈ 0.5). Cells were harvested by centrifugation and immediately 219 frozen at -80°C. CAT (34) and PTS assays (40) were carried out according to previously 220 published protocols and each experiment was repeated with three independent cultures. 221 For the measurement of glucose and fructose in culture supernatant fluids, supernates 222 were first passed through a 0.2 µm filter, followed by incubation in boiling water for 15 223 min to inactivate enzymes, then processed using obtained glucose and fructose assay 224 kits (Sigma). 225 226

Results 227 Growth phenotypes of mutants lacking various sucrose-metabolizing enzymes 228 To begin to investigate the impact of the complement of sucrose-metabolizing enzymes 229 of S. mutans on growth phenotypes, a series of mutants were created employing both 230 marker-dependent and markerless approaches. The panel of mutant strains utilized in 231 this study are described in Table 1 and were first tested for their ability to grow in TV-232 medium supplemented with 0.2% sucrose. 233

GtfBCD and Ftf. Three extracellular glucosyltransferase enzymes produced by S. 234 mutans, GtfB, GtfC and GtfD, convert sucrose into glucans while releasing free fructose. 235 The fructosyltransferase (Ftf) of S. mutans is able to convert exogenous sucrose into 236 fructans and free glucose. Thus, when strains possess these enzymes, extracellular 237


.org/D

ownloaded from

http://jb.asm.org/

11

fructose and glucose are produced for consumption by S. mutans. As shown in Figure 238 1A, loss of gtfD (MMZ948) had no significant impact on the growth of S. mutans in TV 239 containing 0.2% sucrose, whereas a mutant (MMZ945) that was deficient in both gtfB 240 and gtfC showed improved growth on sucrose, compared to the otherwise-isogenic wild-241 type strain UA159. As production of extracellular glucan, especially water-insoluble 242 glucans synthesized by GtfBC, greatly enhances bacterial adherence and clumping, this 243 phenotype of MMZ945 was likely a combination of less aggregation by the cells and less 244 inhibition of growth from accumulation of inhibitory end products within cell aggregates. 245 S. mutans MMZ950, which is deficient in all four exopolysaccharide-producing enzymes 246 (GtfBCD/Ftf), displayed a growth phenotype similar to that of MMZ945 (Figure 1A). 247

EIIScr. S. mutans also possesses a specific sucrose-PTS permease, EIIScr, encoded 248 by a single gene scrA (10). The sucrose-6-PO4 produced by PTS-mediated uptake is 249 cleaved by a sucrose-6-PO4 hydrolase (ScrB) that releases intracellular glucose-6-PO4 250 and fructose. A fructose kinase is encoded immediately downstream of scrA. It has been 251 suggested that the majority of the sucrose that is presented to S. mutans is directly 252 internalized and then catabolized (13, 41). When the entire coding region of scrA was 253 replaced with a non-polar antibiotic marker (em) in the parental strain to create strain 254 MMZ932, there was little change in the ability of the mutant to grow on sucrose (Figure 255 1B). The same was true when the coding sequences of both scrA and scrB were 256 replaced by a nonpolar km or a polar Ωkm cassette (MMZ934 and MMZ957, 257 respectively; data not shown). These results were not surprising, given the presence of 258 multiple GTF and FTF enzymes, as well as FruA and GtfA (sucrose phosphorylase), all 259 of which have been shown to be capable of hydrolyzing sucrose. However, a deletion 260 mutant of scrB alone (MMZ931) presented a sucrose-sensitive phenotype (Figure 1B), 261 consistent with a previous report that suggested that accumulation of sucrose-6- PO4 262 may be toxic to the cells (42). 263


.org/D

ownloaded from

http://jb.asm.org/

12

An interesting phenotype of the scrA mutant MMZ932 was observed when it was 264 growing in TV-base medium supplemented with glucose, fructose, mannose or 265 galactose. In particular, the cells showed excessive clumping in the absence of sucrose 266 (Figure S2 in supplementary material), apparently indicative of pleiotropic effects on the 267 cell that may have influenced envelope composition or cell division in ScrA-deficient 268 strains. 269

An S. mutans strain containing mutations in the gtfBCD/ftf/scrAB genes (MMZ983) 270 was also constructed by introducing a PCR product that contained the replacement of 271 scrAB by the Ωkm cassette into strain MMZ950. Consistent with the current view of the 272 functions of the GTF and FTF enzymes and the ScrAB cluster, very little growth of 273 MMZ983 was noted in TV-sucrose (Figure 1C), indicating that GtfBCD, Ftf and ScrAB 274 are the main contributors to sucrose metabolism by S. mutans. Surprisingly, a mutant 275 strain that lacked only gtfBC and scrAB (MMZ1025) displayed an extended lag phase, 276 ranging from 35 to 45 h, when growing on sucrose (Figure 1D). A previous study 277 indicated that the expression of gtfD is relatively constant between glucose- and 278 sucrose-conditions, and ftf mRNA level is only induced by about 2- to 3-fold by sucrose 279 (32). We confirmed that gtfD and ftf transcript levels were not greatly affected by growth 280 in sucrose (data not shown). Therefore, the behavior of strain MMZ1025 may indicate 281 that, in contrast to GtfB and GtfC, GtfD and Ftf contribute in only minor ways to growth of 282 S. mutans on sucrose under the conditions tested. 283

FruA. When a fruA:km replacement mutation was introduced into S. mutans 284 MMZ950, resulting in strain MMZ984, little change in growth rate on sucrose was noted, 285 although MMZ984 did appear to have a shorter lag phase than MMZ950 (Figure 1C). 286 FruA is an exo-β-D-fructosidase that has been shown to contribute to the virulence of S. 287 mutans in a rat model (9). The preferred substrates of FruA are β2,6-linked levans, but it 288


.org/D

ownloaded from

http://jb.asm.org/

13

is also active on other β-fructosides, including inulins, fructooligosaccharides (FOS), 289 sucrose and raffinose (7). Since expression of the fruAB operon is under tight regulation 290 by CCR (21), it is not surprising that loss of fruA resulted in little change in growth on 291 sucrose. 292

MSM. The multiple-sugar metabolism (msm) pathway in S. mutans has been 293 implicated in the transport and metabolism of sucrose, although this hypothesis has not 294 been directly tested (15, 16). Thus, a new strain (MMZ966) was created in the 295 background of MMZ950 (gtfBCD/ftf) by introducing both the scrAB:Ωkm replacement and 296 a new fruA point mutation that replaced the translational start codon with a stop codon. 297 Subsequently, an msmE:em replacement mutation was introduced into strain MMZ966, 298 resulting in MMZ967. As shown in Figure 2A, similar to strain MMZ983, which produced 299 low levels of growth on sucrose, MMZ966 managed to increase the optical density 300 (OD600) of the culture by only 0.05 units after 48 h of incubation in the presence of 0.2% 301 sucrose. However, strain MMZ967, which further lacks msmE, produced no detectable 302 growth during the same period. Therefore, under conditions used in these growth 303 assays, a minor role was established for the msm system in sucrose catabolism by S. 304 mutans. Furthermore, a mutant lacking the ptsI gene that encodes Enzyme I of the PTS 305 (designated ∆EI)(27) that was constructed in strain UA159 failed to produce substantial 306 growth on sucrose (Figure 2B). Given the fact that glucose but not fructose has been 307 shown to be able to sustain the growth of an EI mutant of S. mutans (43), the largely 308 abolished growth of the ∆EI strain on sucrose is consistent with our view that both FTF, 309 which releases free glucose from sucrose, and the msm pathway contributes very little to 310 sucrose catabolism by S. mutans. 311

FruB. The fruA gene of S. mutans is encoded in an operon with the fruB gene, a 312 predicted β-fructosidase. To date, an activity has not been associated with this ORF. 313


.org/D

ownloaded from

http://jb.asm.org/

14

Since deficiency of gtfBCD/ftf/scrAB/fruA/msmE (strain MMZ967) resulted in complete 314 loss of growth on sucrose, we concluded that FruB, similar to FruA, does not contribute 315 in any major way to sucrose catabolism under the conditions tested. When we further 316 deleted fruB in strain MMZ967 by introducing a fruB:sp replacement, the resultant 317 mutant MMZ968 showed no growth after 48 h of incubation in TV-sucrose (Figure 2A). 318

EIITre. We also tested the hypothesis that the trehalose-PTS pathway can contribute 319 to the catabolism of sucrose by creating strain MMZ993, which was constructed by 320 introducing a scrA deletion into the background of strain MMZ952 (gtfABCD/ftf/fruA) 321 where gtfA had been inactivated to eliminate any contribution of the msm pathway. In 322 comparison with the parental strain MMZ952, loss of scrA led to markedly reduced 323 growth on sucrose, but a significant growth rate and yield were still observed (Figure 3). 324 Also similar to the strain carrying the scrA mutation (MMZ932), strain MMZ993 clumped 325 in TV-medium supplemented with sucrose, glucose or galactose, whereas MMZ952 326 showed no clumping, even in the presence of sucrose. Another strain, MMZ996 that had 327 a nearly identical genetic makeup to MMZ993 except a different antibiotic marker was 328 used to replace the scrA gene (Table 1), also behaved similarly to MMZ993 (data not 329 shown). Importantly, as we demonstrated that a scrAB double mutation in a similar 330 genetic background (gtfBCD/ftf) led to near complete loss of growth on sucrose (Figure 331 1C), these results suggested that additional sucrose-PTS permeases are likely 332 contributing to the residual growth by MMZ993 in the absence of ScrA. A previous study 333 in S. mutans has suggested that EIITre, the PTS permease for trehalose, might function 334 as a low-affinity sucrose permease (14). To test this hypothesis, a treB:sp replacement 335 deletion was constructed using MMZ993 as the base strain, and the resultant mutant 336 (MMZ997) failed to show growth in TV-sucrose (Figure 3). Therefore, the results 337 confirmed that the trehalose-PTS may internalize sucrose as sucrose-6-PO4, which can 338 then be catabolized by ScrB. 339


.org/D

ownloaded from

http://jb.asm.org/

15

Transport of sucrose via the PTS. Despite extensive genetic studies of sucrose uptake 340 and metabolism by S. mutans, obtaining a definitive and accurate assessment of PTS-341 dependent sucrose transport activity has been confounded in part by the presence of the 342 secreted GTF, Ftf and FruA enzymes. Taking advantage of the mutants created for this 343 study, an in vitro PTS assay was carried out using strains UA159, MMZ952, MMZ996 344 and MMZ997. To circumvent the issue of clumping in TV-sucrose by strains UA159 and 345 MMZ996, and of the inability to grow strain MMZ997 in sucrose, cells were cultivated in 346 BHI medium for PTS assays. As shown in Figure 4, strain MMZ952, which lacks the 347 GTFs, Ftf and FruA, showed similar levels of PTS activities to the wild-type strain (P = 348 0.456). However, in the absence of scrA, as in strain MMZ996, significant reductions in 349 sucrose PTS activity were noted (P = 0.034) compared with strain MMZ952, confirming 350 the role of EIIScr in sucrose transport. Additionally, in strain MMZ997, which lacked ScrA 351 and EIITre, a further reduction in PTS activity relative to MMZ996 was observed (P = 352 0.040). It was surprising that MMZ997 still had detectable levels of sucrose-PTS activity. 353 The two most plausible explanations are that other PTS permeases are able to 354 internalize sucrose under our test conditions (e.g. cellobiose PTS) or there are other 355 enzymes produced intracellularly that have low levels of sucrolytic activity, such that they 356 release glucose and/or fructose into the reaction mix, which can then serve as 357 substrates for PTS permeases that act on the monosaccharides released from sucrose. 358 359 Sucrose transport affects fructan catabolism. The production of fructan polymers 360 from sucrose enhances the cariogenic potential of S. mutans by extending the depth and 361 duration of acidification of oral biofilms (9). As part of our ongoing effort to explore the 362 relationship between fructan metabolism and cariogenicity, various S. mutans strains 363 were grown in TV medium containing 0.2% of commercially-obtained 364 fructooligosaccharide (FOS). Two different sources of FOS were tested: one was 365


.org/D

ownloaded from

http://jb.asm.org/

16

derived from inulin via limited acid hydrolysis and contained mostly the Fructosen (Fn) 366 form of FOS, whereas the other was enzymatically synthesized from sucrose and would 367 be composed primarily of the Glucose-Fn (GFn) type, and also would contain significant 368 quantities of sucrose (35). 369

When Fn-FOS was supplied as the sole carbohydrate in TV medium, a non-polar fruA 370 mutant (strain SP8) showed largely abolished growth, consistent with the proven 371 function of FruA to metabolize fructan polymers (Figure 5A). Likewise, when GFn-FOS 372 was used as the carbon source, the fruA mutant showed reduced growth, albeit 373 significantly better than when Fn-FOS was the carbon source (Figure 5B); due to the 374 presence of significant quantities of contaminating sucrose. 375

Interestingly, when the scrA mutant strain MMZ932 was tested for its ability to grow in 376 TV-GFn, improved growth and final yield were noted in comparison to the wild-type 377 strain (Figure 5B). Similar results were obtained with the scrAB mutants (data not 378 shown). However this was not the case in TV-Fn, where little difference was seen 379 between a scrA mutant and UA159 except for a slightly higher yield by the mutant 380 (Figure 5A). Therefore, it appears that loss of ScrA in S. mutans resulted in altered 381 expression or activities of the enzymes responsible for catabolizing certain types of FOS. 382

383 Loss of scrA results in increased activity of the fruA promoter. In order to 384 understand the growth behavior of the scrA mutant on FOS, we examined the 385 expression of the fruA gene using a fruA promoter:cat fusion (24), which was established 386 in the wild-type and scrA mutant backgrounds. The strains were cultivated in TV medium 387 containing 0.5% of FOS. As indicated by the CAT assays (Table 2), fruA promoter 388 activity in the wild-type genetic background was approximately 30-fold higher when 389 growing on the Fn-FOS than on the GFn-FOS, consistent with the fact that the wild-type 390 strain grew better on Fn-FOS than on GFn-FOS (Figure 5C). Introduction of the scrA 391


.org/D

ownloaded from

http://jb.asm.org/

17

mutation resulted in a large increase in fruA promoter activity on both growth substrates, 392 such that equivalent levels of expression were noted in cells growing on Fn-FOS and 393 GFn-FOS (Table 2). Apparently, the baseline level of fruA expression in the wild-type 394 cells growing on Fn-FOS yields sufficient FruA enzyme that there is no further 395 improvement in the growth of cells when the scrA mutation is introduced. In contrast, 396 there is a notable improvement in growth of the scrA mutant on GFn-FOS, presumably 397 because the baseline levels of fruA expression in the wild-type background are so low in 398 GFn-FOS medium. 399

As mentioned above, GFn is likely contaminated by sucrose. To test the hypothesis 400 that increased fruA promoter activity in the scrA mutant background is associated with 401 loss of sucrose transport by EIIScr, the stock solutions of GFn- and Fn-FOS were treated 402 with invertase as detailed in the Materials and Methods section. When the CAT assays 403 were repeated using cells growing on invertase-treated FOS, the PfruA-cat activities in 404 the scrA mutant background were now reduced to the wild-type levels (Table 2), 405 supporting the notion that ScrA-dependent sucrose transport is essential for the 406 observed repressive effect of ScrA on fruA expression. The simplest interpretation of this 407 data is that ScrA is able to effect CCR of the fruA operon through some or all of the 408 mechanisms previously detailed by our laboratory (27). However, further investigation 409 has revealed a novel mechanism whereby sucrose and ScrA can affect gene expression 410 in S. mutans. 411 412 ScrA can affect fruA expression through the LevQRST system. The studies with 413 FOS led us to investigate in more detail how the presence of sucrose and ScrA may 414 influence fruA expression by studying the induction of fruA by the levQRST circuit in the 415 context of sucrose metabolism by S. mutans. We previously established the levD 416 promoter as a useful model system for LevR-dependent activation of gene expression; 417


.org/D

ownloaded from

http://jb.asm.org/

18

particularly because its promoter region has the binding site for LevR; levD promoter 418 activity is minimal in the absence of LevR-dependent activation; and, unlike the fruA 419 promoter, there is no CRE for CcpA binding in the levD promoter and therefore the 420 results would not be confounded by the activity of an additional regulatory pathway (23, 421 24). 422

Since fructose is a potent signal for activation of the LevQRST pathway, a PlevD-cat 423 fusion was introduced into the background of MMZ952 (gtfABCD/ftf/fruA), resulting in 424 strain MMZ998. Similarly created was another PlevD-cat reporter strain, MMZ1002 that 425 also lacks scrA, in addition to the mutations in MMZ952. When these two strains were 426 cultured in TV with 0.5% sucrose, no free glucose was detectable in the culture 427 supernatant fluids (data not shown). Analysis of the CAT activity revealed that 428 expression from the levD promoter was significantly higher in the strain lacking ScrA 429 (MMZ1002; 728 ± 17 CAT units) than in the ScrA-proficient strain MMZ998 (263 ± 4 CAT 430 units). Further tests using two additional strains bearing a PfruA-cat fusion in the same 431 genetic backgrounds showed similarly higher activities in the scrA mutant (data not 432 shown). However, while these results confirmed that sucrose, and not some other 433 compound(s) in FOS preparations, was required for the effects on gene expression, 434 these experiments still did not allow us to distinguish whether CCR alone, or effects 435 mediated through LevQRST, could account for these observations. 436

We previously employed a methodology to study the regulation of the LevQRST 437 regulon by first cultivating cells in medium that did not efficiently induce CCR, i.e. TV-438 galactose, followed by pulsing with inducing substrates to activate expression of the 439 reporter fusion (24). This technique allowed us to successfully discriminate activation of 440 gene expression by LevQRST from effects of catabolite repression by the inducing 441 substrates, which include fructose, fructans and mannose (22, 24, 26). Using this 442 approach, strain MMZ998 was first cultured in TV-galactose to early exponential phase, 443


.org/D

ownloaded from

http://jb.asm.org/

19

then various concentrations of sucrose were added. The cells were incubated for 3.5 h 444 and collected for CAT assays. As shown in Table 3, when strain MMZ998 was pulsed 445 with sucrose, a concentration-dependent activation of the levD promoter was evident. As 446 no extracellular release of free fructose could occur in this particular strain, it appears 447 that sucrose itself has the capacity to contribute to activation of the LevQRST pathway. 448 This conclusion was further supported by the transcript levels of levD in strain MMZ952 449 measured using quantitative RealTime RT-PCR, which showed a 12-fold increase in 450 cells growing in TV containing 0.5% sucrose compared with TV-glucose medium. Also 451 clear was that when sucrose concentrations reached 5 mM or higher, expression of levD 452 was significantly reduced in comparison to that measured at 1 mM, providing evidence 453 of CCR effected by sucrose (Table 3). Interestingly, when a scrA mutant strain 454 MMZ1002 was subjected to the same conditions, there was no evidence of activation of 455 levD expression by sucrose at concentrations of 0.05 to 10 mM (Table 3). Thus the EIIScr 456 permease appears to be required for activation of the LevQRST pathway by sucrose 457 under our pulsing conditions. 458

As reported previously, fructose and mannose are very effective at activating the 459 expression of fruA and levD through the LevQRST complex (22, 24). LevS and LevR are 460 absolutely required for the function of this circuit, while LevQ and LevT are also required 461 for optimal activation and affect the efficiency of induction by specific carbohydrates (23, 462 24). Thus, it was of interest to determine whether the effects of EIIScr on the LevQRST 463 system could modify responses by the complex to the cognate signals, fructose and 464 mannose. As shown in Table 3, when both MMZ998 and MMZ1002 were pulsed with 465 fructose, the levD promoter was activated to essentially the same levels regardless of 466 whether the scrA gene was intact. The same was true with mannose (Table 3). Thus, 467 EIIScr is not required for activation of LevQRST by fructose or mannose. 468


.org/D

ownloaded from

http://jb.asm.org/

20

To further prove that sucrose was the actual inducing reagent and to rule out any 469 effects from the unexpected extracellular hydrolysis of sucrose, we also repeated this 470 pulsing experiment with equivalent concentrations of an equimolar mixture of glucose 471 and fructose. Similar to what was seen with fructose alone (Table 3), the combination of 472 glucose and fructose induced the PlevD-cat fusion in a concentration-dependent fashion 473 regardless of the presence of ScrA. 474

Finally, we investigated the involvement of individual components of the LevQRST 475 circuit in sucrose-dependent activation of the levD promoter. Additional mutations were 476 introduced into the background of MMZ998, using DNA fragments carrying antibiotic 477 insertions (sp marker) into the levQ, levT or levR genes, yielding strains MMZ998Q, 478 MMZ998T and MMZ998R, respectively. These three strains were grown in TV-galactose 479 and pulsed with sucrose as above (Table 3). As expected, loss of the response regulator 480 LevR led to nearly complete loss of expression by the levD promoter fusion. 481 Interestingly, loss of LevQ largely abolished promoter activity, reinforcing our previous 482 conclusion that LevQ could be playing a role in maintaining the structural integrity of the 483 LevQRST four-component system (23). When LevT was deleted, levD promoter activity 484 was shown to be higher without sucrose or when lower concentrations of sucrose were 485 added; however activity again declined when 5 mM sucrose was utilized. Thus, 486 mutations in the carbohydrate binding proteins of the four-component system 487 differentially affect how sucrose and ScrA modulate activation of the LevQRST circuit, 488 and a potential interaction between LevT and ScrA could impact activation of the 489 complex. Clearly, though, the results indicate that ScrA cannot bypass the LevQRST 490 circuit to activate levD through some alternative pathway. 491 492

493


.org/D

ownloaded from

http://jb.asm.org/

21

Discussion 494 We set out to determine the relative contributions of various sucrose-metabolizing 495

enzymes found in S. mutans to growth and to explore the capacity of the sucrose-PTS 496 permease to affect gene expression in a way that the liberation of glucose or fructose 497 from sucrose would not confound the analysis. We have shown that there was no 498 apparent growth defect in various mutants that lack GTFs and FTF (MMZ950 and 499 MMZ952), or ScrA and ScrB (MMZ932, MMZ934 and MMZ957, except MMZ931), yet a 500 deficiency in all three systems (MMZ983) largely abolished the ability to grow on 501 sucrose. We were a bit surprised to find that a mutant deficient in GtfBC and ScrAB 502 (MMZ1025) also failed to yield significant growth on sucrose, since this strain should be 503 able to liberate fermentable carbohydrates from sucrose via GtfD, FTF and FruA, and 504 still had an active Msm pathway. Further analysis of the transcript levels of both gtfD and 505 ftf in strain MMZ1025 showed little change in comparison to that of UA159. In fact, we 506 found that the levels of expression of these genes in strain UA159 did not significantly 507 differ in cells cultured with glucose or sucrose (data not shown). Thus, we conclude that 508 while the EIIScr appears to be the main sucrose transporter, extracellular sucrolytic 509 enzymes, especially GtfBC can release free hexoses at a sufficiently high rate to 510 facilitate wild-type levels of planktonic growth. In fact, when strain UA159 was cultured to 511 OD600 = 0.25~0.3 in TV containing 0.5% sucrose (14.6 mM), glucose concentrations in 512 the supernate were measured at 0.05 ± 0.003 mg/mL (0.28 mM) and fructose at 0.27 ± 513 0.05 mg/mL (1.5 mM). Conversely, but in agreement with previous research indicating 514 that the majority of sucrose encountered by S. mutans is metabolized intracellularly (14), 515 our findings further reveal that the levels of free hexoses in sucrose-grown cultures are 516 probably not high enough to trigger catabolite repression (22, 26). 517

By creating strains lacking multiple sucrolytic enzymes, especially the GTFs and FTF 518 (MMZ952, MMZ998 and MMZ1002) to eliminate the ability for free hexose to be 519


.org/D

ownloaded from

http://jb.asm.org/

22

produced from sucrose in the supernatant fluid, we were able to detect the ability of 520 sucrose both to activate gene expression (levD and fruA) and to trigger CCR when 521 present at relatively high, albeit physiologically-relevant concentrations (> 5 mM). Given 522 that fructose is a potent inducer of expression of levD and fruA and that high 523 concentrations of fructose and glucose can repress expression of these genes, the use 524 of strains lacking exported sucrases was critical for reliable quantitation of the effects of 525 sucrose on gene expression. It is also only by using these mutants that we were able to 526 observe the dependence on ScrA for activation of the LevQRST-controlled genes by 527 sucrose, an unusual and novel observation. Obviously, the control of fruA and levD in 528 this manner is of interest as it adds a new dimension to the regulation of exo-529 polysaccharide metabolism and may influence how S. mutans responds to intermittent 530 introduction of sucrose in the diet. From a mechanistic perspective, we postulate that 531 interactions between ScrA, most likely in its dephosphorylated form, and components of 532 the LevQRST system, in particular LevQ and LevT, influence the signal transduction 533 events that lead to activation of the lev/fru operons. We were not able to include a strain 534 lacking the sensor kinase LevS in our test system for reasons that remain unclear. 535 Specifically, all attempts to create a levS:sp allelic exchange mutation, either in the 536 backgrounds of UA159 or MMZ998, were not successful. This is odd particularly in light 537 of the fact that we had successfully constructed levS deletion mutants using other 538 antibiotic resistance markers. However, since we have already demonstrated that loss of 539 the sensor kinase LevS results in an inability to activate LevR (22), we anticipate that the 540 effects of ScrA in a levS mutant would be minimal. It should also be noted that we 541 examined the ability of three sugars with some similarities to sucrose, isomaltulose (also 542 called palatinose; 6-O-α-D-glucopyranosyl-D-fructose), turanose (3-O-α-D-543 glucopyranosyl-D-fructose) and sucralose (trichlorogalactosucrose) (Sigma) to activate 544 expression of the levD promoter. When used at levels comparable to sucrose to pulse 545


.org/D

ownloaded from

http://jb.asm.org/

23

galactose-grown MMZ998 cultures, the results showed no induction of the levD promoter 546 by any of these compounds. Further, none of these sucrose analogs were able to 547 interfere with induction by sucrose (data not shown). 548

Findings made using these various mutants lacking sucrolytic activities also allowed 549 us to conclude that sucrose efficiently induces CCR without needing to be processed 550 into free hexoses. Interestingly, while both ScrA and the LevQRST system are needed to 551 fully activate the levD promoter, the ability of sucrose to negatively regulate the CCR-552 sensitive targets of LevR remains even in the absence of LevQ and LevT (Table 3). 553 Notably, these observations mirror some of the phenotypes of CCR of the LevQRST 554 system that are elicited by the major glucose-PTS permease, EIIMan (22). Prior studies 555 have indicated that scrA is expressed at high levels in cells growing in a variety of 556 different carbohydrate sources (30), suggesting that ScrA may be constitutively 557 produced in S. mutans and could therefore rapidly detect the presence of sucrose as it is 558 introduced intermittently in the diet of the host. Notably, there are many cases where a 559 bacterial membrane transporter plays a major role as a modulator of gene transcription, 560 imparting regulatory information in response to the very substrate it is transporting (44, 561 45). For example, a recent report described how a maltose-specific ABC transporter, 562 MalFGK2 can regulate the expression of the maltose regulon in E. coli by sequestering 563 the major activator MalT only when not actively engaged in sugar transport (46). Since 564 sucrose can activate levD in a ScrA-dependent fashion at concentrations that are below 565 that which trigger catabolite repression, we hypothesize that ScrA works as a sucrose 566 sensor that transduces regulatory information to other circuits, such as LevQRST, that 567 govern secondary catabolic pathways. Since a clear and significant phenotype of the 568 scrA mutant is its marked clumping in various carbohydrates besides sucrose, the 569 regulation of gene expression by ScrA likely extends beyond the LevQRST regulon. 570 Further transcriptomic studies are planned to investigate the scope of the EIIScr regulon. 571


.org/D

ownloaded from

http://jb.asm.org/

24

It should be noted that while ScrA was required for the activation of the LevQRST 572 system by sucrose in our pulsing experiment, the same strains (MMZ998 and MMZ1002, 573 both deficient in gtfABCD/ftf/fruA) when grown for extended periods in TV-sucrose 574 showed higher fru/lev promoter activities in the scrA mutant than the scrA+ background. 575 Since for the latter experiment, the strains were first cultured in TV-sucrose overnight 576 before sub-culturing again for CAT assays, we tested the fructose concentrations in the 577 supernates of the overnight cultures of MMZ998. Fructose could be detected at low 578 levels (0.65 ± 0.16 mM), yet sufficient to induce the LevQRST system (24). The 579 presence of fructose in these overnight cultures is probably the result of cell lysis. 580 However, we cannot exclude that there are other unknown very low-activity extracellular 581 sucrases or that inducer expulsion may result in transport of fructose into the 582 extracellular environment after it is released from sucrose-6-PO4 (47). In fact, preliminary 583 measurement of fructose in cultures of strain MMZ998 pulsed with various amounts of 584 sucrose, similar to those described above for the CAT assays, have indicated that there 585 may be sufficient fructose present in the extracellular environment to induce gene 586 expression through the LevQRST complex (data not shown). 587

Our previous studies utilizing the LevQRST model system have revealed a dominant 588 role of CcpA-independent CCR mechanisms in regulating carbohydrate metabolism by 589 S. mutans, a process effected primarily by the HPr protein and a number of 590 glucose/fructose-PTS permeases of the PTS (22, 27). With evidence for what appears to 591 be a direct role for the major sucrose-PTS ScrA in transcriptional regulation of 592 exopolysaccharide metabolism, the findings described in this report add to the theme 593 that prioritization by S. mutans of carbohydrate catabolism takes place primarily at the 594 level of substrate sensing and transport, rather than centrally through CcpA, as occurs in 595 many related Gram-positive bacteria. The present study significantly extends our general 596


.org/D

ownloaded from

http://jb.asm.org/

25

knowledge of sucrose metabolism by S. mutans and enhances our understanding of the 597 role of sucrose and S. mutans in the ecology of the oral microbiome and dental caries. 598

599 Acknowledgements 600

This study was supported by funding from National Institute of Dental and Craniofacial 601 Research (DE12236). 602

603 604


.org/D

ownloaded from

http://jb.asm.org/

26

Reference 605 606 1. Woodward M, Walker AR. 1994. Sugar consumption and dental caries: 607

evidence from 90 countries. Br. Dent. J. 176:297-302. 608 2. Burt BA. 1993. Relative consumption of sucrose and other sugars: has it been a 609

factor in reduced caries experience? Caries research 27 Suppl 1:56-63. 610 3. Tanzer JM. 1979. Essential dependence of smooth surface caries on, and 611

augmentation of fissure caries by, sucrose and Streptococcus mutans infection. 612 Infect. Immun. 25:526-531. 613

4. Bowen WH, Koo H. 2011. Biology of Streptococcus mutans-derived 614 glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. 615 Caries research 45:69-86. 616

5. Birkhed D, Rosell KG, Granath K. 1979. Structure of extracellular water-soluble 617 polysaccharides synthesized from sucrose by oral strains of Streptococcus 618 mutans, Streptococcus salivarius, Streptococcus sanguis and Actinomyces 619 viscosus. Archives of oral biology 24:53-61. 620

6. Shiroza T, Kuramitsu HK. 1988. Sequence analysis of the Streptococcus 621 mutans fructosyltransferase gene and flanking regions. J. Bacteriol. 170:810-622 816. 623

7. Burne RA, Schilling K, Bowen WH, Yasbin RE. 1987. Expression, purification, 624 and characterization of an exo-β-D-fructosidase of Streptococcus mutans. J. 625 Bacteriol. 169:4507-4517. 626

8. Miller CH, Somers PJ. 1978. Degradation of levan by Actinomyces viscosus. 627 Infect. Immun. 22:266-274. 628

9. Burne RA, Chen YY, Wexler DL, Kuramitsu H, Bowen WH. 1996. 629 Cariogenicity of Streptococcus mutans strains with defects in fructan metabolism 630


.org/D

ownloaded from

http://jb.asm.org/

27

assessed in a program-fed specific-pathogen-free rat model. J. Dent. Res. 631 75:1572-1577. 632

10. Sato Y, Poy F, Jacobson GR, Kuramitsu HK. 1989. Characterization and 633 sequence analysis of the scrA gene encoding enzyme IIScr of the Streptococcus 634 mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system. J. 635 Bacteriol. 171:263-271. 636

11. Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB, 637 Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai 638 H, White J, Roe BA, Ferretti JJ. 2002. Genome sequence of Streptococcus 639 mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA 640 99:14434-14439. 641

12. Hiratsuka K, Wang B, Sato Y, Kuramitsu H. 1998. Regulation of sucrose-6-642 phosphate hydrolase activity in Streptococcus mutans: characterization of the 643 scrR gene. Infect. Immun. 66:3736-3743. 644

13. Chassy BM, Porter EV. 1979. Initial characterization of sucrose-6-phosphate 645 hydrolase from Streptococcus mutans and its apparent identity with intracellular 646 invertase. Biochem. Biophys. Res. Commun. 89:307-314. 647

14. Poy F, Jacobson GR. 1990. Evidence that a low-affinity sucrose 648 phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity 649 trehalose uptake system. Infect. Immun. 58:1479-1480. 650

15. Russell RR, Aduse-Opoku J, Sutcliffe IC, Tao L, Ferretti JJ. 1992. A binding 651 protein-dependent transport system in Streptococcus mutans responsible for 652 multiple sugar metabolism. J. Biol. Chem. 267:4631-4637. 653

16. Tao L, Sutcliffe IC, Russell RR, Ferretti JJ. 1993. Transport of sugars, 654 including sucrose, by the msm transport system of Streptococcus mutans. J. 655 Dent. Res. 72:1386-1390. 656


.org/D

ownloaded from

http://jb.asm.org/

28

17. Kilic AO, Honeyman AL, Tao L. 2007. Overlapping substrate specificity for 657 sucrose and maltose of two binding protein-dependent sugar uptake systems in 658 Streptococcus mutans. FEMS Microbiol. Lett. 266:218-223. 659

18. Russell RR, Mukasa H, Shimamura A, Ferretti JJ. 1988. Streptococcus 660 mutans gtfA gene specifies sucrose phosphorylase. Infect. Immun. 56:2763-661 2765. 662

19. Burne RA, Penders JE. 1992. Characterization of the Streptococcus mutans 663 GS-5 fruA gene encoding exo-b-D-fructosidase. Infect. Immun. 60:4621-4632. 664

20. Burne RA, Penders JE, Wexler DL, Jayaraman GC, Clancy KA. 1995. 665 Regulation of fructan degradation by Streptococcus mutans. Dev. Biol. Stand. 666 85:323-331. 667

21. Burne RA, Wen ZT, Chen YY, Penders JE. 1999. Regulation of expression of 668 the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and 669 carbon catabolite repression. J. Bacteriol. 181:2863-2871. 670

22. Zeng L, Burne RA. 2008. Multiple sugar: phosphotransferase system 671 permeases participate in catabolite modification of gene expression in 672 Streptococcus mutans. Mol. Microbiol. 70:197-208. 673

23. Zeng L, Das S, Burne RA. 2011. Genetic analysis of the functions and 674 interactions of components of the LevQRST signal transduction complex of 675 Streptococcus mutans. PLoS ONE 6:e17335. 676

24. Zeng L, Wen ZT, Burne RA. 2006. A novel signal transduction system and 677 feedback loop regulate fructan hydrolase gene expression in Streptococcus 678 mutans. Mol. Microbiol. 62:187-200. 679

25. Deutscher J. 2008. The mechanisms of carbon catabolite repression in bacteria. 680 Curr. Opin. Microbiol. 11:87-93. 681


.org/D

ownloaded from

http://jb.asm.org/

29

26. Abranches J, Nascimento MM, Zeng L, Browngardt CM, Wen ZT, Rivera MF, 682 Burne RA. 2008. CcpA regulates central metabolism and virulence gene 683 expression in Streptococcus mutans. J. Bacteriol. 190:2340-2349. 684

27. Zeng L, Burne RA. 2010. Seryl-phosphorylated HPr regulates CcpA-685 independent carbon catabolite repression in conjunction with PTS permeases in 686 Streptococcus mutans. Mol. Microbiol. 75:1145-1158. 687

28. Zeng L, Burne RA. 2009. Transcriptional regulation of the cellobiose operon of 688 Streptococcus mutans. J. Bacteriol. 191:2153-2162. 689

29. Zeng L, Das S, Burne RA. 2010. Utilization of lactose and galactose by 690 Streptococcus mutans: transport, toxicity, and carbon catabolite repression. J. 691 Bacteriol. 192:2434-2444. 692

30. Ajdic D, Pham VT. 2007. Global transcriptional analysis of Streptococcus 693 mutans sugar transporters using microarrays. J. Bacteriol. 189:5049-5059. 694

31. Klein MI, DeBaz L, Agidi S, Lee H, Xie G, Lin AH, Hamaker BR, Lemos JA, 695 Koo H. 2010. Dynamics of Streptococcus mutans transcriptome in response to 696 starch and sucrose during biofilm development. PLoS ONE 5:e13478. 697

32. Shemesh M, Tam A, Feldman M, Steinberg D. 2006. Differential expression 698 profiles of Streptococcus mutans ftf, gtf and vicR genes in the presence of dietary 699 carbohydrates at early and late exponential growth phases. Carbohydr. Res. 700 341:2090-2097. 701

33. Shemesh M, Tam A, Steinberg D. 2007. Expression of biofilm-associated 702 genes of Streptococcus mutans in response to glucose and sucrose. J. Med. 703 Microbiol. 56:1528-1535. 704

34. Shaw WV. 1975. Chloramphenicol acetyltransferase from chloramphenicol-705 resistant bacteria. Methods Enzymol. 43:737-755. 706


.org/D

ownloaded from

http://jb.asm.org/

30

35. Kaplan H, Hutkins RW. 2000. Fermentation of fructooligosaccharides by lactic 707 acid bacteria and bifidobacteria. Appl. Environ. Microbiol. 66:2682-2684. 708

36. Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual, 3rd ed. 709 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 710

37. Petersen FC, Scheie AA. 2010. Natural transformation of oral streptococci. 711 Methods In. Mol. Biol. 666:167-180. 712

38. Cha RS, Zarbl H, Keohavong P, Thilly WG. 1992. Mismatch amplification 713 mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl. 714 2:14-20. 715

39. Ahn SJ, Lemos JA, Burne RA. 2005. Role of HtrA in growth and competence of 716 Streptococcus mutans UA159. J. Bacteriol. 187:3028-3038. 717

40. LeBlanc DJ, Crow VL, Lee LN, Garon CF. 1979. Influence of the lactose 718 plasmid on the metabolism of galactose by Streptococcus lactis. J. Bacteriol. 719 137:878-884. 720

41. Tanzer JM, Chassy BM, Krichevsky MI. 1971. Sucrose metabolism by 721 Streptococcus mutans, SL-I. Biochim. Biophys. Acta 261:379-387. 722

42. Macrina FL, Jones KR, Alpert CA, Chassy BM, Michalek SM. 1991. Repeated 723 DNA sequence involved in mutations affecting transport of sucrose into 724 Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase 725 system. Infect. Immun. 59:1535-1543. 726

43. Cvitkovitch DG, Boyd DA, Thevenot T, Hamilton IR. 1995. Glucose transport 727 by a mutant of Streptococcus mutans unable to accumulate sugars via the 728 phosphoenolpyruvate phosphotransferase system. J. Bacteriol. 177:2251-2258. 729

44. Liu Y, Zeng L, Burne RA. 2009. AguR is required for induction of the 730 Streptococcus mutans agmatine deiminase system by low pH and agmatine. 731 Appl. Environ. Microbiol. 75:2629-2637. 732


.org/D

ownloaded from

http://jb.asm.org/

31

45. Kuper C, Jung K. 2005. CadC-mediated activation of the cadBA promoter in 733 Escherichia coli. J. Mol. Microbiol. Biotechnol. 10:26-39. 734

46. Richet E, Davidson AL, Joly N. 2012. The ABC transporter MalFGK(2) 735 sequesters the MalT transcription factor at the membrane in the absence of 736 cognate substrate. Mol. Microbiol. 85:632-647. 737

47. Egan JB, Morse ML. 1966. Carbohydrate transport in Staphylococcus aureus. 3. 738 Studies of the transport process. Biochim. Biophys. Acta 112:63-73. 739

740 741

Figure legends 742 Figure 1. Growth curves of the mutants of major sucrose-metabolizing pathways in S. 743 mutans. All strains were grown in BHI medium overnight, sub-cultured in BHI to mid-744 exponential phase and then diluted 1:300 into TV medium containing 0.2% sucrose. 745 Growth was monitored using a Bioscreen C reader set at 37°C with readings (OD600) 746 taken every 30 min. A, UA159 (wild type), MMZ948 (gtfDM9stop), MMZ945 (gtfBC) and 747 MMZ950 (gtfBCD/ftf). B, UA159, MMZ932 (scrA) and MMZ931 (scrB). C, MMZ950, 748 MMZ983 (gtfBCD/ftf/scrAB) and MMZ984 (gtfBCD/ftf/fruA). D, UA159 and MMZ1025 749 (gtfBC/scrAB). Two isolates were included for MMZ1025 as variability in lag phase was 750 noted. 751 752 Figure 2. Minor contributions from the Msm pathway to growth on sucrose. Growth tests 753 were performed same as in Figure 1. A, strains MMZ950 (gtfBCD/ftf), MMZ966 754 (gtfBCD/ftf/scrAB/fruA), MMZ967 (gtfBCD/ftf/scrAB/fruA/msmE) and MMZ968 755 (gtfBCD/ftf/scrAB/fruAB/msmE). B, stains UA159 and ∆EI (ptsI). 756 757


.org/D

ownloaded from

http://jb.asm.org/

32

Figure 3. Role of EIITre in sucrose metabolism by S. mutans. Strains UA159, MMZ952 758 (gtfABCD/ftf/fruA), MMZ993 (gtfABCD/ftf/fruA/scrA) and MMZ997 759 (gtfABCD/ftf/fruA/scrA/treB) were grown in TV medium containing 0.2% sucrose. 760 761 Figure 4. Sucrose transporting activities measured in strain UA159, MMZ952, MMZ996 762 and MMZ997. Cells were harvested from exponentially-growing cultures in BHI, washed 763 in Na/K-phosphate buffer, permeablized with toluene:acetone and tested in PTS assays. 764 The results are the average activities measured using three independent cultures. Units 765 are expressed as nmol NADH oxidized (mg of protein)-1 (min)-1 in a PEP-dependent 766 manner. The error bars represent standard deviations and the asterisks indicate P 767 values less than 0.05 (Student t-test). 768 769 Figure 5. Growth of UA159 and fruA and scrA mutants on fructooligosaccharides (FOS). 770 Strains UA159, SP8 (fruA) and MMZ932 (scrA) were cultured to mid-exponential phase 771 in BHI medium and used to inoculate TV-base medium containing 0.2% of Fn-FOS (A) 772 or GFn-FOS (B). Also compared was the growth of UA159 in both FOS preparations (C). 773 774


.org/D

ownloaded from

http://jb.asm.org/

33

Table 1. Bacterial strains used in this study. 775 776 Strains Description Source or reference

UA159 Wild type University of Alabama

MMZ108 UA159/PfruA-cat (24)

∆EI ptsI:em (27)

MMZ931 scrB:km From UA159

MMZ932 scrA:em From UA159

MMZ934 scrAB:km From UA159

MMZ957 scrAB:Ωkm From UA159

MMZ945 gtfBC:Tc From UA159

MMZ948 gtfDM9stop From UA159

MMZ950 gtfBC/gtfDM9stop/ftfM9stop From MMZ945

MMZ952 gtfA-em/gtfBCD/ftf/fruAM12stop From MMZ950

MMZ966 gtfBCD/ftf/scrAB:Ωkm/fruAM12stop From MMZ950

MMZ967 gtfBCD/ftf/scrAB/fruA/msmE:em From MMZ966

MMZ968 gtfBCD/ftf/scrAB/fruA/fruB:sp/msmE From MMZ967

MMZ974 scrA/PfruA-cat From MMZ932

MMZ983 gtfBCD/ftf/scrAB:Ωkm From MMZ950

MMZ984 gtfBCD/ftf/fruA:km From MMZ950

MMZ993 gtfABCD/ftf/fruA/scrA:km From MMZ952

MMZ996 gtfABCD/ftf/fruA/scrA:sp From MMZ952

MMZ997 gtfABCD/ftf/fruA/scrA/treB:sp From MMZ993

MMZ998 gtfABCD/ftf/fruA/PlevD-cat From MMZ952

MMZ1002 gtfABCD/ftf/fruA/scrA/PlevD-cat From MMZ996

MMZ998Q gtfABCD/ftf/fruA/PlevD-cat/levQ:sp From MMZ998

MMZ998T gtfABCD/ftf/fruA/PlevD-cat/levT:sp From MMZ998

MMZ998R gtfABCD/ftf/fruA/PlevD-cat/levR:sp From MMZ998

MMZ1025 gtfBC/scrAB:Ωkm From MMZ945

SP8 fruA:km From UA159 777


.org/D

ownloaded from

http://jb.asm.org/

34

Table 2. CAT activities of PfruA-cat in the background of wild-type UA159 and its scrA 778 mutant. Cells were growing exponentially in TV media containing 0.5% of 779 fructooligosaccharide (GFn or Fn), with or without prior treatment of the FOS with 780 invertase. 781 782 Avg CAT sp activitya [nmole (mg protein)-1min-1]

Background GFn Fn

untreated + invertase untreated + invertase

UA159 0.14 (0.25) 0.47 (0.31) 4.40b (2.02) 2.58b (0.43)

scrA 63.96c (4.52) 0.74 (0.30) 62.65c (14.2) 2.88b (0.20)

783 a Values represent averages (standard deviation, SD) of three separate experiments. 784 b The data are statistically different from that in corresponding GFn conditions as verified 785 by Student t-test (P < 0.05). 786 c The data are statistically different from that of UA159 in corresponding conditions as 787 verified by Student t-test (P < 0.05). 788


.org/D

ownloaded from

http://jb.asm.org/

35

Table 3. PlevD-cat activities measured in various strains under carbohydrate pulsing a. 789 790

Strain

Avg CAT sp activity (SD) when pulsed with indicated concentrations of sugar

0 mM 0.05 mM 0.2 mM 1 mM 5 mM 10 mM

Pulsed with sucrose

MMZ998 15.8 (5.9) 22.4 (4.9) 117.3 (12) 308.7 (37) 169.4 (18) 185.1 (12)

MMZ1002 17.3 (6.2) 14.2 (5.7) 12.5 b (4.4) 16.4 b (5.0) 24.0 b (7.1) 31.0 b (7.3)

MMZ998Q 16.3 (1.3) ND 9.3 b (0.5) 9.6 b (1.1) 2.2 b (0.3) ND

MMZ998T 242 b (6.4) ND 247 b (8.5) 242 (43) 37.1 b (1.1) ND

MMZ998R 0 b (0) ND 0.23 b (0.2) 0.25 b (0.2) 0.18 b (0.04) ND

Pulsed with fructose

MMZ998 ND 190 (6.8) 387 (7.5) 511 (12) ND

MMZ1002 ND 180 (15) 388 (38) 486 (93) ND

Pulsed with mannose

MMZ998 ND 251 (6.5) 564 (30) 359 (6.3) ND

MMZ1002 ND 241 (29) 557 (97) 471 (81) ND

Pulsed with glucose and fructose

MMZ998 ND 258 (11) 556 (2.1) 677 (33) ND

MMZ1002 ND 281 (46) 651 (114) 692 (179) ND

791 a The cells were cultured in TV containing 0.5% galactose to an OD600 of 0.2, then the 792 indicated concentrations of carbohydrates were added. Cells were then incubated for 3.5 793 h before CAT assays. CAT activity is expressed as nmol of chloramphenicol acetylated 794 (mg of protein)-1 (min)-1, followed by the standard deviation in parentheses. The data are 795 averages from three independent cultures. ND, not determined. 796


.org/D

ownloaded from

http://jb.asm.org/

36

b The data are statistically different from that of MMZ998 in corresponding conditions as 797 verified by Student t-test (P < 0.05). 798 799


.org/D

ownloaded from

http://jb.asm.org/

Fig. 1A

0 5

0.6

Fig. 1A

0.4

0.5

UA159

0.3

OD

600 MMZ948

MMZ945

MMZ950

0.1

0.2MMZ950

00 10 20 30 40 50

Ti (h)Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 1B

0.6

Fig. 1B

0.4

0.5

0.3

OD

600 UA159

MMZ932

MMZ931

0.1

0.2

00 5 10 15 20

Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 1C

0 5

0.6

0.4

0.5

MMZ950

0.3

OD

600

MMZ950

MMZ983

MMZ984

0.1

0.2

00 10 20 30 40 50

Time (h)Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 1D

0.5

g

0.3

0.4

UA159

0.2

0.3

OD

600 UA159

MMZ1025-1

MMZ1025-2

0.1

00 10 20 30 40 50

Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 2A

0.5

0.6

0.4

0

MMZ950

0.2

0.3OD

600

MMZ966

MMZ967

MMZ968

0.1

0.2

00 10 20 30 40 50

Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 2B

0.6

0.4

0.5

0.3

OD

600 UA159

ΔΕΙ

0.1

0.2

00 10 20 30 40 50

Time (h)( )


.org/D

ownloaded from

http://jb.asm.org/

Fig. 3

0.6

0.4

0.5

UA159

0.3

OD

600 MMZ952

MMZ993

MMZ997

0.1

0.2MMZ997

00 10 20 30 40 50

Time (h)Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 4

200 00

*

*

150.00

200.00

activ

ity

*

100.00

ende

nt P

TS

*

50.00

PEP-

depe

0.00UA159 MMZ952 MMZ996 MMZ997


.org/D

ownloaded from

http://jb.asm.org/

Fig. 5A

0.6

Fig. 5A

0.4

0.5

0.3

OD

600 UA159

SP8

MMZ932

0.1

0.2

00 10 20 30 40 50

Time (h)Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 5B

0.6

0.4

0.5

UA159

0.3

OD

600

UA159

SP8

MMZ932

0.1

0.2

00 10 20 30 40 50

Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Fig. 5C

0.6

0.4

0.5

0.3OD

600 UA159 GFn

UA159 Fn

0.1

0.2

0

0.1

0 10 20 30 40 50Time (h)


.org/D

ownloaded from

http://jb.asm.org/

Documents

revised comprehensive mutational analysis of sucrose-metabolizing