Modification of Gene Expression and Virulence Traits in Streptococcusmutans in Response to Carbohydrate Availability

Zachary D. Moye, Lin Zeng, Robert A. Burne

Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA

The genetic and phenotypic responses of Streptococcus mutans, an organism that is strongly associated with the development ofdental caries, to changes in carbohydrate availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL,which is the EIIAB component (EIIABMan) of a glucose/mannose permease of the phosphoenolpyruvate:sugar phosphotransfer-ase system (PTS) and a dominant effector of catabolite repression, was grown in continuous culture to steady state under condi-tions of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans UA159 revealed that 174 geneswere differentially expressed in response to changes in carbohydrate availability (P < 0.001). Glucose-limited cells possessedhigher PTS activity, could acidify the environment more rapidly and to a greater extent, and produced more ManL protein thancultures grown with excess glucose. Loss of ManL adversely affected carbohydrate transport and acid tolerance. Comparison ofthe histidine protein (HPr) in S. mutans UA159 and the manL deletion strain indicated that the differences in the behaviors ofthe strains were not due to major differences in HPr pools or HPr phosphorylation status. Therefore, carbohydrate availabilityalone can dramatically influence the expression of physiologic and biochemical pathways that contribute directly to the viru-lence of S. mutans, and ManL has a profound influence on this behavior.

Dental caries develops as a result of changes in the microbialcomposition and biochemical activities of oral biofilms in

response to alterations in environmental conditions, includingnutrient source and availability and repeated acidification (1).When the host is fasting, the oral flora catabolizes nutrients de-rived from host secretions and sloughed cells (1, 2). When the hostconsumes large quantities of sugary foodstuffs, multiple cario-genic species, including Streptococcus mutans, ferment these car-bohydrates to organic acids that damage the tooth enamel (3–5).Analysis of the human oral microbiota in health and disease hasprovided evidence that a diverse community of organisms con-tributes to the initiation and progression of dental caries, althoughstrong associations of elevated levels of S. mutans with the pres-ence of active disease are consistently observed (3, 6). Dissectionof the virulence of individual organisms or combinations of oralbacteria has been complicated by the fact that phenotypic behav-iors can be greatly impacted by environmental conditions (7). Anunderstanding of how specific pathogens, such as S. mutans, copewith fluctuations in the chemical composition of the environmentto compete with commensal members of the flora is essential tounderstanding the cariogenicity of the oral microbiome and fordesigning interventions to eliminate or reduce the proportions ofcariogenic organisms in oral biofilms (8, 9).

S. mutans relies on the fermentation of dietary sugars to gen-erate energy for growth, as it lacks a complete trichloroacetic acidcycle and respiratory chain (10). The organism expresses severalpathways for the internalization of carbohydrates, including themultiple sugar metabolism (msm) ABC transport system that iscapable of internalizing raffinose, melibiose, and isomaltosaccha-rides (11). However, most of the carbohydrates catabolized by S.mutans are internalized by the phosphoenolpyruvate:sugar phos-photransferase system (PTS) (12). The general PTS proteins en-zyme I (EI) and histidine protein (HPr) participate in the trans-port of all PTS sugars. A variety of sugar-specific enzyme II (EII)permeases are commonly present in S. mutans (10, 13–15) and arecomposed of cytoplasmically located A and B subunits, as well as C

and, in some cases, D domains embedded in the cell membrane(12). More than 14 unique PTS permeases that transport a spec-trum of mono- and disaccharides, including glucose, sucrose,mannose, fructose, cellobiose, lactose, and maltose, are present inthe reference strain S. mutans UA159 (13, 15). Rapid fermentationof sugars by S. mutans can cause a substantial drop in the pH oforal biofilms, from neutral pH to values well below 5 (16). Theacidic environment created by carbohydrate fermentation favorsthe growth of S. mutans over that of health-associated, acid-sen-sitive commensal organisms, because S. mutans is well adapted tosurvive and continues to metabolize carbohydrates at low pH(17).

In low-G�C-content Gram-positive bacteria, the regulation ofcarbohydrate catabolite repression (CCR), the ability to selectivelycatabolize preferred carbohydrates when nonpreferred carbohy-drates are also present, is typically controlled by HPr of the PTSand the LacI/GalR-type catabolite control protein A (CcpA) (18).Interestingly, it has been demonstrated that CcpA has very littleinfluence on CCR in S. mutans (19–21), whereas transcriptomicanalyses have revealed a prominent role for CcpA in the regulationof global carbon flow, control of the transition between homo-and heterofermentative growth, and regeneration of NADH, asfailure to maintain adequate NADH levels in S. mutans can havedetrimental effects (14, 22). While it has been shown that HPr canexert effects on CCR in S. mutans, CCR in this organism is dom-inantly controlled by the glucose/mannose EIIAB permease,

Received 31 October 2013 Accepted 18 November 2013

Published ahead of print 22 November 2013

Address correspondence to Robert A. Burne, rburne@dental.ufl.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03579-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03579-13

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ManL (EIIABMan), but also the FruI and FruCD fructose per-meases, the EIILev fructose/mannose permease, and the sucrosePTS permease have been shown to be capable of influencing CCRin this cariogenic pathogen when their cognate sugars are present(23, 24). Notably, deletion of manL resulted in a loss of diauxicgrowth in medium containing a combination of glucose and thefructose homopolymer inulin as the carbohydrate sources, and ageneral dysregulation of many genes involved in carbohydratetransport, catabolite repression, and energy metabolism wasnoted in a manL deletion mutant compared with the gene regula-tion in the parental strain UA159 (25). Thus, the current workingmodel for CCR in S. mutans posits that information on carbohy-drate flow through the glycolytic pathway is transduced by phos-phorylated derivatives of HPr, while PTS permeases monitor thesource and availability of specific carbohydrates in the environ-ment (26).

The use of continuous culture has proven to be a powerful toolfor dissecting the impact of carbohydrate availability, growth rate,and pH on the physiology of S. mutans, although most of thestudies were done when technologies were not available to corre-late the results with gene expression profiles (27–32). For example,when cultures of S. mutans strain Ingbritt were grown to steadystate with excess glucose, the cells displayed a slight drop in glyco-lytic rate when cultured at a lower pH or higher growth rates. Asimilar pattern was observed for strain Ingbritt grown under glu-cose limitation, although the decrease in the glycolytic rate wasmore pronounced (27, 30). The glycolytic rate for glucose, fruc-tose, and sucrose was higher in cells of S. mutans Ingbritt grownunder glucose limitation than glucose excess, whereas the rate ofglycolysis of endogenous stores of carbohydrates was higher incells grown with excess glucose. Likewise, cells limited for carbo-hydrate were able to lower the pH faster using exogenous sources,while cells grown in excess glucose contained greater endogenousstores of carbohydrates (28). When sugar transport was assayed inS. mutans Ingbritt and the clinical isolate 123.1, PTS-dependenttransport of glucose, mannose, and 2-deoxyglucose was lowerwhen cells were grown at higher dilution rates, at a lower pH, orwith excess glucose (27, 29, 31), although fructose PTS activity didnot follow the same trend (31). A decrease in EII activity was notedas strain Ingbritt or strain 123.1 was grown in increasing concen-trations of glucose (31, 32). Similarly, EI protein levels were foundto be lower under conditions of glucose excess than under condi-tions of glucose limitation, except when cells where grown at avery low growth rate at pH 7 (31, 32). In S. mutans Ingbritt, theamount of HPr was shown to decrease slightly with increasingconcentrations of glucose (32), whereas others have reported thatthe levels of HPr in S. mutans 123.1 remain relatively constant(31). Multiple studies demonstrated that PTS-dependent trans-port did not always correlate with the total growth rate of cells, andthis effect was more pronounced for cells grown at high growthrates (27, 33, 34).

Clearly, the availability of carbohydrates has substantial ef-fects on the physiology and pathogenic potential of S mutans,and in a few instances, these adaptations have begun to beunderstood at the biochemical level. However, the genetic reg-ulatory circuits underlying these adaptations are only begin-ning to be appreciated, and a limited understanding of thescope and complexity of the responses is presently available.The goals of this study were to more fully characterize themechanisms utilized for adaptation to carbohydrate availabil-

ity by S. mutans in the context of the major regulator of CCR,ManL, and to begin to integrate genetic data with physiologicbehaviors. The results provide new insights into how this dentalpathogen alters its virulence potential in response to the majordriver of dental caries, carbohydrate availability.

MATERIALS AND METHODSBacterial strains and growth conditions. Streptococcus mutans strainUA159 was maintained using brain heart infusion (BHI) medium androutinely cultured in a tryptone-vitamin (TV) base medium supple-mented with glucose (35). For continuous culture studies, cells were cul-tivated in a Biostat i Twin chemostat system (B. Braun Biotech, Inc.,Allentown, PA) with a working volume of 0.5 liter. Cultures were grown inTV base medium supplemented with either 10 mM (limiting) or 100 mM(excess) glucose (35). In all cases, concentrated glucose solutions werebriefly autoclaved or filter sterilized, combined with a filter-sterilizedvitamin mixture, and aseptically added to a vessel containing sterile tryp-tone base medium. To begin each experiment, a chemostat vessel contain-ing 500 ml of sterile TV medium with 0.5% glucose was inoculated with 5ml of an overnight culture of S. mutans grown in BHI broth. Cells weregrown to an optical density at 600 nm (OD600) of 0.7, at which timemedium was pumped into the vessel at a dilution rate (D) equal to 0.3 h�1,corresponding to a generation time of 2.3 h at steady state (36). Steadystate was assumed to have been reached after 10 generations (36). Theavailability of glucose was monitored by measuring the free glucose in theculture supernatant using a glucose oxidase assay (Sigma, St. Louis, MO)with a lower limit of detection of approximately 1 �g ml�1. The pH of themedium was maintained at 7.0 by the addition of 1 M KOH, the temper-ature was kept at 37°C, and the culture was stirred at 200 rpm. Once steadystate was established, 250 ml of the cell culture was removed from thevessel and immediately chilled on ice. Cells were quickly aliquoted intosmaller volumes and centrifuged at 4°C. In some instances, cells wereresuspended and utilized immediately in assays, while others were snap-frozen in a dry ice-ethanol bath and stored at �80°C. In other cases,pellets were treated with RNAprotect Bacteria reagent (Qiagen, Inc.,Chatsworth, CA), snap-frozen, and stored at �80°C to preserve RNA formicroarray and quantitative reverse transcription-PCR (qRT-PCR) stud-ies. Three replicates of cultures grown in 10 mM or 100 mM glucose weregenerated.

pH drop assays. Bacterial cultures were subjected to pH drop experi-ments as previously described (22, 37). Briefly, samples of the cultureswere collected and centrifuged at 4°C. Cells were resuspended in colddeionized water, and the OD600 of the suspension was adjusted to 0.5.Aliquots (50 ml) of the normalized cell suspensions were then centrifugedat 4°C and resuspended in 4.75 ml of 50 mM KCl, 1 mM MgCl2. A smallstir bar was added, and the pH of the cell suspension was adjusted to 7.2 bythe addition of 0.1 M KOH. Once a stable pH was established, 0.25 ml ofa 1 M glucose solution was added and the decrease in the pH was moni-tored continuously for 30 min using a pH meter connected to a computer.For assays of acid production from endogenous sources, cells were har-vested and resuspended in cold deionized water, and the OD600 values ofthe cell suspensions were adjusted to 0.5 with cold water. Cells were col-lected as described above and resuspended in 5 ml of 50 mM KCl, 1 mMMgCl2, the pH of the suspension was rapidly adjusted to pH 7.2 withKOH, and the pH of the cell suspension was monitored over time withoutaddition of glucose.

PTS assays. The ability of cells to transport sugars by the PTS wasassessed as described elsewhere (38, 39), with minor modifications.Briefly, frozen cell pellets were thawed on ice, and the OD600 of the sus-pensions was adjusted to 0.5. The cells were washed twice with 0.1 Msodium-potassium phosphate buffer and resuspended in 0.1 volume ofthe same buffer. Previous analyses conducted in our laboratory showedthat snap-freezing and storage at �80°C do not affect PTS activity. Cellswere permeabilized by addition of 0.05 volume of a toluene-acetone (1:9,vol/vol) solution and vortexing twice for 2 min each time. The reaction

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mixtures included permeabilized cells, 100 �M NADH, 10 �M NaF, 10�M the desired carbohydrate, and 10 �l of a lactate dehydrogenase solu-tion (13.1 kU/ml; Sigma) in 0.1 M sodium potassium phosphate buffer.The reaction was conducted at 37°C and initiated by the addition of 5 mMphosphoenolpyruvate (PEP). The rate of PEP-dependent oxidation ofNADH was monitored over time and used to calculate reaction rates,which were then normalized to the concentration of protein measuredusing a bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL)with bovine serum albumin as the standard.

Immunoblot assays. A polyclonal antibody was raised against a puri-fied, recombinant ManL protein. To express ManL, oligonucleotideprimers were synthesized to PCR amplify the manL coding sequence andto introduce 5= BamHI and 3=HindIII restriction sites. The resulting PCRproduct was digested with BamHI and HindIII and ligated into plasmidpQE-30 carrying a 6-histidine N-terminal tag in frame with the manLgene. The ligation mixture was transformed into Escherichia coli strainM15. DNA sequencing was used to verify that the fusion protein wasintact. The recombinant strain was grown in 1 liter of L broth to an OD600

of 0.5. IPTG (isopropyl-�-D-thiogalactopyranoside) was then added to afinal concentration of 1 mM, and the culture was incubated for an addi-tional 4 h. Following purification by metal affinity chromatography, ap-proximately 1 mg of purified, recombinant ManL was electrophoresed inan SDS-polyacrylamide gel. Slices of the gel containing ManL were sent toLampire Biologicals (Pipersville, PA), where a rabbit polyclonal antise-rum was generated. Antisera generated against the EI and HPr proteinsfrom S. mutans strain DR0001 (40) were a kind gift from ChristianVadeboncouer.

S. mutans cell lysates were generated in a Bead Beater apparatus(Biospec, Bartlesville, OK) using 0.5 ml of glass beads (average diam-eter, 0.1 mm) in 750 �l of 1� phosphate-buffered saline, the lysateswere clarified by centrifugation at 16,000 � g at 4°C for 10 min, and theprotein concentration of clarified cell lysates was measured using acommercially available Bradford assay (Bio-Rad, Hercules, CA). Equalamounts of protein from each cell lysate were boiled for 5 min in SDSsample buffer, separated by SDS-PAGE, and transferred to a polyvi-nylidene difluoride (PVDF) membrane using a Trans-Blot SD appara-tus (Bio-Rad). Membranes were blocked with Tris-buffered saline (10mM Tris-HCl, pH 7.4, 0.9% NaCl, 0.05% Tween 20) containing 5%nonfat dried milk and then incubated with primary antisera, washed,and incubated with a goat antirabbit peroxidase-conjugated antibody(1:4,000 dilution; KPL, Gaithersburg, MD). After additional washing,the membranes were developed using SuperSignal West Pico chemi-luminescent substrate (Thermo Scientific). The different species ofHPr (26) were detected in cell lysates that had been either kept on iceor boiled to remove the phosphate moiety from histidine-15. Afterseparation of the proteins by nondenaturing PAGE and transfer to aPVDF membrane, HPr was detected by immunoblotting using thepolyclonal anti-HPr antiserum described above. Quantification of sig-nals was performed using densitometry.

RNA isolation. RNA was extracted from cells as described previously,with some modifications (22). Briefly, 50 ml of cells from the chemostatwas pelleted, resuspended in 1 ml of RNAprotect Bacteria reagent, andincubated at room temperature for 10 min. The cell suspensions were thenpelleted, the supernatant was removed, and cell pellets were quickly frozenusing dry ice and stored at �80°C. Frozen cells were thawed on ice, resus-pended in 5 ml of 50:10 TE buffer (50 mM Tris, 10 mM EDTA), anddiluted to equal cell densities. Then, 250 �l of each cell suspension wasmixed with 300 �l of acidic phenol and placed in 1.5-ml screw-cap tubeswith 0.2 ml of glass beads (average diameter, 0.1 mm). Cells were homog-enized by bead beating two times for 30 s each time, with placement on icefor 2 min between cycles. The lysates were centrifuged for 10 min, 150 �lof the aqueous phase was removed, and RNA was extracted using anRNeasy extraction kit (Qiagen, Inc.). Extracts were then subjected to twoseparate treatments with RNase-free DNase I (Qiagen, Inc.). RNA was

transferred to an RNase-free column, washed, and eluted in 30 �l ofRNase-free water.

Microarray experiments. Microarray slides containing 1,948 70-meroligonucleotide open reading frames of S. mutans UA159 printed 5 timeson glass slides were obtained from the Pathogen Functional GenomicsResource Center (PFGRC; http://pfgrc.jcvi.org) (22). In order to accountfor biases arising from array data due to differences in the fluorescentsignal intensity of the two dyes used, reference RNA was prepared from a50-ml culture of S. mutans UA159 grown to mid-exponential phase inBHI broth (22). RNA that was purified as described above was used togenerate aminoallyl-labeled cDNA using protocols provided by thePFGRC (http://pfgrc.tigr.org/protocols.shtml). Briefly, 2 �g of RNA fromeach of the three replicates from chemostat cells and samples of refer-ence RNA was used to generate cDNA by amplification with randomhexamer primers using SuperScript III reverse transcriptase (Invitro-gen, Gaithersburg, MD). To label the cDNA, aminoallyl-dUTP (aa-dUTP; Sigma) was added to the nucleotide pool during the reversetranscription reaction at a ratio of aa-dUTP to dTTP of 2:1. The fluo-rescent molecule indocarbocyanine Cy3 was used to label the experi-mental group cDNA, while indocarbocyanine Cy5 (Amersham Biosci-ences, Piscataway, NJ) was coupled to the reference group cDNA. Onthe basis of the amount of dye incorporation measured, experimentalgroup cDNA was mixed with reference cDNA possessing similar dyeincorporation and hybridized to prepared slides using a Maui four-chamber hybridization system (BioMicro Systems, Salt Lake City, UT)for 16 h at 42°C. The slides were washed according to the PFGRCprotocols and prepared for scanning using a GenePix scanner (AxonInstruments Inc., Union City, CA).

Scanned slides were analyzed using PFGRC Spotfinder software (http://www.tm4.org/spotfinder.html), and the Cy3 and Cy5 images were over-laid. Using the software, a spot-locating grid was created and used toidentify spots and determine spot intensity. The Microarray Data AnalysisSoftware (MIDAS) from PFGRC was used to normalize data collectedfrom the overlaid spot grids (http://www.tm4.org/midas.html). BRBarray tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) were used togenerate the statistical data, with a P value of 0.001 used as the cutoff.

Real-time quantitative RT-PCR. RNA was isolated from experimen-tal samples as described above. Using 1 �g of RNA, SuperScript III reversetranscriptase was utilized to generate cDNA using gene-specific antisenseprimers. Gene-specific PCR products were created and used as standards.The preparation of standards and data analysis were conducted as de-scribed elsewhere (25).

Microarray data accession number. Microarray data have been de-posited in the NCBI Gene Expression Omnibus (GEO) database(GSE51362).

RESULTSAcid production. The ability of S. mutans strains to lower the pHwhen provided with excess exogenous glucose was monitored by apH drop assay, as detailed in the Materials and Methods section.Cell suspensions of S. mutans UA159 that had been cultured tosteady state under glucose-limiting (10 mM glucose) conditionsrapidly lowered the pH to an average value of 3.50 � 0.08 after 30min (Table 1). In contrast, wild-type cells that had been grownwith excess glucose (100 mM glucose) lowered the pH to an aver-age of 3.70 � 0.01 during the same time interval (Fig. 1A; P �0.047). Moreover, glucose-limited cells were able to lower the pHat a higher rate, as illustrated by the fact that the pH of cell sus-pensions fell to an average of 5.39 � 0.31 after 2 min, whereaswild-type cells grown in excess glucose reached a pH of 6.23 �0.08 over the same time period (Fig. 1A; P � 0.035). It was alsonoted that cells grown in excess glucose required substantiallymore KOH to equilibrate the pH before beginning the assay thanglucose-limited cells, presumably due to metabolism of endoge-

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nous stores of glycogen-like polysaccharides. To confirm this, werapidly neutralized fresh cell suspensions to a pH of 7.2 and mon-itored the pH for 30 min without adding exogenous glucose.These results demonstrated that S. mutans UA159 grown withexcess glucose lowered the pH to an average of 6.39 � 0.20,whereas cells grown under glucose-limiting conditions loweredthe pH to 6.68 � 0.06 (Fig. 1B; P � 0.119).

Interestingly, cell suspensions of the manL mutant culturesthat had been grown to steady state in limiting glucose were able tolower the pH after 30 min to an average of 3.88 � 0.12, whereascells grown in excess glucose lowered the pH to an average of3.76 � 0.05 (Fig. 1C; P � 0.220). Additionally, glucose-limited

manL mutant cells consistently lowered the pH at a higher rate(6.03 � 0.21 after 2 min) than cells grown in excess glucose(6.30 � 0.20 over the same time period), although the differenceswere not statistically significant between cells grown in excess glu-cose and cells grown with glucose limitation (P � 0.195). As men-tioned above, glucose-limited wild-type cells were able to lowerthe pH to an average of 5.39 � 0.31 after 2 min, and this wassignificantly different from the results for the manL mutant grownin limiting glucose (P � 0.047) or excess glucose (P � 0.018),although there were no significant differences in the pH valuesattained by the wild-type and manL mutant cell suspensions whengrown in excess glucose (P � 0.616). Thus, the loss of ManL dra-matically impacts glycolytic rates when cells are grown in limitingglucose. Also of note, the terminal pH achieved in the pH dropassay is strongly correlated with acid tolerance (17, 41, 42), withthe lower pH being associated with greater acid tolerance. The factthat cells that lacked ManL and that were limited for glucose couldnot achieve as low a terminal pH as the wild-type strain grownunder the same conditions implicates ManL as a potential con-tributor to or regulator of acid tolerance. We also attempted toquantify the acid tolerance of cells using a typical acid killing assay(37). However, exposure to pH 2.8 of fresh chemostat cultures ofboth the wild-type and manL deletion strains grown with excessglucose caused the cells to aggregate into large clumps very rap-idly. Despite multiple attempts to disperse the cells, it was notpossible to reliably enumerate the CFU in these populations of

TABLE 1 Comparison of pH values achieved by S. mutans UA159 and astrain bearing a deletion of manL grown in continuous culture using 10mM versus 100 mM glucose

Strain(glucoseconcn[mM])

Exogenous glucose addedEndogenous carbohydratestores

pH after2 min

pH after30 min

pH after2 min

pH after30 min

UA159 (10) 5.39 � 0.31 3.50 � 0.08 7.13 � 0.00 6.68 � 0.06UA159 (100) 6.23 � 0.08 3.70 � 0.01 6.99 � 0.02 6.39 � 0.20manL (10) 6.03 � 0.21 3.88 � 0.12 NDa NDmanL (100) 6.30 � 0.20 3.76 � 0.05 ND NDa ND, not determined.

FIG 1 Glycolytic acidification by S. mutans UA159 and a manL mutant strain grown in TV base medium supplemented with either 10 mM or 100 mM glucose(glc) to steady state in continuous culture. Experiments were conducted as described in the Materials and Methods section, and each data set represents an averageof the results from three independent experiments. The pH of the cell suspensions was neutralized to 7.2, and the change in pH for S. mutans UA159 (A) and amanL mutant strain (C) was monitored over time after the addition of glucose to a final concentration of 50 mM. *, P 0.05 (by the Student t test). Additionally,to assess the metabolism of intracellular stores of carbohydrate, the pH of wild-type cell suspensions was rapidly neutralized, and the change in pH was monitoredwithout the addition of exogenous sugar (B).

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cells following an acid challenge. Initial investigation into thecause of the clumping at low pH suggested that it was associated, atleast in part, with enhanced cell surface hydrophobicity (data notshown).

PTS activity. To test whether the enhanced generation of acidby glucose-limited cells could be associated with the more efficientinternalization of carbohydrates by the PTS, the PTS-dependenttransport of carbohydrates by cells grown under conditions ofglucose limitation or excess was compared. The transport of man-nose, fructose, or glucose by the PTS was markedly higher in wild-type cells grown under conditions of glucose limitation than incells grown with excess glucose (Fig. 2A). This finding is consistentwith and may in large part explain the higher rate at which cellsgrown under conditions of limiting glucose can lower the pH (Fig.1A). Conversely, the manL mutant strain displayed very low levelsof PTS-dependent transport of glucose and mannose. These re-sults agree with those of the pH drop experiments, in that themanL mutant strain showed a consistently lower rate and extent ofacidification when grown under glucose limitation. Notably, thetransport of fructose by cells grown under glucose limitation was

also lower for cells lacking ManL than for cells of the wild-typestrain, but no difference in PTS-dependent fructose transport wasobserved between wild-type and manL mutant cells grown withexcess glucose.

To determine if the effects on PTS-dependent carbohydratetransport in the manL mutant were associated with a general de-fect in the PTS or were associated only with the loss of ManL,which has been shown to transport glucose, mannose, and galac-tose, PTS-dependent transport assays were performed using malt-ose, cellobiose, or lactose as the substrate. For all three disaccha-rides, cells grown with glucose limitation were more efficient attransporting these carbohydrates than cells grown with glucoseexcess (Fig. 2B). Cells bearing a deletion in manL displayed a re-duced capacity for PTS-dependent transport of cellobiose andmaltose (Fig. 2B). The PTS-dependent transport of lactose wasgenerally lower than that for the other sugars tested, probably dueto the requirement for lactose in the growth medium to induce thelac operon in S. mutans (43, 44).

PTS enzyme levels and carbohydrate availability. To probe inmore detail the basis for the decreased PTS-dependent carbohy-

FIG 2 S. mutans UA159 and a manL mutant strain grown in continuous culture were assessed for PTS activity as described in the Materials and Methods sectionusing glucose (glc), fructose (fru), or mannose (man) (A) or maltose (malt), lactose (lac), or cellobiose (cel) (B) as the substrate. Each bar represents the averageresults of three independent experiments. *, P 0.05; **, P 0.01; ***, P 0.001 (by the Student t test).

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drate transport observed in cells grown in excess glucose, immu-noblotting was used to compare the levels of selected PTS proteinsobtained under different growth conditions and with differentstrains. To perform this analysis, we generated in a rabbit antiseraagainst a full-length, histidine-tagged S. mutans ManL proteinthat was expressed in E. coli, purified by nickel affinity chromatog-raphy, and further purified by SDS-PAGE and excision of gelslices. Immunoblotting confirmed that the antibody specificallyrecognized a protein band with the predicted size of ManL (�35kDa) in batch-grown cells cultured in glucose or fructose and thatthis band was absent in cells carrying a nonpolar allelic exchangemutation of the manL gene (see Fig. S1 in the supplemental ma-terial) (45). We next probed cell lysates of S. mutans UA159 fromsteady-state cultures grown under glucose-limited conditions orwith excess glucose and consistently found more ManL protein incells grown under glucose-limited conditions than in cells grownwith excess glucose (Fig. 3A). Immunoblotting of the strain bear-ing a manL deletion grown in continuous culture in limiting orexcess glucose was performed and revealed no protein bands un-der either condition (data not shown).

In contrast to the results obtained with ManL, when immuno-blots were performed using antibodies against the general PTSproteins EI and HPr, very little change in the amount of theseproteins in the wild-type cells grown under glucose-limiting and-excess conditions was evident (Fig. 3A). Similar results were seenfor the amounts of EI and HPr in the manL deletion strain (Fig.3B). Immunoblot analysis was performed using whole-cell lysatesof at least three separate replicates, and by using densitometry, itwas determined that the levels of EI and HPr never differed bygreater than 40% between cells grown in limiting or excess glu-cose. We also performed qRT-PCR to compare the expressionlevels of manL, ptsI (EI), and ptsH (HPr) using RNA generated

from chemostat samples of wild-type cultures. We observed con-sistently lower levels of manL mRNA by real-time PCR in cellsgrown with excess glucose (data not shown), in agreement withour microarray results (see Table 3). In contrast, the transcriptlevels of EI (ptsI) and HPr (ptsH) did not differ between the twowild-type cell populations (see Fig. S2 in the supplemental mate-rial).

Our previous studies with S. mutans UA159 have providedevidence that HPr phosphorylated on serine 46 [HPr(Ser-P)] canregulate carbohydrate transport (26). To probe whether the phos-phorylation state of HPr was altered by the deletion of manL andcould therefore be a potential contributor to the observed de-crease in overall PTS activity, we subjected cell lysates to nonde-naturing PAGE and performed immunoblotting with HPr antise-rum. The wild-type and manL mutant strains grown underconditions of glucose excess displayed prominent bands corre-sponding to HPr(Ser-P) and the dually phosphorylated form ofHPr [HPr(Ser-P)(His�P)], whereas cells grown under glucoselimitation completely lacked these bands and contained mostlyHPr(His�P) (Fig. 4). Although we consistently observed slightlylower levels of the serine-phosphorylated forms of HPr in themanL mutant, HPr(Ser-P) and HPr(Ser-P)(His�P) were nearlyas abundant and readily detected in the manL mutant grown un-der conditions of glucose excess.

The transcriptome of Streptococcus mutans UA159 is alteredin response to carbohydrate availability. The transcriptionalchanges present in steady-state planktonic cultures of S. mutansUA159 were monitored as a function of glucose availability. Com-parison of the transcriptomes of cells grown in limiting versusexcess glucose revealed that 174 genes were differentially ex-pressed (P 0.001), with 81 transcripts being upregulated and 93transcripts being downregulated under conditions of limiting glu-

FIG 3 Immunoblotting of lysates to determine the levels of ManL, EI, and HPr. (A) S. mutans UA159 grown in continuous culture using TV mediumsupplemented with 10 mM or 100 mM glucose was lysed by mechanical disruption, and equal amounts of protein were separated by SDS-PAGE. Immunoblottingwas performed using ManL, EI, and HPr antibodies at a previously determined optimal dilution of primary antibody. (B) The manL deletion strain grown incontinuous culture using TV medium supplemented with 10 mM or 100 mM glucose was lysed by mechanical disruption, and equal amounts of protein wereseparated by SDS-PAGE. Immunoblotting was performed using EI and HPr antibodies. The images are representatives of at least three independent replicates,with each replicate showing similar results.

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cose compared to the gene regulation under growth conditionswith excess glucose (Table 2). The validity of the microarray re-sults was confirmed by subjecting a subset of genes to real-timequantitative PCR (Table 3).

Cells grown in limiting carbohydrate showed higher expres-sion of genes associated with carbohydrate transport and energymetabolism, suggestive of a broad relief of catabolite repression.Interestingly, we observed elevated level of transcripts of EII forfructose (SMU.115) and the cotranscribed fructose-1-phosphatekinase; a second fructose PTS operon, including EII (SMU.872);an associated transcriptional repressor and a second fructose-1-phosphate kinase; the predicted ribulose-monophosphate PTS EIIenzyme; and an associated metabolic gene encoding hexulose-6-phosphate isomerase, as well as glucose/mannose and trehalosePTS EII permeases. Also of note, the gene for the transcriptionalregulator CcpA was more highly expressed in glucose-limitedcells. Again, in relation to sugar metabolism, we noted an elevatedlevel of transcripts encoding ScrB, sucrose-6-phosphate hydro-lase, and several transcripts of the msm ABC transporter system,including msmE, msmF, msmG, gtfA, msmK, and dexB. Collec-tively, these results provide evidence that glucose-limited cells ad-just gene expression to optimize the capacity for metabolism ofother carbohydrates, even in the absence of inducing sugars.

One of the most striking differences between conditions oflimiting and excess glucose was found in the category of energymetabolism. Under conditions of limiting glucose, a transcript ofthe Leloir pathway (galT) and a galactose-6-phosphate isomerase(lacB) of the tagatose pathway for galactose metabolism were ele-vated. Notably, the genes for the metabolism of glycogen, glyco-gen phosphorylase (phsG), glycogen synthase (glgA), and otherglycogen metabolic genes (glgB, glgC, glgD) were upregulated.Two genes encoding components of the pyruvate dehydrogenase(PDH) complex, SMU.1422 and SMU.1423, were also upregu-lated in glucose-limited cells and represented the most highly up-regulated genes. Also in relation to energy metabolism, transcriptsfor a putative phosphoglucomutase (pgm), adhE encoding a pu-tative alcohol/acetaldehyde dehydrogenase, the water-formingNADH oxidase, pyruvate formate-lyase, and a putative succinatesemialdehyde dehydrogenase were elevated under conditions ofglucose limitation. Finally, we noted the upregulation of severalgenes related to cell processes that were of interest, including the

gene for the molecular chaperone DnaK and SMU.1425 (clpB),which encodes the ATP-binding subunit of a putative Clp protei-nase.

Cells grown under conditions of excess carbohydrate displayeda transcriptome very different from that of cells cultured in limit-ing glucose. Of particular interest, we noted the upregulation ofthe transcriptional regulator codY and the associated transcriptpncA. Genes for two putative amino acid permeases (SMU.1450and SMU.951), a subunit (OpuCa) of the putative osmopro-tectant amino acid ABC transporter, and a subunit of an unrelatedputative amino acid ABC transporter were upregulated. We alsonoted that the transcription of a predicted zinc-dependent pro-tease, SMU.1784c, which encodes a potential homolog of theEnterococcus faecalis membrane-associated protease Eep (46), aswell as two proteases resembling collagenase, SMU.759 andSMU.761, was elevated under conditions of glucose excess. In ad-dition, we observed the upregulation of several genes related toamino acid biosynthesis, including threonine dehydratase, glu-tamine synthetase, threonine synthase, argininosuccinate lyase,and a putative NADP-specific glutamate dehydrogenase. The in-creased expression of this group of genes may signal the activationof amino acid-scavenging pathways, consistent with the idea thatcells growing with excess glucose are likely limited for an aminoacid or group of amino acids (27, 28). Finally, in relation to signalsof depleted resources, we noted elevated transcription of phoH,which encodes a protein associated with phosphate starvation.

Our microarray results also revealed the upregulation of genesinstrumental in the transport of solutes across the membrane andthe biosynthesis of nucleotide precursors and components neededfor cell wall integrity in cells grown in excess glucose. Genes relatedto transport included a glycerol uptake facilitator permease, theammonium transporter NrgA, a putative multidrug efflux pump,a putative sodium-dependent transporter, and a subunit of anABC transporter of unknown function (SMU.1315c). With regardto the metabolism of nucleotides and nucleotide precursors, anumber of genes displayed elevated transcription, including guaB,involved in GTP biosynthesis; pyrD for pyrimidine biosynthesis;purL and purC for purine metabolism; add for adenosine metab-olism; and kitH, which encodes a putative thymidine kinase. Alsoof interest, transcripts for several cell wall biosynthesis gene prod-ucts, including undecaprenyl pyrophosphate synthetase, UDP-N-

FIG 4 S. mutans UA159 (wt) and a manL deletion strain grown in continuous culture using TV medium supplemented with 10 mM or 100 mM glucose werelysed by mechanical disruption, and equal amounts of protein were either boiled (indicated by the plus symbol) at 100°C for 5 min or immediately subjected tonondenaturing PAGE (indicated by the minus symbol) to separate the various species of HPr on the basis of charge. Immunoblotting was performed using anHPr antibody, and the image is a representative of more than three independent replicates, with each replicate showing similar results.

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TABLE 2 Microarray comparing continuous cultures of S. mutans UA159 grown in 10 mM versus 100 mM glucose

Gene Product descriptiona Functional categoryFoldchangeb P value

SMU.1422 Putative pyruvate dehydrogenase E1 component beta subunit Energy metabolism 133.8 4.74E�04SMU.1423 Putative pyruvate dehydrogenase, TPP-dependent, E1 component alpha subunit Energy metabolism 124.93 4.50E�05SMU.1495 Galactose-6-phosphate isomerase subunit LacB Energy metabolism 53.9 2.47E�04SMU.1539 Glycogen branching enzyme Energy metabolism 30.91 6.00E�07SMU.113 Putative fructose-1-phosphate kinase Energy metabolism 30.5 2.50E�04SMU.1538 Glucose-1-phosphate adenylyltransferase Energy metabolism 29.33 5.20E�06SMU.179 Hypothetical protein Unknown 27.2 4.20E�06SMU.115 Putative PTS system, fructose-specific IIA component PTS 25.67 1.93E�04SMU.2038 Putative PTS system, trehalose-specific IIABC component PTS 25.3 1.29E�04SMU.1537 Putative glycogen biosynthesis protein GlgD Energy metabolism 24.42 6.50E�06SMU.1425 Putative Clp proteinase, ATP-binding subunit ClpB Protein synthesis and fate 23.25 1.47E�04SMU.1340 Putative surfactin synthetase Cellular processes 16.67 2.94E�04SMU.1536 Glycogen synthase Energy metabolism 16.25 2.29E�05SMU.148 Bifunctional acetaldehyde-CoA/alcohol dehydrogenase Energy metabolism 15.08 6.50E�06SMU.180 Putative oxidoreductase, fumarate reductase Unknown 14.77 2.12E�05SMU.252 Hypothetical protein Hypothetical 14.41 2.38E�04SMU.1116c Hypothetical protein Hypothetical 13.69 8.89E�05SMU.1879 Putative PTS system, mannose-specific component IID PTS 12.01 8.07E�04SMU.270 Ascorbate-specific PTS system enzyme IIC PTS 11.59 4.55E�05SMU.1535 Glycogen phosphorylase Energy metabolism 10.34 4.54E�05SMU.870 Putative transcriptional regulator of sugar metabolism Transcription regulator 9.48 8.40E�06SMU.1117 NADH oxidase (H2O forming) Energy metabolism 8.78 3.03E�04SMU.290 Putative L-ascorbate 6-phosphate lactonase Unknown 8.67 2.52E�04SMU.402 Pyruvate formate-lyase Energy metabolism 8.56 8.21E�05SMU.500 Putative ribosome-associated protein Protein synthesis and fate 8.34 8.72E�05SMU.881 Sucrose phosphorylase GtfA Transport and binding 8.1 5.10E�06SMU.1411 Hypothetical protein Unknown 7.61 2.27E�05SMU.1878 Putative PTS system, mannose-specific component IIC PTS 7.45 6.79E�04SMU.2127 Putative succinate semialdehyde dehydrogenase Central intermediary metabolism 7.41 5.06E�05SMU.1843 Sucrose-6-phosphate hydrolase Energy metabolism 7 3.27E�05SMU.879 Multiple sugar-binding ABC transporter, permease protein MsmF ABC transporter 6.91 4.55E�04SMU.871 Putative fructose-1-phosphate kinase Energy metabolism 6.56 8.07E�04SMU.878 Multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE ABC transporter 6.54 1.52E�05SMU.880 Multiple sugar-binding ABC transporter, permease protein MsmG ABC transporter 6.49 3.94E�05SMU.882 Multiple sugar-binding ABC transporter, ATP-binding protein, MsmK ABC transporter 6.46 3.26E�05SMU.1158c Hypothetical protein Unknown 6.44 3.10E�04SMU.1344c Putative malonyl-CoA acyl carrier protein transacylase Fatty acid and phospholipid

metabolism6.37 3.65E�04

SMU.1877 Putative PTS system, mannose-specific component IIAB PTS 5.93 5.50E�04SMU.883 Dextran glucosidase DexB Energy metabolism 5.82 6.10E�06SMU.447 Hypothetical protein Unknown 5.79 8.70E�04SMU.1077 Putative phosphoglucomutase Energy metabolism 5.18 1.23E�05SMU.1574c Hypothetical protein Unknown 4.93 2.47E�04SMU.887 Galactose-1-phosphate uridylyltransferase Energy metabolism 4.71 1.88E�04SMU.1088 Putative thiamine biosynthesis lipoprotein Biosynthesis of cofactors, prosthetic

groups, and carriers4.38 3.17E�05

SMU.2155 Hypothetical protein Unknown 4.35 7.50E�04SMU.872 Putative PTS system, fructose-specific enzyme IIABC component PTS 4.08 3.87E�04SMU.550 Putative cell division protein FtsQ (DivIB) Cellular processes 3.89 7.10E�04SMU.1591 Catabolite control protein A, CcpA Transcription regulator 3.65 8.13E�05SMU.1090 Hypothetical protein Unknown 3.6 1.42E�04SMU.1644c Hypothetical protein Unknown 3.48 8.21E�05SMU.549 Undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase Cell envelope 3.47 7.84E�04SMU.82 Molecular chaperone DnaK Cellular processes 3.2 8.73E�04SMU.131 Putative lipoate-protein ligase Protein synthesis and fate 3.17 8.36E�04SMU.1023 Oxaloacetate decarboxylase Amino acid biosynthesis 3.06 2.79E�05SMU.1389 Hypothetical protein Energy metabolism 3.03 4.50E�04SMU.2035 Bacteriocin immunity protein Cellular processes 2.9 1.54E�04SMU.271 Putative PTS system, enzyme IIB component PTS 2.82 5.86E�05SMU.129 Branched-chain alpha-keto acid dehydrogenase subunit E2 Energy metabolism 2.77 4.81E�04SMU.1089 Hypothetical protein Unknown 2.77 4.16E�04SMU.241c Putative ABC transporter, ATP-binding protein, amino acid transport system ABC transporter 2.76 2.63E�05SMU.20 Putative cell shape-determining protein MreC Cell envelope 2.57 7.70E�06SMU.1737 (3R)-Hydroxymyristoyl-ACP dehydratase Fatty acid and phospholipid

metabolism2.55 2.96E�04

SMU.274 Putative L-xylulose 5-phosphate 3-epimerase Energy metabolism 2.55 6.42E�04SMU.2005 Adenylate kinase Biosynthesis of nucleotides and

precursors2.52 4.56E�05

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TABLE 2 (Continued)

Gene Product descriptiona Functional categoryFoldchangeb P value

SMU.963c Putative deacetylase Energy metabolism 2.52 5.74E�04SMU.2053c Hypothetical protein Hypothetical 2.44 4.14E�04SMU.259 Putative oligopeptide ABC transporter, ATP-binding protein OppF ABC transporter 2.43 4.27E�04SMU.1462c Putative oxidoreductase Unknown 2.38 6.91E�04SMU.2052c Hypothetical protein Hypothetical 2.38 1.95E�05SMU.1619c Putative metalloprotease Unknown 2.24 4.02E�04SMU.580 Exodeoxyribonuclease VII large subunit Biosynthesis of nucleotides and

precursors2.24 1.26E�04

SMU.812 Hypothetical protein Hypothetical 2.24 4.49E�05SMU.1428c Hypothetical protein Unknown 2.23 5.57E�04SMU.844 Hypothetical protein Unknown 2.23 7.17E�04SMU.1020 Putative citrate lyase CilB, citryl-CoA lyase, beta subunit Energy metabolism 2.22 1.15E�04SMU.1679c Hypothetical protein Unknown 2.21 1.48E�04SMU.458 Putative ATP-dependent RNA helicase Transcription 2.14 2.35E�05SMU.1542c Putative lipid kinase Unknown 2.07 5.12E�04SMU.1011 Putative CitG protein Energy metabolism 2.06 2.10E�04SMU.1207 Mobilization/cell filamentation proteins Unknown 2.04 5.04E�04SMU.1229 Purine nucleoside phosphorylase Biosynthesis of nucleotides and

precursors2.02 3.68E�04

SMU.517 Phosphopantetheine adenylyltransferase Cell envelope 0.49 4.26E�04SMU.848 Hypothetical protein Unknown 0.49 5.87E�05SMU.1784c Membrane-associated Zn-dependent protease Unknown 0.48 8.82E�04SMU.712 Phosphoenolpyruvate carboxylase Central intermediary metabolism 0.47 9.51E�04SMU.318 Putative hippurate hydrolase Central intermediary metabolism 0.47 8.98E�04SMU.234 Threonine dehydratase Amino acid biosynthesis 0.46 4.47E�04SMU.1623c Hypothetical protein Unknown 0.46 5.44E�04SMU.509 Hypothetical protein Unknown 0.46 3.48E�04SMU.841 Putative aminotransferase Biosynthesis of cofactors, prosthetic

groups, and carriers0.45 2.37E�04

SMU.707c Putative endolysin Cell envelope 0.45 8.01E�04SMU.1076 Hypothetical protein Unknown 0.45 3.15E�04SMU.1745c Putative transcriptional regulator Transcription regulator 0.44 1.11E�04SMU.1191 6-Phosphofructokinase Energy metabolism 0.44 6.13E�04SMU.320 Putative 5-formyltetrahydrofolate cyclo-ligase Biosynthesis of cofactors, prosthetic

groups, and carriers0.44 4.40E�04

SMU.1325 Putative ABC transporter, ATP-binding component ABC transporter 0.44 3.63E�04SMU.759 Putative protease Unknown 0.43 7.03E�04SMU.321 Hypothetical protein Unknown 0.43 4.92E�05SMU.364 Glutamine synthetase type 1, glutamate-ammonia ligase Amino acid biosynthesis 0.42 8.31E�05SMU.330 Glutamyl-tRNA synthetase Protein synthesis and fate 0.41 2.37E�04SMU.572 Putative tetrahydrofolate dehydrogenase/cyclohydrolase Biosynthesis of cofactors, prosthetic

groups, and carriers0.4 1.00E�07

SMU.2121c Hypothetical protein Unknown 0.4 6.18E�04SMU.1685c Hypothetical protein Unknown 0.4 5.04E�04SMU.1930 Putative cytoplasmic membrane protein, LemA-like protein Unknown 0.39 3.16E�04SMU.648 Foldase protein PrsA Cellular processes 0.39 9.99E�05SMU.235 Hypothetical protein Unknown 0.39 1.85E�04SMU.429c Hypothetical protein Hypothetical 0.39 2.74E�04SMU.337 Hypothetical protein Unknown 0.38 7.49E�04SMU.647 Putative methyltransferase Unknown 0.37 3.86E�05SMU.1824c Transcriptional repressor CodY Transcription regulator 0.37 5.24E�04SMU.70 Threonine synthase Amino acid biosynthesis 0.36 2.56E�04SMU.1789c Hypothetical protein Unknown 0.36 8.32E�05SMU.2067 Putative stress response protein, glycosyltransferase involved in cell wall biogenesis Unknown 0.36 8.49E�04SMU.761 Putative protease Unknown 0.35 3.38E�04SMU.1140c Hypothetical protein Unknown 0.34 2.72E�05SMU.2032 30S ribosomal protein S2 Protein synthesis and fate 0.34 5.67E�04SMU.1620 Putative phosphate starvation-induced protein PhoH Unknown 0.33 5.00E�07SMU.22 Putative secreted antigen GbpB/SagA, putative peptidoglycan hydrolase Cell envelope 0.33 2.10E�04SMU.1823 Putative pyrazinamidase/nicotinamidase Biosynthesis of cofactors, prosthetic

groups, and carriers0.32 7.48E�04

SMU.2116 Putative osmoprotectant amino acid ABC transporter, ATP-binding protein ABC transporter 0.32 7.36E�04SMU.469 Holliday junction-specific endonuclease DNA metabolism and repair 0.32 5.87E�04SMU.568 Putative amino acid ABC transporter, ATP-binding protein ABC transporter 0.32 2.59E�04SMU.1086 Thymidine kinase Biosynthesis of nucleotides and

precursors0.31 3.90E�04

SMU.1450 Putative amino acid permease Unknown 0.31 4.27E�04SMU.1786 Putative undecaprenyl pyrophosphate synthetase Cell envelope 0.3 2.79E�04SMU.676 NADP-dependent glyceraldehyde-3-phosphate dehydrogenase Energy metabolism 0.3 2.19E�04

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acetylmuramyl tripeptide synthetase, UDP-N-acetylglucosamine2-epimerase, a putative peptidoglycan hydrolase, and a putativeglycosyltransferase (csbB) involved in cell stress, were elevated un-der conditions of glucose excess. Finally, a few genes related to themetabolism of cofactors were upregulated; the most notableamong these genes were a putative biotin biosynthesis protein(SMU.1827) and thiD, which is involved in thiamine metabolism,as well as other genes associated with energy metabolism, includ-

ing gapN (SMU.676), capP (SMU.712), and a putative proteinmaturation peptidase precursor (prtM).

DISCUSSION

The intermittent introduction of and variety in the types of car-bohydrates in the diet of the human host presents multiple chal-lenges to members of the oral microbiota, including S. mutans (7,9). Of particular relevance to this study is that a delay in the in-

TABLE 2 (Continued)

Gene Product descriptiona Functional categoryFoldchangeb P value

SMU.474 S-Ribosylhomocysteinase Cellular processes 0.29 4.80E�04SMU.1189c Hypothetical protein Unknown 0.28 4.26E�05SMU.1429 Putative UDP-N-acetylmuramyl tripeptide synthetase MurC Cell envelope 0.28 6.99E�04SMU.369c Hypothetical protein Unknown 0.28 5.50E�05SMU.1476c Putative GTP-binding protein Unknown 0.27 2.13E�04SMU.1326 Peptide chain release factor 2 Protein synthesis and fate 0.26 3.73E�04SMU.1437 Putative UDP-N-acetylglucosamine 2-epimerase Central intermediary metabolism 0.25 3.07E�04SMU.611 ATP-dependent RNA helicase Transcription 0.24 5.39E�05SMU.516 Hypothetical protein Unknown 0.23 4.47E�05SMU.530c Hypothetical protein Unknown 0.22 2.29E�05SMU.335 Argininosuccinate lyase Amino acid biosynthesis 0.22 1.30E�04SMU.210c Hypothetical protein Hypothetical 0.22 6.82E�04SMU.1627 50S ribosomal protein L11 Protein synthesis and fate 0.21 3.00E�06SMU.283 Hypothetical protein Hypothetical 0.21 9.62E�04SMU.1200 30S ribosomal protein S1 Protein synthesis and fate 0.2 3.94E�04SMU.595 Dihydroorotate dehydrogenase 1A Biosynthesis of nucleotides and

precursors0.2 9.10E�06

SMU.204c Hypothetical protein Hypothetical 0.19 6.65E�04SMU.2157 IMP dehydrogenase Biosynthesis of nucleotides and

precursors0.18 6.29E�04

SMU.951 Putative amino acid permease Transport and binding 0.17 2.42E�04SMU.396 Putative glycerol uptake facilitator protein Transport and binding 0.17 4.60E�06SMU.1315c Putative ATP-binding protein ABC transporter 0.17 4.12E�05SMU.195c Hypothetical protein Hypothetical 0.17 7.42E�04SMU.277 Hypothetical protein Hypothetical 0.16 1.63E�05SMU.1658 Putative ammonium transporter, NrgA protein Transport and binding 0.15 3.14E�04SMU.336 RNase P Transcription 0.14 7.47E�04SMU.1098c Putative oxidoreductase Unknown 0.14 2.00E�06SMU.87 Hypothetical protein Unknown 0.14 1.51E�04SMU.30 Putative phosphoribosylformylglycinamidine synthase, (FGAM synthase) Biosynthesis of nucleotides and

precursors0.14 4.47E�04

SMU.850 Hypothetical protein Unknown 0.13 4.87E�04SMU.285 Hypothetical protein Hypothetical 0.13 4.28E�04SMU.278 Hypothetical protein Hypothetical 0.12 9.40E�06SMU.1626 50S ribosomal protein L1 Protein synthesis and fate 0.12 7.59E�04SMU.1477 tRNA delta(2)-isopentenylpyrophosphate transferase Protein synthesis and fate 0.12 3.66E�04SMU.1946 Hypothetical protein Hypothetical 0.12 3.84E�04SMU.670 Aconitate hydratase Energy metabolism 0.12 9.51E�04SMU.71 Putative cation efflux pump (multidrug resistance protein) Transport and binding 0.11 6.97E�04SMU.281 Hypothetical protein Hypothetical 0.11 9.34E�04SMU.913 Glutamate dehydrogenase Amino acid biosynthesis 0.11 5.83E�05SMU.86 Hypothetical protein Unknown 0.11 9.10E�06SMU.196c Putative transfer protein Unknown 0.098 7.66E�04SMU.85 Phosphomethylpyrimidine kinase Biosynthesis of cofactors, prosthetic

groups, and carriers0.089 5.37E�05

SMU.600c Hypothetical protein Unknown 0.077 2.02E�04SMU.1295 Adenosine deaminase Biosynthesis of nucleotides and

precursors0.067 3.72E�04

SMU.29 Phosphoribosylaminoimidazole-succinocarboxamide synthase Biosynthesis of nucleotides andprecursors

0.049 6.79E�04

SMU.962 Putative dehydrogenase Fatty acid and phospholipidmetabolism

0.024 2.59E�04

SMU.961 Hypothetical protein Unknown 0.023 4.49E�04SMU.602 Putative sodium-dependent transporter Transport and binding 0.021 8.57E�04SMU.1827 Putative biotin biosynthesis protein Biosynthesis of cofactors, prosthetic

groups, and carriers0.02 4.48E�04

a TPP, thiamine pyrophosphate; CoA, coenzyme A; FGAM, formylglycinamidine ribonucleotide.b A fold change above 1.00 indicates upregulation under glucose-limiting conditions.

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duction of expression of transporters and catabolic pathways forparticular carbohydrates that are consumed by the host could ren-der S. mutans less competitive with commensal bacteria and im-pede its ability to contribute to the caries process. In our study,cells grown under conditions of glucose limitation displayed ageneral trend of increased PTS-dependent transport efficiencyacross all sugars tested (Fig. 2). These results agree with those ofprevious studies and significantly extend the findings of previousstudies that showed enhanced transport of glucose, mannose, and2-deoxyglucose in carbohydrate-limited cells, although the glu-cose concentrations, dilution rates, and the strain utilized in ourstudy differed from those utilized in previous studies (31, 32).

The diminished level of PTS-dependent sugar transport de-tected in both the wild-type and manL deletion strains when cellswere grown with excess glucose could be related to the accumula-tion of HPr(Ser-P) and the dually phosphorylated form of HPr,HPr(Ser-P)(His�P) (Fig. 4). These observations are consistentwith those presented in a previous report where an increase inHPr(Ser-P)(His�P) and HPr(Ser-P) species was evident when S.mutans strain Ingbritt was grown in continuous culture in me-dium containing excess glucose (47). HPr is a central effector ofcatabolite repression, and its phosphorylation state can have asignificant impact on growth and carbohydrate transport, as wellas on the expression of virulence-related genes, by enhancing thebinding of CcpA to catabolite-responsive elements (CREs) (18,26, 48). We suggest that the repression of PTS-dependent trans-port noted when cells were grown with excess glucose could beattributed in part to the interference of HPr(Ser-P) with carbohy-drate transporters, which would prevent the internalization of theinducing sugar, a phenomenon known as inducer exclusion (48).Evidence for HPr-dependent interference with carbohydratetransport has been observed in genetically modified derivatives ofS. mutans UA159 expressing elevated levels of HPr(Ser-P) or anHPr(Ser-P) mimic, with the latter being an HPr carrying an aspar-tic acid residue in place of serine 46. Strains carrying this serine-to-aspartic acid mutation displayed repression of PTS-dependentuptake of glucose, fructose, and mannose, as well as diminishedgrowth on lactose and cellobiose (26). Thus, HPr phosphorylationstatus, perhaps coupled with changes in ccpA expression, must beconsidered a possible explanation for the phenotypic and tran-scriptomic differences in cells cultured in limiting versus excessglucose. In contrast, the fact that the pools of modified and un-modified HPr in the wild-type and manL mutant strains weregenerally similar (Fig. 4) supports the notion that differences inthe phenotypic behaviors of the wild-type and manL mutant

strains arise primarily from the loss of ManL rather than frommajor changes in the amount or phosphorylation status of HPr.

Previous studies have demonstrated that cells of S. mutansgrown with excess glucose under continuous culture conditionswere limited for aspartate and asparagine at high dilution rates,whereas only cysteine was found to be limiting at low dilutionrates (27–29). Our microarray data revealed elevated transcrip-tion of genes related to the transport of amino acids and aminoacid biosynthesis when cells were grown in excess glucose. Fur-ther, we noted the upregulation of the transcriptional regulatorCodY, which is a global regulator of gene expression that respondsto the levels of branched-chain amino acids. These data providestrong evidence that the organism is indeed limited for aminoacids during periods of growth with excess carbohydrate. Severalof the genes that were upregulated when cells were grown withexcess glucose were annotated as amino acid or ion transporters,as well as multiple uncharacterized transporters. Given the poten-tially important role that these transporters play under conditionsthat are conducive to the development of caries, i.e., when excesscarbohydrates are available, future studies identifying ways to in-hibit the acquisition of key nutrients by S. mutans during carbo-hydrate excess may discourage the emergence of this caries patho-gen in oral biofilms.

Previous investigators have shown that there is insufficientPTS-dependent transport in cells growing with excess carbohy-drate to account for the total amount of glucose internalized, andit has been suggested that an alternative glucose uptake systemmust exist (27, 33, 49, 50). The most convincing evidence for thistheory is that a ptsI mutant strain lacking enzyme I was able togrow when glucose was present in the growth medium, but nogrowth could be observed on any other PTS sugar. Further, non-PTS sugars, such as raffinose and mellibiose, could stimulate thegrowth of the ptsI mutant (50). It has been suggested that glucoseuptake at high growth rates occurs through an ATP-dependentmechanism, and the msm ABC transporter has been proposed tobe an alternative glucose transporter (49, 50). However, our re-sults demonstrate that the msm genes are downregulated, alongwith PTS activity, when S. mutans UA159 is grown with excessglucose. Functional analysis of the remaining uncharacterizedtransport systems upregulated under glucose excess may elucidatewhether any of these transporters can participate in PTS-indepen-dent internalization of glucose or other carbohydrates.

In an attempt to ascertain the influence of carbohydrate avail-ability on global transcriptional regulation, we searched our mi-croarray data for genetic alterations to the CcpA regulon, which isknown to include a broad range of transcripts encoding proteinsimportant for the uptake of carbohydrates and pyruvate metabo-lism. In Gram-positive organisms, CcpA orchestrates gene regu-lation by binding to a CRE consensus sequence located near thepromoter region of target operons (22, 48). Our results alsoshowed that the cofactor that is known to stimulate CcpA binding,HPr(Ser-P), was abundant in cells grown under conditions of glu-cose excess, whereas we were unable to detect HPr(Ser-P) andHPr(Ser-P)(His�P) in cultures of S. mutans grown with limitingcarbohydrate (Fig. 4). Using the online bioinformatics tool Reg-Precise (http://regprecise.lbl.gov/RegPrecise/), we identified 32genes within operons downstream of presumptive CREs amongthe genes that were found to be aberrantly regulated in our mi-croarray, which indicated that changes in expression of approxi-mately 18% of the genes affected in this study may be attributed, at

TABLE 3 RT-quantitative PCR confirmation of microarray results for S.mutans UA159 grown in 10 mM versus 100 mM glucose

Gene

Array RT-quantitative PCR

Foldchange P value

Foldchange P value

SMU.1424 (SMU.1422) 133.8 4.74E�04 3,626.28 1.50E�03SMU.114 (SMU.113) 30.5 2.50E�04 128.36 3.38E�02SMU.2038 25.3 1.29E�04 14.31 1.61E�02SMU.1591 3.65 8.13E�05 4.37 4.85E�03SMU.1437 0.25 3.07E�04 0.15 4.45E�02SMU.335 0.22 1.30E�04 0.12 4.55E�02SMU.29 0.049 6.79E�04 0.03 3.41E�02

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least in part, to CcpA (Table 4). Thus, HPr by itself and in con-junction with CcpA may contribute to the changes in the tran-scriptome and phenotype of the cells in response to carbohydrateavailability. However, it should be emphasized that the lack ofmajor differences in HPr levels or phosphorylation status betweenthe wild type and manL mutant means that ManL is a major con-tributor to modification of the transcriptome and phenotypic be-havior of S. mutans in response to glucose availability. We werealso able to identify several genes from our microarray study bear-ing predicted CodY binding sites after searching through RegPre-cise and previously collected microarray data, though there werefar fewer genes than the number predicted for the CcpA regulon(Table 4). Interestingly, our study of the affected regulons revealedthat the majority of genes of the CcpA regulon were upregulatedunder glucose-limiting conditions, whereas the majority of genespredicted to be under CodY regulation displayed elevated tran-scription when cells were grown with excess glucose. These dataindicate that CcpA and CodY may coordinate responses to carbo-hydrate availability and amino acid limitation.

It would be of interest to perform a comprehensive transcrip-tional analysis of the manL deletion strain grown under continu-

ous culture conditions with limiting and excess carbohydrate con-centrations. Unfortunately, the Pathogen Functional GenomicsResource Center no longer provides microarrays for S. mutans.With the remaining set of microarray slides that we did have onhand, we used RNA extracted from the manL mutant strain grownin continuous culture with 10 mM and 100 mM glucose to seewhether we could find significant changes in the transcriptome inthe mutant background in response to carbohydrate availability.We were not satisfied with the quality or with the coverage in theseexperiments. Consequently, we could not develop a comprehen-sive comparison of the parental and manL mutant strains andhave not included the data in this communication. However, incases where a sufficient amount of quality data was obtained for asubset of genes, a small set of genes was found to be differentiallyregulated (P 0.001) in the manL mutant compared to the wild-type strain in response to carbohydrate availability, and the resultswere confirmed using qRT-PCR. Since the results are of interest,the genes found to be differentially regulated by the microarrayand confirmed by qRT-PCR have been included in Table S1 in thesupplemental material, with the caveat that this does not representthe totality of differences in the transcription profiles of UA159

TABLE 4 Genes identified in a microarray of S. mutans UA159 grown in 10 mM versus 100 mM glucose that reside within an operon predicted topossess a CRE

Gene Product description Functional categoryFoldchange P value

SMU.1422 Putative pyruvate dehydrogenase E1 component beta subunit Energy metabolism 133.8 4.74E�04SMU.1423 Putative pyruvate dehydrogenase, TPPa-dependent E1 component alpha subunit Energy metabolism 124.93 4.50E�05SMU.113 Putative fructose-1-phosphate kinase Energy metabolism 30.5 2.50E�04SMU.179 Hypothetical protein Unknown 27.2 4.20E�06SMU.115 Putative PTS system, fructose-specific IIA component PTS 25.67 1.93E�04SMU.2038 Putative PTS system, trehalose-specific IIABC component PTS 25.3 1.29E�04SMU.180 Putative oxidoreductase, fumarate reductase Unknown 14.77 2.12E�05SMU.1879 Putative PTS system, mannose-specific component IID PTS 12.01 8.07E�04SMU.270 Ascorbate-specific PTS system enzyme IIC PTS 11.59 4.55E�05SMU.870 Putative transcriptional regulator of sugar metabolism Transcription regulator 9.48 8.40E�06SMU.402 Pyruvate formate-lyase Energy metabolism 8.56 8.21E�05SMU.500 Putative ribosome-associated protein Protein synthesis and fate 8.34 8.72E�05SMU.1878 Putative PTS system, mannose-specific component IIC PTS 7.45 6.79E�04SMU.2127 Putative succinate semialdehyde dehydrogenase Central intermediary metabolism 7.41 5.06E�05SMU.1843 Sucrose-6-phosphate hydrolase Energy metabolism 7 3.27E�05SMU.879 Multiple sugar-binding ABC transporter, permease protein MsmF ABC transporter 6.91 4.55E�04SMU.871 Putative fructose-1-phosphate kinase Energy metabolism 6.56 8.07E�04SMU.878 Multiple sugar-binding ABC transporter, sugar-binding protein precursor MsmE ABC transporter 6.54 1.52E�05SMU.880 Multiple sugar-binding ABC transporter, permease protein MsmG ABC transporter 6.49 3.94E�05SMU.1877 Putative PTS system, mannose-specific component IIAB PTS 5.93 5.50E�04SMU.1077 Putative phosphoglucomutase Energy metabolism 5.18 1.23E�05SMU.1088 Putative thiamine biosynthesis lipoprotein Biosynthesis of cofactors, prosthetic

groups, and carriers4.38 3.17E�05

SMU.872 Putative PTS system, fructose-specific enzyme IIABC component PTS 4.08 3.87E�04SMU.1591 Catabolite control protein A, CcpA Transcription regulator 3.65 8.13E�05SMU.1389 Hypothetical protein Energy metabolism 3.03 4.50E�04SMU.271 Putative PTS system, enzyme IIB component PTS 2.82 5.86E�05SMU.274 Putative L-xylulose 5-phosphate 3-epimerase Energy metabolism 2.55 6.42E�04SMU.1191 6-Phosphofructokinase Energy metabolism 0.44 6.13E�04SMU.1824c Transcriptional repressor CodY Transcription regulator 0.37 5.24E�04SMU.1823 Putative pyrazinamidase/nicotinamidase Biosynthesis of cofactors, prosthetic

groups, and carriers0.32 7.48E�04

SMU.611 ATP-dependent RNA helicase Transcription 0.24 5.39E�05SMU.396 Putative glycerol uptake facilitator protein Transport and binding 0.17 4.60E�06a TPP, thiamine pyrophosphate.

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and its manL mutant. Recently, our laboratory demonstrated thattranscriptome sequencing (RNA-Seq) is well suited for transcrip-tion profiling in S. mutans (14), so we plan to more thoroughlyprobe the extent of the ManL regulon as a function of carbohy-drate availability using chemostat culture and RNA-Seq.

The levels of EI and HPr changed very little in both wild-typeand manL deletion strains in response to the conditions that wetested (Fig. 3). We noted that the total protein level of HPr re-mained relatively stable between the conditions and the strainstested, similar to what was reported previously (31), and that thelevels of the ptsH transcript were not significantly altered (see Fig.S2 in the supplemental material). Although some studies havedemonstrated a 4- or 5-fold drop in EI levels when cultures weregrown in continuous culture with nitrogen limitation (carbohy-drate excess) (31, 32), we observed that the amounts of EI proteinand ptsI mRNA were not substantially altered in response tochanges in carbohydrate availability (see Fig. S2 in the supplemen-tal material). Thus, data from our study do not support the ideathat major changes in the amount of EI or HPr influence theregulation of carbohydrate transport. The differences between ourresults and those of others may arise from the use of differentculture conditions or strains.

In summary, by using steady-state continuous culture, we wereable to examine the effects of carbohydrate availability on the car-iogenic human pathogen S. mutans in the absence of confoundingeffects from pH, growth rate, and other influences that are knownto affect gene expression in this organism. Moreover, by usingstrain UA159, we were able to integrate the physiologic data withdata from complete genome sequence and transcriptomic analysisby use of a microarray. In addition, we further characterized thephysiology of a strain bearing a deletion in the glucose/mannoseEIIAB permease gene, manL, which has been shown to play adominant role in CCR of genes in S. mutans and to regulate mul-tiple virulence attributes of the organism (20, 25, 45, 51). Theresults reveal that there are profound changes in the expression ofgenes and the phenotypic properties of the organism that areknown to affect its establishment, persistence, and virulence inresponse to the amount of carbohydrate in the environment. Thestudies also enhance the body of evidence that ManL plays majorand direct roles in the control of key virulence attributes in re-sponse to carbohydrate source and availability.

ACKNOWLEDGMENTS

This study was supported by grant DE12236 from the National Institute ofDental and Craniofacial Research. Z.D.M. was supported by a Universityof Florida Alumni Fellowship.

We thank Matthew Watts, Kinda Seaton, and Jeong Nam Kim forhelpful discussions and technical advice while performing microarrays.

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